CA2618690A1 - Hemoglobin mutants with increased soluble expression and/or reduced nitric oxide scavenging - Google Patents

Abstract

A hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the alpha subunit: E11(Val-- > Leu) and E7(His--> Gln);
E11(Val-- > Phe or Trp) and E7(His-- > Gln); E11(Val-- > Phe or Trp or Leu) and E7(His--- > Gln) and G8(Leu-- > Phe or Trp); B10(Leu-- > Phe) and E4(Val-- > Leu); B10(Leu-- >
Trp) and E4(Val-- > Leu); B10(Leu-- > Trp) and E7(His-- > Gln); B10(Leu-- > Trp) and E11(Val--- > Phe);
B10(Leu-- > Trp) and E11(Val-- > Trp); B10(Leu-- > Trp) and E11(Val-- > Leu) and G8(Leu-- > Trp); B10(Leu-- > Trp) and E11(Val-- > Leu) and G8(Leu-- > Phe);
B10(Leu--- > Trp) and E11(Val-- > Phe) and G8(Leu-- > Trp); B10(Leu-- > Trp) and E11(Val-- >
Phe) and G8(Leu-- > Ilc); B10(Leu-- > Trp) and E7(His-- > Gln) and E11(Val-- > Leu) and G8(Leu-- > Trp); B10(Leu-- > Trp) and E11(Val-- > Trp) and G8(Leu-- > Trp);
E11(Val--- > Leu) and G8(Leu-- > Phe); E11(Val-- > Leu) and G8(Leu-- > Trp); B13(Met-- > Phe or Trp); G12(Leu-- > Phe or Trp); or B14(Phe-- > Trp).

Description

DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

CECI EST LE TOME DE _2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

JUMBO APPLICATIONS / PATENTS

THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.

THIS IS VOLUME OF ,2 NOTE: For additional volumes please contact the Canadian Patent Office.

HEMOGLOBIN MUTANTS WITH INCREASED SOLUBLE
=EXPRESSION AND/OR REDUCED NITRIC OXIDE SCAVENGING
Field of the Invention The invention relates to novel hemoglobin mutants having one or more desired functions, including reduced nitric oxide scavenging and incmased. soluble expression.
The invention further relates to methods of increasing soluble expression of recombinant hemoglobin by the addition of excess heme.
BackQround of the Invention Hemoglobin (Hb) is the oxygen-carrying component of blood that circulates through the bloodstream inside small enucleate cells known as erythrocytes or red blood cells. It is a protein comprised of four associated polypeptide chains that bear prosthetic groups known as hemes. The structure of hemoglobin is well known and described in Bunn & Forget, eds., Himoglobin: Molecular. Genetic and Clinical A== (W.B. Saunders Co., Philadelphia, PA: 1986) and Fermi & Perntz "Hemoglobin and Myoglobin," in Phillips and Richards, Atlas of Molecular Structures in B_iologv (Clarendon Press: 1981).
Expression of varioos recombinant hemoglobins containing naturally-occurring and non-naturally occurring globin mutants has been achieved. Such expression methods include individual globin expression as described, for example, in U.S.
Patent. No. 5,028,588, and di-alpha globin expression created by joining two alpha globins with a glycine linker through genetic fusion coupled with expression of a single beta globin gene to produce a pseudotetrameric hemoglobin molecule as described in WO 90/13645 and Looker et al., Nature 356:258-260 (1992). Other modified recombinant hemoglobins are disclosed in PCT Publication WO 96/40920. Similar to other heterologous proteins expressed in E. coli, recombinant hemoglobins have N-tcnninal methionines, which in some recombinant hemoglobins replace the native. N-terminal valines.
The process cost of producing recombinant hemoglobin is affected by the yield of soluble protein. Economic production of heterologous protein in E.coli is especially challenging when the protein must not only be soluble and functional, and it is also composed of multiple subunits as for recombinant hemoglobin. In addition, recombinant hemoglobins require the enhanced presence of essential co-factors (prosthetic groups) such as heme and flavins through supplementation or increased endogenous production. In E.coli, soluble accumulation of necombinant henzoglobin is limited by heme availability as indicated by the fact that without heme supplementation, addition of S-ALA, a heme precursor, increases heme and protein accumulation.
Consequently, methods of increasing soluble yield are highly desirable.
However, prior to the present invention, the effect of mutations on soluble E. coli expression had not been studied nor were the detenliinants of soluble yield thoroughly understood.
Thus, a need exists for methods of increasing soluble yield of recombinant hemoglobin.
A need also exists for methods of reducing scavenging of nitric oxide ("NO") by extracellular hemoglobin. Mild hypertension has sometimes been observed following administration of certain extracell ular hemoglobin solutions. It is believed by many that the hypertension is due to depletion of nitric oxide ("NO") in the wall of the vasculature based in part on the known high affinity of deoxyhemoglobin for NO
(Schultz et al., J. Lab. Clin. Med.122:301-308 (1993); Thompson et al., J.
Anpl.
Physiol. 77:2348-2354 (1994); Rooney et al., Anesthesiology 79:60-72 (1993)).
Extravasation of the hemoglobin into endothelial cells or interstitial spaces may _ cause significant consumption of NO (Gould et al., World J. Sur~. 20: 1200-1207 (1996)).
-A recent study also suggests that the oxidative reaction of NO with the bound OZ of oxyhemoglobin may be of gre,ater significance in vivo than simple binding to the iron .20 atom as reported in Eich et al., Biochemistrv 35: 6976-6983 (1996). Eich et al.
showed that steric hinderance introduced by substitution of amino acids adjacent to bound oxygen can markedly lower the rate of NO-induced oxidation.
Nitric oxide acts as a chemical messenger in the control of many important processes in vivo, including neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. The biological actions of nitric oxide are mediated by binding to and activation of soluble guanylyl cyclase, which initiates a biochemical cascade resulting in. a variety of tissue-specific responses (Feldman et al., Chem. En~.News Dec: 26-38 (1993)).
Elucidating the functions of nitric oxide has depended largely on inhibition of the NO-generating enzyme, nitric oxide synthase. Most conclusions about the effects of cell-free hemoglobin'have been drawn based on experiments involving NO
synthase inhibitors and/or NO donors.
While the rapid, high-affuuty binding of nitric oxide to deoxyhemoglobin. is well known, the importance of the oxidative reaction between NO and oxyhemoglobin is not as. widely appreciated. In this reaction, the NO molecule does not bind to the heme, but reacts directly with the bound oxygen of the Hb02 complex to form methemoglobin and nitrate (Doyle et al., J. Inorg, Biochem. 14: 351-358 (1981)). The chemistry is analogous to the rapid reaction of NO with free superoxide in solution (Huie et al., Free Rad. Res. Comms. 18: 195-199 (1993)). Both the heme iron and nitric oxide become oxidized by the bound oxygen atonis, and the reaction occurs so rapidly that no replacement of 02 by NO is observed (Eich et al., su ra.).
Since nitric oxide is produced and consumed on a continuous basis; there is a natural tumover of NO in vivo. When a cell-free hemoglobin is administered, the balance between NO production and consumption is altered by reactions with hemoglobin. The most relevant parameter for NO scavenging by oxyhemoglobin is the rate of reaction with NO, not the position of the Hb allosteric (R/T) equilibrium. The oxidative reaction is irreversible, and NO binding to deoxyhemoglobin is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours (Moore et al., J. Biol. Chem. 251: 2788-2794 (1976).
Once an NO molecule reacts with oxyhemoglobin or deoxyhemoglobin, it is eliminated from the pool of signal molecules causing cxrtain adverse conditions. For example, hemoglobin can bi~d nitric oxide causing the prevention of vascular relaxation and potentially leading to hypertension that is sometimes observed after administration of certain extracellular hemoglobin solutions. In addition, the ability of NO
to oxidize oxyhemoglobin producing peroxynitrite and methemoglobin could also lower free concentrations of NO and lead to hypertension.
Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation.
Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A2 and serotinin. These compounds nmy act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in an significant vasoconstriction.
- In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn can lead to ceU wall damage.
Endothelial ceU
wall damage has been observed with the infusion of certain hemoglobin solutions (White et al., J. Lab. Clin. Med. 108:121-181 (1986)).

Accordingly, a need exists for new hemoglobin mutants with decreased NO-scavenging while still functioning as an effective oxygen carrying agent. The present invention satisfies this need as well as the need for increased soluble yield of recombinant hemoglobin.
Summarv of the Invention The invention relates to hemoglobin mutations that confer certain desired functions, such as increased yield of soluble Hb and 'reduced NO scavenging.
The hemoglobin mutants can be designed to include these functions separately or in combination.
Therefore, in one aspect, the invention is directed to introducing a mutation into one or more beta and/or alpha subunits of recombinant hemoglobin to increase the yield of soluble recombinant hemoglobin compared to the unmodified protein. The methods are accomplished by:
(a) incorporating into a host cell a vector containing a gene encoding a globin having at least one mutation that directs high soluble expression of the recombinant hemoglobin;
(b) inducing the host cell to express soluble recombinant hemoglobin; and (c) purifying the soluble recombinant hemoglobin.
Useful mutations in the beta subunit include D73I, D73M, D73E, D73T, D73Y, K82D, K82E, K82G, K82P, K82Q, K82S, K82N, N 102A and N 102V.
Altematively, or in_ combination with the above method, high expression levels of soluble hemoglobin can be obtained by adding excess hemin, preferably at least a 2.5-fold molar excess of hemin.
In another aspect, the invention further relates to novel hemoglobin mutants having reduced rates of reaction with nitric oxide. Mutations in the alpha subunit having desired rates of reaction include:
El l (Val-4Leu) B 10(Leu-->Trp) + E7(His--+Gln) B 10(Leu-+Trp) + El l(Val-Whe) B l 0(Leu-.)Trp) + E 11(Val-+Leu) + G8(Leu-->Trp) B 10(Leu-4Trp) + El l(Val-+Phe) + G8(Leu--~Trp) B 10(L,eu--)Trp) + El l(Val-4Phe) + G8(Leu-Ile) B 10(L.eu4Trp) + E7(His-->Gln) + El 1(Val-+I.eu) + G8(Leu-+Trp) B 10(Leu-4Trp) + El 1(Val-4Tip) + G8(Leu-+Trp) El l (Val-+Leu) + G8(Leu--Whe) Ell(Val-iNLeu) + G8(Leu-+Trp) B 13(Met-+Phe or Trp) G12(Leu-*Phe or Trp), and B 14(Phe-~Trp) Mutations in the beta subunit having desired rates of reaction include:
El I (Val--4Leu) B 13(1.eu-->Phe or Trp) G 12(Leu-4Phe or Trp) B 14(L.eu--fte or Trp) G8(Leu-4Phe) + G 12 (Leu-+Trp) El 1( V al-+Leu) + G 8(Leu-+Trp) El l (Va)-->Trp) + G8(Leu-+Met) El 1(Val-+L,eu) + G8(Leu-->Phc) El 1(Val-+Leu) + G8(Lx.u-+Met) El l (Val-+Phe) + G8(Leu--+De) El I (Val-+Phe) + G8(Leu--+Phe) El l (Val-Phe) + G8(Leu-;Trp) El l (Va]-Rhe) + G8(Leu-+Met) El 1(Val-->Met) + G8(Leu--+Trp) E11(Ya1-+Met) + G8(Leu-ffrp) + E7(His-->Gln) El 1(Va1-+Trp) + G8(Leu-4Ile) E7(His-+Gln) E7(His--*Gln) + El I (Va1-+Trp) E7(His-+Gln) + El ] (Val--+I,eu) E7(His-+Gln) + E11(Val-Whe) E7(His-+G1n) + E11(Val-Whe) + G8(I.eu-+Phe or Trp) E7(His-+Gln) + El l(Val-.)Leu or Trp) + G8(Leu-fte or Trp) E7(His-+Phe) . ' E l l(Val-4Tip or Phe) + G 12 (Leu-+Trp or Met) El 1(Val-4Trp or Phe) + B 13 (Leu-Mp or Met) B 10(L,eu-4Trp) + B 13 (Leu-~Trp or Met) B 10(Leu-+Phe) + B 13 (Leu-4Trp) B l 0(Leu-4Trp or Phe) + G 12 (Leu-+Trp) B 10(Leu-+Phe) + G 12 (Leu-+Met) E11(VaI-4Phe, Met or Leu) + G8(Leu-+Trp, Met or Phe) G8(I.eu-+Phe) + G12(Leu-+Trp) G8(I,eu-+Trp) + G12 (Leu-4Trp or Met), and G8(Leu-4Trp) + B 13 (Leu-+Trp or Met) Preferably, such mutations have rate constants for reaction of NO with oxyhemoglobin of less than 25 M-ls 1, more preferably between 12 and 15 1Vrls'', and most preferably less than 5 M"ls"l.
According to an aspect of the present invention, there is provided a hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the alpha subunit:
E11(Val-->Leu) and E7(His-->Gln);
E11(Val->Phe or Trp) and E7(His->Gln);
E11(Val-->Phe or Trp or Leu) and E7(His->Gln) and G8(Leu-->Phe or Trp);
B10(Leu-->Phe) and E4(Val->Leu);
BlO(Leu-->Trp) and E4(Val-->Leu);
BlO(Leu-->Trp) and E7(His-->Gln);
BlO(Leu-->Trp) and E11(Val-->Phe);
B10(Leu->Trp) and E11(Val-->Trp);
BlO(Leu-->Trp) and E11(Val->Leu) and G8(Leu->Trp);
BlO(Leu-->Trp) and E11(Val-->Leu) and G8(Leu-->Phe);
BlO(Leu-->Trp) and E11(Val-->Phe) and G8(Leu->Trp);
BlO(Leu-->Trp) and E11(Val-->Phe) and G8(Leu->Ilc);
BlO(Leu-->Trp) and E7(His->Gln) and E11(Val->Leu) and G8(Leu->Trp);
BlO(Leu-->Trp) and E11(Val-->Trp) and G8(Leu-->Trp);
E11(Val->Leu) and G8(Leu-->Phe);
E11(Val-->Leu) and G8(Leu-->Trp);
B13(Met-->Phe or Trp);
G12(Leu->Phe or Trp); or B14(Phe->Trp).

According to another aspect of the present invention, there is provided a hemoglobin mutant with reduced rate of reaction with nitric oxide selected. from the following mutation in the beta subunit:
B13(Leu->Phe or Trp);
G12(Leu->Phe or Trp);
B10(Leu->Phe) and E4(Val->Leu);
B10(Leu->Trp) and E4(Val->Leu);
B14(Leu->Phe or Trp);
G8(Leu->Phe) and G12(Leu->Trp);

Ell(Val-->Leu) and G8(Leu->Trp);
E11(Val-->Trp) and G8(Leu-->Met);
Ell(Val-->Leu) and G8(Leu->Phe);
E11(Val-->Leu) and G8(Leu-->Met);
Ell(Val-->Phe) and G8(Leu-->Ile);
E11(Val-->Phe) and G8(Leu-->Phe);
E11(Val-->Phe) and G8(Leu->Trp);
E11(Val-->Phe) and G8(Leu-->Met);
E11(Val-->Met) and G8(Leu-->Trp);
E11(Val-->Met) and G8(Leu-->Trp) and E7(His->Gln);
E11(Val-->Trp) and G8(Leu-->Ile);
E7(His-->Gln) and E11(Val-->Trp);
E7(His-->Gln) and Ell(Val-->Leu);
E7(His-->Gln) and E11(Val-->Phe);
E7(His-->Gln) and Ell(Val-->Phe) and G8(Leu-->Phe or Trp);
E7(His-->Gln) and E11(Val-->Leu or Trp) and G8(Leu->Phe or Trp);
E11(Val-->Trp or Phe) and G12(Leu-->Trp or Met);
E11(Val-->Trp or Phe) and B13(Leu-->Trp or Met);
B10(Leu-->Trp) and B13(Leu-->Trp or Met);
B10(Leu-->Phe) and B13(Leu->Trp);
B10(Leu-->Trp or Phe) and G12(Leu->Trp);
B10(Leu-->Phe) and G12(Leu-->Met);
G8(Leu-->Trp) and G12(Leu-->Trp or Met); or G8(Leu-->Trp) and B13(Leu->Trp or Met).
According to yet an aspect of the present invention, there is provided a hemoglobin mutant having the following mutations:
alph aB 10(Leu-->Trp), alphaE7(His-->Gln) and beta E11(Val-->Trp); or alpha B10(Leu-->Phe), alpha E7(His-->Gln) and beta G8(Leu->Trp).
7a Brief Description of the Drawings Figure 1 shows the accumulation of solube hemoglobin (Hb) for three variants of soluble rHb with a di-a K158C mutation up to 16 hours post induction. Theses are: rHb di-a K158C/DN1O8K (SGE3OS3; circles), rHb di-a. K158C/OK82D (SGE3084; squares) and), rHb di-a K158C/DN108K,K82D (SGE3172; triangles) accumulation for 16 hours after induction. Heme supplementation was made at 0, 3, 6, 9 and 12 ours post induction to a final concentration of 0.63mM hemin. Each symbol represents the average from four fermentations except for SGE3083 data, which are the average of two.
Figure 2 shows the accumulation of soluble rHb9. 1 (expressed as total hem concentration) as a function of the hemin supplied to the fermentations.
Filled circles represent each of 70 individual samples. One heme site is present in each alpha and beta subunit resulting in four per hemoglobin molecule. The heavy line represents a 3-fold molar excess of hemin to heme binding sites in hemoglobin. The dashed line represents a 2.5-fold molar excess of hemin.
Figure 3 shows hemin uptake and hemoglobin production. The filled squares show independent measurements of hemin loss from the fermentation broth in pMoles of hemin required for each level of rHb accumulation, assuming 4 heme molecules per rHb molecule.
Figure 4 shows the plasmid pSGE728 which was used for certain mutagenesis and expression experiments.

Figure 5 shows the partial restriction map of the alpha gene inserted into pSGE728.
Figure 6 shows the partial restriction map of the beta gene inserted into pSGE728.
Figure 7 shows the normalized time, course of the NO-induced oxidation of recombinant oxyhemoglobin. The recombinant hemoglobin constructs rHbO.0, rHb0.1, rHbl.1 and rHb Bethesda all have wild-type amino acids in their heme pockets and an NO-oxidation rate constant of 60 M'ls 1. Distal h , 7b rHb3 and rHb4 have rate constants of 24, 15 and 2 M-'s'', respectively.
Conditions after mixing were 0.2 M oxyhemoglobin (heme), 1.0 pM NO, 20 C. The heme pocket substitutions in rHb2 are aEl l(Val-+Phe), .PE11(Val-4Phe), PG8(Leu-+1Ie).
In rHb3 the substitutions are a El 1(Val-Leu), PEl l(Val-+Phe) and in rHb4 they are a B l0(I.eu-+Phe), ~ El 1(Vai-+Trp).
Figure 8 shows the pressor effects of recombinant hemoglobins and human serum albumin (HSA). Changes in mean arterial pressure from pre-administradon values are plotted versus time from administration. All hemoglobins contained a genetically fused dialpha subunit to prevent dissociation into ap dimers. The rate constant for reaction of nitric oxide with each rHb is noted beside each data set. HSA
indicates the pressor data collected when 5% HSA was administered as a volume control. Protein doses were 350 mg/kg for each rHb. rHb l. l(0 , n=6), rHb2 (O
, n=6), rHb3 (0 , n=6), rHb4 (0, n=6), 5% HSA (A , n=9).
Figure 9 shows the pressor responses to rHb1.1 and rHb Bethesda. Both molecules contained a genetically fused dialpha subunit to prevent dissociation into , alpha/beta dimers. Meai arterial pressure data are plotted versus time from administration. -'rHbl.l has a Pso of 32 mmHg, and rHb Bethesda has a P50 of 2.7 mmHg. Both hemoglobins have a value of about 60 M's" for the. rate of NO
oxidation, k'. x. Circles (r) reprosent data for rHbl.l (n=6), and squaros (M) denote data for rHb Bethesda (n=6). The two hemoglobins have identical rates of reaction with NO but significantly different P50.
Figure 10 shows the direct relationship between the magnitude of the pressor response and the rate of NO oxidation. The magnitude of the pressor response was calculated by averaging the post-infusion values for each time course in Figure 8, and plotted against the measured value of k',,,o, o,, for the corresponding hemoglobin. The data point at the y-intencept was collected following administration of an equivalent volume of 5% HSA.
Figure 11 shows the average total peripheral resistance (TPR) response to recombinant hemoglobins. The average TPR was calculated from data collected starting 20 minutes and ending 90 minutes following administration. The dose used in each case was 350 mg Hb/kg body weight. "Mono Hb" refers to single-tetramer Hb species (MW about 64,000), and "di Hb" indicates two Hb tetramers that are.

genetically fused (MW about 128,000). The rate constant for NO-oxidation of each rHb is noted on the abscissa.
Figure 12 shows the TPR responses to thnx recombinant hemoglobins.
Changes in TPR are plotted as percent of pre-administration values versus time from administration. All hemoglobins contained a genetically fused dialpha subunit to prevent dissociation into ap dimers. The rate constant for reaction of nitric oxide with each rHb is noted beside each data set. Hemoglobin doses were always 350 mg/kg.
Figure 13 shows the results of the gastric emptying model of GI motility.
Greater scavenging of nitric oxide, or inhibition of synthesis of nitric oxide, decreases emptying of the stomach. The dose used for each protein was 750 mg Hblkg body weight (10% solution) and the dose for L-NAME was 10 mg/kg.

Detailed Descrintion of the Invention , The invention generally relates to hemoglobin mutations that, either separately or in combinantion, confer increased soluble expression or reduced NO
scavenging.
Thus, in one aspect, the invention relates to methods of producing high soluble expression of recombinant hemoglobin in a host cell. It has now beendiscovered that high soluble expression of recombinant hemoglobin ("rHb") can be obtained by introducing certain mutations into the globin subunits separately or in combinantion with the addition of excess heme. As used herein, the term "high soluble expression"
means soluble expression -levels of recombinant hemoglobin that are greater than expression levels obtained without an alteration that improves soluble expression and/or the addition of at least about a 2.5-fold molar excess of hemin. Preferably, high soluble expression levels include an increase of at least 5%, more preferably at least 10%, and even more preferably at least 50%.
The methods of the present invention are generally accomplished by:
(a) incorporating into the host cell a gene encoding a globin having at least one mutation that directs high soluble expression of the recombinant hemoglobin;
(b) culturing the host cell to express soluble recombinant hemoglobin with or without the addition of excess hemin; and (c) purifying the soluble recombinant hemoglobin.
In one embodiment of the invention, a desired hemoglobin or hemoglobin-like molecule can be altered or modified to contain one or more mutations to enhance its soluble yield. Preferably, the non-modified hemoglobin is human hemoglobin.

However, hemoglobin from other species are also candidates for increased soluble expression including, for example, primate, bovine, fowl, fish, shark and other invertebrates such as Ancniia, earthworm, sea lamprey, moiluse and marine bloodworms.
Suitable recolnbinant hemolgobin candidates for incrr.ased soluble expnrssion include wholly or partially wildtype hemoglobins. In another embodiment, the recombinant hemoglobin can be naturally or non-naturally oeewring hemoglobin mutants containing mutations in the alpha and/or. beta globins as described, for example, in U.S. Patent No. 5,028,855. Such mutations can confer other desired characterisitics in addition to enhancement of soluble expression, such as high or low ogygen affinity resistance or cooperativity, enhancements in stability and assembly rates, decreased heme loss rates or autooxidation rates, or resistance to proteolytic degradation and aggregation.
Other recombinant hemoglobins suitable for increased soluble expression includes the hemoglobin-like molecules (pseudotetramers) disclosed in U.S. Patent Nos. 5,449,759 and 5,545,727. The mutant and modified hemoglbins described in U.S. Patent No. 5,559,907 and WO 96%40920 and WO
97/23631, are also suitable target proteins for enhanced solution expression.
Mutations that diroct high soluble expression can appear in the alpha and/or beta globin. Although not wishing to be bound by any particular theory, it is believed a.
mutation that stabilizes hemoglobin or increases assernbly or folding rates -can also lead' to improved- soluble yield. Accordingly, mutations that stabilize alpha helices, such as alanine substitutions, or that increase hydrophobic core packing, for example, by .25 substituting intcrnal' hydrophilic amino acids with hydrophobic amino acids, are generally useful mutations for the methods of the present invention: Other generally useful mutations include: (1) those that stabilize the alphalbetaz intcrface, such as naturally occurring mutant Hb. Kansas; (2) substitutions with charged amino acids at surface sites, such as the naturally occunring mutant Hb Vancouvet; and (3) substitutions with polar or charged amino acids at the diphosphoglycerate-binding site.
Amino acid substitutions at beta surface site D73 that have a volunp- between that of aspartate and lysine ane also generally -useful mutations.. Such antino acids include aspartate, valine, asparagine, leucine, isoleucine, glutamine, methionine, glutamate, histidine and lysine.

For example, the naturally occunring Providence mutant occurs at a key residue in the DPG binding cleft (K82D or Lys82--*Asp) between the two beta subunits in the hemoglobin tetramer. This anion binding cleft is lined with at least six positively charged residues, three from each beta globin (His2, His143, and Lys82). In the absence of DPG or inositol hexaphosphate, such as during accumulation of rHbs in E.
coli, an electrostatic repulsion between these residues may destabilize or even partially denature the beta globin structure. Substitution of the normally occurring positively charged lysine by a negatively charged aspartate introduces a counterion to the DPG
pocket, which may form an electrostatic interaction with His 143, is likely to stabilize the beta chain. This substitution could account for the improved soluble accumulation of rHb9. l versus rHb0.1, which contains the wild type lysine at position 82.
The charge substitution may also increase the rate of hemoglobin assembly by reducing the electrostatic repulsion in this region. Differences in accumulation of stable hemoglobin variants in humans appears largely correlated with differences in subunit assembly rates.

It is likely that the same Lys82-~Asp (Providence) substitution could stabilize and improve soluble accumulation of other recombinant hemoglobin molecules, since t all are typically expressed without DPG present. This was observed with a di-a mutant rHb, Lys 158-4Cys, in which Providence ("Prov") more than doubled soluble accumulation. The addition of the Providence mutation to rHbl.1, to create rHb9+1.1, achieved a greater than 2-fold increase in soluble expression, rescuing the Presbyterian mutant by restoring the soluble expression to wild type levels. In addition to improving soluble accumulation, rHb variants also increased total globin accumulation implying that the soluble hemoglobin is more stable than the insoluble protein. This result is consistent with pulse chase experiments in which insoluble globin was observed to have a much shorter half life than soluble globin. Thus the greater amount of globin maintained in soluble form, the greater the total accumulation expected, since the insoluble globin is more rapidly removed.
Useful beta mutations for high soluble expression include, for example, D731, D73M, D73E, D73T, D73Y, K82D, K82E, K82G, K82P, K82Q, K82S, K82N, N 102A and N 102V. A number of these variants have soluble expression yields gn rater than the corresponding recombinant hemoglobins with the wild-type amino acid at the target mutation site. The nomenclature used in referring to a particular mutation first identifies 'the wild-type amino acid, followed by the residue number and finally the substitute amino acid. For example, D731 means that aspartate has been substituted with isoleucine at residue number 73.
Combinations of mutations am also useful for high soluble expression.
Examples of useful beta globin mutant combinations are listed in Tables I and 2. In Table 2, certain strains and their descriptions are listed to provide information about the basic strains that can be altered with useful beta mutations. For example, SGE3011 is the basic strain used to produce SGE3010 in addition to several others.

Table I
Beta Mutant Combinations Providence + Tilburg Providence + Vancouver SGE3011-beta (V67W) + D73E
SGE301 l beta *(V67W) + D731 SGE3011 beta (V67W) + D73Y (Vancouver) SGE3011 beta (V67W) + D73E + K82D (Providence) SGE3011 beta (V67W) + D731 + K82D (Providence) SGE301 l beta (V67W) + D73Y (Vancouver) + K82D (Providence) SGE3011 beta (V67W) + K82E
SGE3Q11 beta (V67W) + K82G

Table 2 Description Strain SGE3004 diaV62LIPV67F
SGE3005 3004 + St. Mande SGE3263 3004 + Providence + Presbyterian SGE3173 di-dia 3004 SGE2821 diaL29F, H58Q/PL106W
SGE1737 2821 + Presbyterian SGE3006 2821 + Prov + St. Mande SGE 3007 2821 + Prov + Pres SGE2822 di-dia 2821 SGE1738 2822 + Pres SGE3008 2822 + Prov + St. Mande SGE3009 2822 + Prov + Pres .
SGE3001 diaL29F/PV67W
SGE1728 3001 + Pres SGE1729 3001 + Prov di-dia 3001 SGE301l diaL29W,H58Q/PV67W
SGE3010 3011 + Prov SGE2966 3011 + Pres SGE2968 3011 + Ptov + Pres SGE2967 3011 + St. Mande SGE2963 di-dia 3011 SGE2971 di-dia 3011 +.Prov SGE2969 di-dia 3011 + Pres SGE2972 di=dia 3011 + Prov + Pres SGE2970 di-dia 3011 + St. Mande 3rR3012 diaL29W. H58O/BL106W
Zlie prewnt invention further provides novel DNA sequences that encode the desired mutations that enhance soluble expression, some of which are identified in Table 16. For example, DNA that ultimately results in the Providence mutation is K82N, but this residueundergoes hydrolysis to yield K82D in vivo. Therefore, DNA
directly encoding for aspartate is novel.
In general, those skilled in the art can constroct genes encoding a mutant globin having at least one mutation that directs high soluble expression of the recombinant hemoglobin by techniques well-known in the art, including by complete DNA;
synthesis or by standard recombinant DNA methods.(see, for example., Sambrook, et aL,.
Molecular Cloning: A Labo'ratorv Manual. 2nd ed. Cold Spring Harbor lraboratory Press, Cold Spring Harbor, NY. (1989)). Methods for incorporating the desired mutations are well known in the art and include, for example, site-directed mutagenesis. Random mutagenesis is -also useful for generating a number of mutants at a particular site. Other recombinant techniques are also known, such as those described in U.S. Patent No. 5,028,588, U.S. Patent No. 5,545,727, U.S. Patent No.
5,599,907, PCT Publications WO 96/40920 and WO 97/04110.

The genes can be used to construct plasmids that are inserted into appropriate host cells according to conventional methods or as described in WO 96/4092Q
Any suitable host cell can be used to express the novel polypeptides. Suitable host cells include, for example, bacteria, yeast, mannnalian, plants and insect cells and transgenic animals. E. coli cells are particularly useful for expressing desired recombinant hemoglobin.
The transformed host cell is then cultured or fermented until soluble hemoglobin is harvested for purification. Purification of the soluble fraction can be accomplished using the methods described in WO 95/14038, or by following the guidance provided in the examples below.
In a further embodiment of the invention, exoess hemin can be added to obtain high soluble expression of recombinant hemoglobin. As used herein, "excess heme"
means at least a 2.5-fold molar excess of hemin to heme binding sites in hemoglobin.
Measurements of the insoluble protein content in fermentations yielded evidence that reduction in soluble hemoglobin was due to conversion into insoluble globin. Loss of heme is a mechanism for turnover of soluble hemoglobin into insoluble globins that has been well-characterized as described in Mitraki .& King, Bio/Tch.. 7:690-(1989). The rate of turnover was consistent with an 11 hour half-life of rHb1.1 in E.
coli flask cultures (Weickert & Curry, Arch. Biochem. Biophvs., 348:337-346 (1997)), raising the possibility that when heme becarne limiting (dropped below a required stoichiometric level), soluble accumulation ceased, and turnover of rHb was driven primarily by heme loss, followed by insoluble aggregation.
Many unstable mutant human Hbs found in vivo in inclusion bodies contain heme, as do in vitro heat precipitated forms of mutant Hbs (Rachmilewitz, Seminars in Hematology, 11:441-462 (1974)). Heme may stabilize some globin conformations even in insoluble aggregates, and its absence from one or mone of the four hemoglobin subunits, may lead to insolubility and rapid turnover of the subunits (Weickert &
Curry, suFra). Although globin can accept heme before it is released from the ribosome in eukaryotic cell free translation systems, it is unclear whether this occurs in bacteria. Unlike in eukaryotes, globin probably cannot complete folding to its native configuration until its release from the ribosome in E. coli (Netzer & Hartl, IVature.
388:343-349 (1997)). Therefore, without heme, apo-globin can be likened to a trapped folding intermediate.
In the experiments described in the Examples below, the expression strains and plasmids were identical, as were the fermentation and induction conditions.
Therefore, approximately the same amount of protein synthesis occurred for each rHb variant. No significant differences in cell growth were observed suggesting that rHb variants did not have different toxic effects on the cells, which could have accounted for the differences in the percent of the soluble protein accumulating as soluble rHb.
The extracellular heme concentration was manipulated to study the influence on intracellular heme concentration. The use of identical expression strains imposes the same heme transport (diffusion) and biosynthetic capacity for cells expressing each rHb variant. The E. coli strains were sufficiently heme permeable to allow a heme protein, rHb9.1, to accumulate to almost 40% of the soluble protein in the cell. Since very high soluble expression of rHbs requires more than a 2.5-fold molar excess of heme, this excess is believed to be required for sufficient diffusion across the cell membrane to maintain an intracellular heme concentration high enough to drive the bimolecular reaction of heme with globin. Fermentations not supplemented with heme accumulated only. about 12-15% of the soluble rHb0.1 achieved when hemin was supplemented, indicating that biosynthesis of heme contributes little to the soluble globin and therefore to the heme pool within the cell.
In another aspect, the present invention also relates to novel mutant hemoglobins that have significantly reduced rates of reaction with nitric oxide or NO
scavenging. As such, the mutant hemoglobins of this aspect of the present invention are also referred to herein as "NO mutants."

The NO mutants have one or more mutations in or around the heme pocket.
Heme is an iron-containing porphyrin that serves as a prosthetic group in proteins such as hemoglobin, myoglobin and the cytochromes. In hemoglobin, the heme appears in a cleft between the E and F helices in the globin subunits. The heme iron is linked covalently to the imidazole nitrogen of the "proximal" F8 histidine, while the distal E7 histidine and El I valine appear near the access of oxygen to the heme pocket.
The residues of the heme pocket include those residues that are on a nearest atom-to-nearest atom basis within 6 angstroms, and preferably within 4 angstroms, of the heme moiety (Fermi, et al. (1984) J. Mol. Biol. 175: 159-174). For alpha globin, the heme pocket residues include:

Distal residues:

First shell Second Shell B l 0 Lzu B 13 Met CEl Phe CE3 His E7 His. CE4 Phe El 1 Val El0 Lys G8 Leu E14 Ala 0 12 Leu B 14 Phe Proximal residues:
First shell Second Shell F8 His C7 Tyr F4 Leu F7 Leu . FG3 Leu FG5 Val G4 Asn G5 Phe H15 Val H19 Leu and for beta globin:

Distal residues:

First shell Second Shell B 10 Lzu B13 Leu CD I Phe . CD3 Ser E7 His B 14 Leu El l Val CD4 Phe G8 Leu ElOLys E14 Ala G12Lzu Proximal residues:
First shell Second Shell F8 His C7 Phe F4 Leu F7 Leu FG3 Lzu FG5 Val G4 Asn G5 Phe G12 Lzu H15 Val H19 Lzu The first shell residues are those residues in close or direct contact with the heme iron atom and/or the bound ligand, while second shell, residues are those amino acids which are not in direct contact with the heme or the bound ligand, but are in direct contact with first shell residues. The term "heme pocket residues" include these, first and second shell residues.
Various terms are used herein in describing this aspect of the present invention.
Unless defined otheiwise, all technical and scientific terms used herein have the same 35. meaning as commonly understood by those skilled in the art.
For the purposes of this invention, "naturally occurring human hemoglobin,"
"native hemoglobin," "wild type hemoglobin" or "conventional hemoglobin" refer to human hemoglobin A. whose alpha and beta globin amino acid sequences are given in Figure 1 of U.S. Patent No. 5,028,588. Note that it is conventional to identify the helical segments of the globin subunits by letters, for example, the proximal histidine of the alpha chain or the beta chain is termed F8 (residue 8 of helix F). The non-helical segments are identified by letter pairs, indicating which helical segments they connect, for example, non-helical segment BC connects helix B and helix C. The helical notation and corresponding amino acids for alpha and beta globin are shown in Table 4 of U.S. Patent No. 5,028,588.

'Recombinant hemoglobin" means hemoglobin, whether native or mutant, comprising alpha-like globin proteins and/or beta-like globin proteins, at least one of which is obtained by expression of a globin gene carried by a recombinant DNA
molecule in a cell other than the cell in which ihat hemoglobin gene and/or' hemoglobin protein is naturally found. In other words, the hemoglobin gene is heterologous to the host in which it is expressed. For example, the expression of any human hemoglobin gene in any cell other than a human blood cell would be considered to be a recombinant hemoglobin.
As used herein, an "alpha globin" has at least about 75% sequence idendty with native human alpha globin. However, a polypeptide of lesser sequence identity may still be considered substantially homologous with native human alpha globin, and thus may be an alpha globin, if it has a greater sequence identity than would be expected from chance and also has the characteristic higher, structure of native hunian alpha globin and similar biological activity. Likewise, a "beta globin" has. at ieast about 75%
sequence identity with narive human beta globin. However, a polypeptide of lesser sequence identity may still be considered substantially homologous with native human beta globin, and thus may be a beta globin, if it has a grrater sequence identity than would be expected from chance and also has the characteristic higher structure of native human beta globin and similar biological activity.
"Liganded hemoglobin" means hemoglobin to which a ligand is bound at the heme groups. Common preferred ligands include, but are not limited to 02, CO, NO
.25 and the like.
"Oxyhemoglobin" means hemoglobin in which an oxygen molecule is bound to the functional oxygen binding sites in each subunit.
"Deoxyhemoglobin" or "unliganded hemoglobin" rneans any hemoglobin -to which no ligand is bound to the alpha globin, the beta globin, and/or any functional heme prosthetic group.
"Methemoglobin" or "oxidized hemoglobin" means any hemoglobin in which the iron has been oxidized to the ferric state.
"R state hemoglobin" is the high affmity stat,e of hemoglobin and is the dominant form of hemoglobin when a]igand is bound at the heme pockets. The ligand is typically oxygen, thus this state is known as the "oxy" or 'R" (for relaxed) state. In the R state, intersubunit distances are increased relative to the distances in T-state hemoglobin.
'T-state hemoglobin" is the low affinity state of hemoglobin and is the dominant form of hemoglobin when it is deoxygenated ("deoxy", or 'T" for "tense").
"Heme pocket" means that pocket formed around the heme of each globin subunit described by the surrounding residues and is meant to include residues within about 6 A of the heme moiety and as described above.
'Vistal heme pocket" means that portion of the heme pocket above the plane of the heme that contains the free coordination site of the iron where ligand inolecules can combine reversibly with the iron atnm and which contains such residues as histidine E7 and valine E11. Likewise, the proximal side of the heme pocket is described by those residues below the plane of the heme and contains such residues as the proximal histidine at position F8.
"Oxidation" means the oxidation of the iron in the heme of any or all of the subunits making up the hemoglobin tetramer from the ferrous (Fe+2) to the ferric form (Fe+3). Autooxidation occurs spontaneously without the addition of exogenous oxidants, however oxidation can be induced by the presence of exogenous oxidizing agents, most notably NO and;hydrogen peroxide.
"Mutations" are substitutions, deletions or additions of one or more amino acids to the amino acid sequence that constitutes naturally occurring human hemoglobin.
"Affinity" refers to the equilibrium binding of a ligand to hemoglobin, and is described by the thermodynamic equilibrium constant, Keq. Affiniry is the ratio of the ligand association rate and the hemoglobin - ligand dissociation rate, and thus changes in association rate, dissociation rate or both can lead to changes in ligand affinity.
"Altered affinity" means the affinity of a recombinant hemoglobin for a gaseous ligand that is at least 10% different from the affinity of naturally occurring human hemoglobin for that same gaseous ligand under the same measurement conditions.
' In designing the NO mutants of the present invention, two independent protein engineering strategies were used to vary oxygen affinities and rate of reacktion with nitric acid: (l) manipulation of the intrinsic kinetics and affuuties of NO
and 02, respcctively, by making novel substitutions in the distal pockets of the subunits, and (2) adjustment of the position of the allosteric R/T equilibrium with mutations at key points away from the heme groups as described in Olson et al., Art. Cells.
Blood Subs.. and Immob. Biotech. 25: 227-241 (1997). Changes in hemoglobin allostery are specific for oxygen binding kinetics an,d affini'ty, while the R/T equilibrium has no effect on NO scavenging. The distal pocket substitutions affect entry and exit rates of NO and 02 by varying steric hindrance and hydrogen bonding (Olson et al., su ra.).
Thus, the general strategy for identifying NO mutants of the present invention is set forth as follows:
a) making mutations that cause one or more amino acid substitutions in the distal heme pockets of a and P globin subunits;

b) incorporating DNA fragments containing these : mutations into a suitable expression vector then introducing this vector into an appropriate host cell;
c) culturing the host cell, followed by a screening assay (e.g. ELISA) with antibodies to hemoglobin,-to identify host cells expressing soluble hemoglobin;
d) culturing the host cell to express soluble hemoglobin followed by purification of the hemoglobin;
e) conducting in vitro testing to verify the decrease in NO-scavenging activity and netention of oxygen delivery as needed.
f) selecting the proper a mutations to pair with the proper ~i mutations;
g) as needed, selecting other mutations to - adjust the Pso or rate of 02 dissociation; and _ h) in vivo testing for 02 delivery and hemodynamic data In following this strategy, mutant alpha subunits were first paired with wild-type beta subunits and vice versa. Additional mutants fonmed by combination of mutations in the alpha and beta globins were also constructed.
Any of the mutations described herein can be accomplished by a number of methods that are known in the art. Mutations can be made at the codon level by alteration of the nucleotide sequence that codes for a given anuno acid.
Substitution of an amino.acid at any given position in a protein can be achieved by altering the codon for that particular amino acid. This substitution can be accomplished by site dirocted mutagenesis using, for example: (1) the Amershani technique (AmershamPmutagenesis kit, Amersham'; Inc., Cleveland, Ohio) based on the methods of Taylor et al., Nucl.
Acids Res. (1985)1a: 8749-8764; Taylor et al.,- (1985) Nucl. Acids Res. j1.

8785; Nakamaye and Eckstein, (1986) Nucl. Acids Res. 1A: 9679-9698; and Dente ot al., in DNA ~, Glover, Ed., IRL Press (1985) pages 791-802, (2),the Promega*
kit (Promega Inc., Madison, Wisconsin) or (3) the Biorad kit (Biorad Inc., Richmond, California), based on the methods of Kunkel, (1985) Proc. Natl. Acad. Sc'r.
USA

488; Kunkel et al., (1987) Meth. Enzymol. 154: 367; Kunkel, U.S Patent 4,873,192.
It can also be accomplished by other commercially available or non-commercial means which incorporate the technique of site-directed mutagenesis using mutant oligonucleotides to achieve mutagenesis or as described in the Examples below.
Site directed mutagenesis can also be aocomplished using PCR based mutagenesis such as that described in Zhengbin et al., pages 205-207 in PCR
)Vlethods and Applications. Cold Spring Harbor Laborato Press, New York (1992); Jones and Howard, (1990) BioTechniques $(2):178 (1990); Jones and Howard, BioTechniques 10: 62-66 (1991), or as described in the Examples below. Site directed mutagenesis can also be accomplished using cassette mutagenesis with techniques that are known to those of skill in the art.
Any suitable host cell can be transformed with a plasmid containing the desired mutation(s) by methods known to those skilled in the art or as described in the Examples below. Suitable host cells include, for example, bacterial, yeast, plant, mammalian and insect cells. E. coli cells are particularly useful for expressing the novel mutant hemoglobins. Preferably, when multiple subunits are expressed in bacteria, it is desirable, but not required, that the subunits be co-expressed in the same cell polycistronically as desrribed in WO 93/09143. It is preferable in E.
coli to use a single promoter to drive the expression of the genes encoding the desired proteins.
The reaction of NO with oxyhemoglobin forms of the mutants often yielded biphasic reaction timecourses due to different reactivities of the two subunit types in the hemoglobin t.etramer. Fitting these reaction timecourses to a two-exponential function yielded the reaction rates of both the wild-type and mutant subunits. By repeating this process for a large number of mutant constructs, a number of mutants in each subunit type were identified exhibiting a wide range of rate constants for NO
reactivity (k'NO. ox) were obtained. After purifying and screening the large number of mutants, the following alpha and beta globin mutants and combinations thereof were identified as having reduced NO reacdvity compared to conventional hemoglobin:

acglobin mutations:
Ell(Val-+Leu) E l 1(Val-4Leu) + E7(His-+Gln) E11(VahPhe or Trp) + E7(His-+Gln) El l(Va1-Whe or Trp or Leu) + E7(His-+G1n) + G8(Leu-Whe or Trp) B I 0(Leu-4Trp) + E7(His-+Gln) B lU(Ireu-*Trp) + El l(Val-+Phe) B 10(I.eu-4Trp) + El 1(Val-Mp) B 10(Leu-4Tip) + El I(Val-+l.eu) +. G8(Leu--+Trp) B I 0(I.eu-~Trp) + El l(Val-4Leu) + G8(Leu-+Phe) B 10(L,eu-+Trp) + El l(Va1-+Phe) + G8(I.eu-4Trp) B 10(Leu-4Trp) + El 1(Val-+Phe) + G8(Leu--+De) B 10(I.eu--~Trp) + E7(His-+Gln) + E 11(Va1-+Leu) + G8(Leu-Trp) B 10(L,eu-4Trp) + El l jVal-+Trp) + G8(Leu-+Trp) El l (VaI--+Leu) + G8(Leu-+Phe) f Ell(Val-+Leu) + G8(Leu.:-+Trp) B 13 (Met-Phe or Trp) G I. 2(Leu-4Phe or Trp) B 14(Phe-4Trp) globin mutations:
Ell(VaI-4L,eu) B 13(Leu--+Phe or Trp) G12(Leu-+Phe or Trp) B 14(Leu-)Phe or Trp) G8(Leu-4Phe) + G12 (Leu-+Trp) E 11(Va1-+Leu) + G8(Leu-+Trp) E 11(Val--4Trp) + G8(Leu-4Met) El l (Val-4Leu) + G8(I,eu-4Phe) El 1(Val-+Leu) + G8(Leu--~Met) El l (Val-4Phe) + G8(Leu-+Ile) El 1(Vai-4Phe) + G8(Leu--*Phe) El l(Va}-Whe) + G8(Leu-~Ttp) E11(Va1-Phe) + G8(Leu-~Met) El l (Val-4Met) + G8(Leu--~Trp) El l (Val--+Met) + G8(Leu-4Trp) + E7(His-+Gln) El I (Val--)Trp) + G8(Leu~lle) E7(His-+Gln) E7(His-4Gln) + El 1(Val4Trp) E7(His-+Gln) + E 11(Val-~Leu) E7(His-~Gln) + E11(Val-+Phe) E7(His-4Gln) + El 1(Va1-4Phe) + G8(Leu-Phe or Trp) E7(His-+Gln) + El l(Va1-Leu or Trp) + G8(Leu-->Phe or Trp) E7(His-4Phe) El 1(Val--+Trp or Phe) + G12 (Leu-+Trp or Met) El 1(Val-4Trp or Phe) + B 13 (Leu-->Trp or Met) B 10(Leu-4Trp) + B 13 (Leu->Trp or Met) B 10(Leu-fte) + B 13 (Leu-4Trp) B 1 U(L.eu-+Trp or Phe) + G12 (Leu-+Trp) B 10(Leu-Whe) + G 12 (Leu-4Met) G8(Leu-~Trp) + G12 (Leu-+Trp or Met) G8(Leu-~Trp) + B 13 (Leu-+Trp or Met) El l(Val-4Phe, Met or Leu) + G8(Leu-.)Trp, Met or Phe) G8(Leu--Whe) + G 12(Leu-+Trp) -The above list of representative NO mutants is not intended to be all inclusive.
The designations used to reference the mutations first identify the helix, then the residue number within the helix, followed by the wildtype amino acid and the substituted amino acid. For example, E11(Val--+Leu) refers to the eleventh residue of the E
helix in which wildtype valine is substituted with leucine.
If desired, any of the above alpha globin mutations can be combined with any of the above beta globin mutations, or any of the above alpha globins can be combined 15. with a known beta globin and vice versa to obtain desirable properties. In addition, other mutations may be added to adjust oxygen affinity. For example, the following combinations have the desired NO reaction kinetics:
1. aB 10(Leu-4Phe) + PE11(Val-->Trp) + PPresbyterian (pres) 2. aEl l(Val-4Leu) +PEl l(Val-+Phe) + St. Mande 3. aB 10(Leu-4Phe) + aE7(His-~Gln) +PG8(Leu-~Trp) + St. Mande +
OProvidence (prov) 4. aB 10(Leu-Phe) + ocE7(His-+Gln) + PG8(Leu--+Trp) + Pprov + Ppres 5. ocB 10(Leu--)Trp) + aE7(His-+Gln) + PE11(Val-+Trp) 6. aB10(Leu-4Trp) + aE7(His-+Gln) + aE11(Val-Leu) +aG8(Leu-+Trp) +
PE7(His-+Gln) + PE11(Val-+Met) + PG8(L.eu-+Trp) 7. aB 10(L.eu--)Trp) + aEl l(Val-+Leu) +aG8(Leu-+Trp) +PE7(His-+Gln) +
PE 11(Val-+Met) + OG8(Leu-+Trp) 8. aB10(Leu-~Trp) + aEl I(Val-Phe) + aG8(Leu-4Trp) +PE11(Val--~Leu) +
OG8(Leu-4Trp) 9. aB l0(Leu-+Trp) + aEl I(Val-+Phe) +PEl l(Val-+Met) +PG8(Leu-4Trp) 10. aB 10(L.eu-+Trp) + aEl I (Val-+Phe) + PE7(His-~Gln) + PE11(Val-4Met) +
PG8(Leu-4Trp) 11. aB10(Leu-4Trp) + aEl l(Val-fte) + aG8(Leu-+Ile) +PEl l(Val-41.eu) +
OG8(Leu-4Trp) 12. aB 10(Leu---)Trp) + aEl l(Val-4Phe) + aG8(Leu-Ile) +PE11(Val-~Met) +
PG8(Leu-4Trp) 13. aB 10(Leu-4Phe) + aE7(His-+Gln) +PG8(Leu-~Trp) 14. aB ] 0(Leu-+Phe) + aE7(His-+Gln) +PE 11(Val-aTrp) 15. aB 10(Leu-4Phe) + aE7(His-~Gln) + aG8(Leu--~Trp) +PE11(Val-~Trp) 16. aB 10(Leu-Phe) + aE7(His-+Gln) + PG8(Leu-+Trp) 17. aB 10(Leu-~Trp) + aE7(His--~Gln) +(3E1 I(VaI-+Trp) +Pprov 18. aB 10(Leu-+Phe) + aE7(His--+Gln) + j3G8(Leu-*Trp) Although the following mutations were introduced into di-alpha constructs, the described combinations can be placed into mono-alpha constructs as well:

di a constructs (substitutions in both a subunits):

1. aB l0(Leu-->Trp) + aE7(His-4Gln) + PE7(His-~Tyr) + PG8(Leu-4Trp) 2. aB 10(L.eu--4Trp) + aE7(His-+Gln) +PE7(His-+Phe) +(3G8(Leu-+Trp) 3. aB I 0(Leu-4Trp) + aE7(His-+Gln) +PE7(His-+Tyr) +PE11(Val--)Tip) 4. aB10(Leu-->Trp) + aE7(His-+Gln) + PE7(His-4Phe) + PEl 1(Val-+Trp) 5. aB 10(Leu-4Trp) + aE7(His-4GIn) +(3E11(Val---)Trp) [3011]

6. aB l 0(Leu-4Trp) + aE7(His-~Gln) +PG8(Leu-+Trp) [3012]

7. aBIO(Leu-+Trp) + orE7(His-+Gln) + fEll(Va1-+Met) + PG8(Leu-+Trp) [3017]
8. aB 10(Leu-+Trp) + ocE7(His-4Gln) + PE7(His-4Gln) + [iE11(Val-4Met) +
PG8(Leu-+Trp) [3019]

9. aB10(Leu-Arp) + PEl l(VaI-+Trp) 10. aB 10(Leu-+Phe) + aE7(His-~Gln) + (3G8(Leu-aTrp) [2821]
11. aB10(L.eu--+Phe) + aE7(His-4Gln) + PEl 1(Val-+Trp) 12. aB I U(Leu--->Phe) + PE11(Va1-~Trp) [30011 13. aEl ](Val-+Phe) + PE11(Val-Phe) + PG8(Leu-+Ile) [3002]
l0 14. aEl l (VaI-4Leu) + 0E11(Va1--fte) [3004]

di-a constructs containing the following g]-obin-substi tutionsl:
B l 0(L.eu-+Phe) + B 13(Leu-+Trp) B 10(Leu-4Phe) + B 13%.eu--+Met) B l 0(Leu-Phe) + G 12(Leu-4Trp) B 10(Leu-Phe) + G 12(Leu-~Met) B 13(Leu-->Trp) + El 1(Val-+Phe) B 13(Leu-->Trp) + E11(Val-+Trp) B 13(Leu-~Met) + El l (Val--+Phe) B 13(Leu-+Met) + El l (Val-4Trp) El I(Val-Whe, Met or Leu) + G8(Leu-Trp, Met or Phe) G8(Leu-+Phe) + B 13(l.eu-+Met) G8(i.eu--+Phe) + B 13(Leu--+Trp) G8(Leu-+Phe) + G 12(Leu-+Met) G8(Leu-+Phe) + G12(Leu-+Trp) G8(Leu-+Trp) + B 13(Leu-+Met) G8(Leu-4Trp) + B 13(L.eu-+Trp) G8(Leu-4Trp) + G 12(Leu-+Met) G8(l.eu-+Trp) + G12(I,eu-4Trp) El l(Val-+Phe or Trp) + G 12(Leu-+Met or Trp) Combinations of these mutations can also be placed in larger sized hemoglobins having more - than one tetramer such as, for example, di-hemoglobins (di-di alpha constructs).
In general, substitution of large hydrophobic residues at key positions in the distal heme pockets of alpha and beta globins can substantially reduce the rate of NO
catalyzed oxidation (NO scavenging). However, there were exceptions to this general observation. For example, substitutions of Trp and Phe at Leu-B 10 in alpha subunits profoundly lowered the NO scavenging rate (about 20 times slower) but Trp and Phe substitutions at Leu-B 10 in beta did not measurably alter the NO scavenging rate.
Conversely, Trp and Phe substitutions at Leu-G8 significantly reduced the NO
scavenging rate in beta (6 times slower for Trp) but had no detectable effect on the NO
scavenging rate in alpha. In addition, while substitutions at Val-El I gave reduced NO
scavenging rates forboth alpha and beta subunits the results were not strictly consistent with the size of the substituted amino acid (Table 3).

Table 3 Nlutant Subunit Val-E11 (wild-type) a 70 Trp at Val El l a 22 Phe at Val-El 1 a 26 LaiatValEll a 16 Val-E11 (wild-type) 60 Trp at Val E11 5 Phe at Val El 1 12 Leu at Val-El l 16 In alpha subunits the smallest amino acid (leucine) produces the greatest reduction in NO scavenging rate. In beta subunitsthe results are more consistent with the prediction that increasing bulk and hydrophobicity will reduce the NO
scavenging rate.
Amino acid substitutions obtained from the mutant libraries were also generally consistent with the theme of large hydrophobic residues promoting reduced rates of NO
-scavenging. Among mutants identified in this screen as having significantly reduced rates of NO catalyzed oxidation, there was a preponderance of substitutions such as Trp, Phe, Leu, Met, Val and De. However, some mutant heme pockets containing unusual hydrophilic residues such as Thr, Ser, Tyr and His were also observed to have substantially reduced NO scavenging rates. While such substitutions might be expected to lead to unstable molecules due to rapid autooxidation, it is possible that some of these molecules could exhibit novel and useful properties.
The NO mutants of the present invention should have a rate constant for reaction of NO with oxyhemoglobin (k'r4Oox) less than conventional hemoglobin, preferably in the range of about 0.1 M''s' to less than about 60 M"'s''.
Preferably, the rate constant is less than 25 M"'s'', more preferably between 12 and 15 M''s"', and most preferably less than 5 M''s'.
Of the NO mutantsproduced that were tested in vivo, the results demonstrate a linear correlation between the magnitude of the pressor effect in vivo and the in vitro rate of NO scavenging. For example, reducing the NO reactivity of the heme groups significantly reduced the magnitude of the pressor response in conscious rats.
Accordingly, the data support the hypothesis that the pressor response to extracellular hemoglobins is due to a decrease in steady-state levels of NO in the region of the endothelial and smooth muscle cells lining the vasculature. A decrease in NO
concentration results in lesser activation of guanylyl cyclase, which ultimately increases the tone of vascular smooth muscle. It is believed that following administration of cell-free hemoglobin, a competition exists for the available NO between guanylyl cyclase and ferrous oxy- and deoxyhemoglobin (extravasation of the hemoglobin into or through the endothelium may be required). Those recombinant hemoglobins that.
have lower rates of reaction with NO are less potent competitors for nitric oxide.
Consequently, the NO mutants of the present invention are useful for a variety of applications because due to their intrinsic lower reactivities, they produce little or no perturbation in the natural turnover of nitric oxide, but remain capable of binding and delivering oxygen.

The results also demonstrate that if the amino acid substitutions tised to mduce NO scavenging have an undesirable effect on oxygen delivery, the P30 and rate of Oz dissociation can be "corrected" by other strat,egicaAy placed amino acid substitutions.
02kinetics and equilibria can be manipulated by changing the position of the.
allosteric equilibrium; or by altering the intrinsic binding kinedcs and affinity of the subunits.
Mutations away from the . distal heme pockets can be used to change. the relative stabilities of the high-affinity "R" and low-aftinity 'T' allosteric states, Hemoglobin allostery has a significant effect on oxygen binding, but no effect on oxidatioh by NO.
The intrinsic Oz-binding properties of each subunit can be modified by varying the degree of steric hindrance" near the iron atom, or by varying the strength of hydrogen bonding between the E7 residue and the bound oxygen. Steric hindrance affects 'the association and dissociation rate constants by altering thc entry and exit of oxygen to and from the heme pocket. Similarly, steric hindrance also affects the rate of oxidation by NO. The E7 histidine stabilizes bound oxygen through hydrogen bonding interactions. Substitution of the E7 histidine can decrease the.degree of stabilization of bound oxygen and enhance oxygen dissociation kinetics. E7 substitutions can also affect reactiviry of NO with Hb.
The results further demonstrate the beneficial effects of increasing the size of the . , .
hemoglobin molecule, which'is additive to the effects of amino acid substitutions used to reduce NO scavenging as shown in Figure 11.
The re.combinant hemoglobins of the prrsent invention can be used for- a number of in vitro or in vivo applications. Such in vitro applications include, for example, the delivery of oxygen by compositions of the instant invention for the enhancement. of cell growth in cell culture. by maintaining oxygen levels in vitro (DiSorbo. and Reeves, PCT publication WO 99/22482). Moreover, the hemoglobins of the instant invention can be used to remove oxygen from solutions requiring the removal of oxygen (Bonaventura and Bonaventura, US Patent 4,343,715) and as reference standards for analytical assays and Instrumentation (Chiang, US
Patent 5,320,965) and other such in vitro applications known to those of skill In the art.

In addition, the recombinant hemoglobins can be formulated for use in various therapeutic applications. Example formulations suitable for the tccombinant hemoglobin of the instant invention are described.in Milne, et al., WO
95/14038 and 35. . iCerber_et a1..TWO. 96/2?3.88.. MPharmaceutical compositions can be administered by, for example, subcutaneous, intravenous, or intramuscular injection, topical or oral. administration, large volume parenteral solutions, aerosol, transdernnal or mucus membrane adsorption and the like.
For example, the recombinant hemoglobins of the present invention can be used in compositions useful as substitutes for red blood cells in any application that red blood cells are used or for any application in which oxygen delivery is desired. The recombinant hemoglobins can also be formulated as oxygen canying therapeutics and used for the treatment of hemorrhages, traumas and surgeries where blood volume is lost and either fluid volume:or oxygen carrying capacity or both must be replaced.
Moreover, because the, recombinant hemoglobins of the instant invention can be made pharmaceutically acceptable, they can be used not only as blood substitut.es that deliver oxygen but also as simple volume expanders that provide oncotic pressure due to the presence of the large hemoglobin protein molecule.
In a further embodiment, the recombinant hemoglobins of the instant invention can be crosslinked by methods known in the art and used in situations where it is desirable to limit the extravasation or reduce the colloid osmotic pressure of the hemoglobin-based blood substitute. Thus, the recombinant hemoglobins can act to transport oxygen as a red blood cell substitute, while reducing the adverse effects that can be associated with excessive extravasation.
A typical dose of recombinant hemoglobin as an oxygen delivery agent can be from 2 mg to 5 grams of hemoglobin per kilogram of patient body weight. Thus, a typical dose for a human patient might be from a few grams to over 350 grams.
It will be appreciated that the unitcontent of active ingredients contained in an individual dose of each dosage fomm need not in itself constitute an effective amount since the necessary effective amount could be reached by administration of a number of administrations.
The selection of dosage depends upon the dosage fonm utilized, the condition being treated, and the particular purpose to be achieved according to the detemnination of those skilled in the art.
Administration of recombinant hemoglobin can occur for a period of seconds to . hours depending on the purpose of the hemoglobin usage. For example, as an oxygen carrier, the usual time course of administration is as rapid as possible.
Typical infusion rates for hemoglobin solutions as oxygen therapeutics can be from about l00 ml to 3000 mUhour.
In a further embodiment, the hemoglobins of the instant invention can be used to treat anemia, both by providing additional oxygen carrying capacity in a patient that is suffering from anemia, and/or by stimulating hematopoiesis as described in PCT publication WO 95/24213.. When used to stimulate hematopoiesis, administration rates can be slow because the dosage of haemoglobin is much smaller than dosages that can be required to treat hemorrhage. Therefore the 5. recombinant hemoglobins of the instant invention can be used for applications requiring administration to a patient of high volumes of hemoglobin as well as in situations where only a snWl volume of the hemoglobin of the instant invention is administered.
Because the distribution in the vasculature of extraeelluar hemoglobins is not limited by the size of the red blood cells, the hemoglobins of the present invention can 10, be used to deliver oxygen to areas that red blood cells cannot penetrate.
These areas can include any tissue areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of thrombi, sickle cell occlusions, arterial occlusions, angioplasty balloons, surgical instrumentation, any tissues that are suffering from oxygen starvation or are hypoxic, and the like. Additionally, all types 15 of tissue ischemia can be treated using the hemoglobins of the instant invention. Such tissue ischemias include, for example, stroke, emerging stroke, transient ischemic attacks, myocardial stunning and . hibernation, acute or unstable angina, emerging angina, infarct, and the like. Recombinant hemoglobin can also be used as an adjunct with radiation or chemotherapy for the treatment of cancer.
20 . Because of the broad distribution in the body, the recombinant hemoglobins of the instant invention can also be used to deliver drugs and for in vivo imaging as described in WO 93/08842. .
Recombinant hemoglobins can also be used as replacement for blood that is removed during surgical procedures where the patient's blood is removed and saved for 25 reinfusion at the end of surgery or during recovery (acute normovoleniic hemodilution or hemoaugmentation). In addition, the recombinant hemoglobins of the instant invention can be used to increase the amount of blood that can be pn:donated prior to surgery, by acting to replace some of the oxygen carrying capacity that is donated.
The following Examples are intended to illustrate, but not limit, the .30 present invention. Examples 1-16 are studies relating to increasing soluble yield of recombinant hemoglobins expressed in Ecoli, while Examples 17-22 are studies relating to hemoglobin mutants having reduced NO scavenging.

Example 1 Genetic Construction PCR based cassette mutagenesis was used to incorporate desired amino acid substitutions. DNA sequence for variant codons was based on codons used in proteins that are highly expressed in E.coli as described in Sharp et aL, Nuel. Acids Res., 16:8207-8211 (1988). In general, strains were constructed by transformation of the plasmid DNAs into E. coli strains lacking a plasmid using the procedure of Chung, ei al.. Proc. Natl. Acad. Sci. USA, 86:2172-2175 (1989), or Hanahan, DNA Cloning:
A
Practical AI2proach, V: 1, pp. 109-135 (IRL Press, 1985).
] 0 A variety of E.coli strains were used, including those listed in Tables 4 and 5.
The construction of these strains, especially those containing different oligomerizing mutants or domains, is described in WO 97/04110.
Tables 4 and 5 summarize the strain background and plasmid backbone used for the expression of these molecules. Plasmid copy number has been. shown to influence soluble expression yields as well. The medium copy plasmid pSGE705 served as a platform for some of the molecules tested. Plasmids.pSGE7l5 and pSGE720 are high .
copy number plasmids,, and expression from these plasmids should exceed that observed from pSGE705-based plasmids. pSGE720 contains a synthetic operon composed of the di-a-globin and OPresbyterian-globin genes transcribed from the. tac promoter on a tetracycline resistant plasmid with the pUC high copy number origin of replication (Weickert & Curry, Arch. Biochem. Biophys. 348:337-346 (1997)).

Table 4 SGE lasmid bk rd. strain alpha beta 1464 pSGE720 1675 di- resby 2784 SGE720 1675 di- .. prov 2706 : SGE720 1675 di- wt 2782 SGE72 1675 di- resb y+ rov 939 pSGE705 1661 di-di-1 resb 2791 SGE705 1661 di-di-.1 wt 946 SGE720 1675. di-di-1 resb p 3085 p SGE720 1675 di-di-1 prov 948 SGE705 1661 di-di-2 presby 2951 pSGE720 1675 di-di-2 prov 953 pSGE715 1661 di-di-3 presby 2952 pSGE720 1675 di-di-3 prov 955 pSGE715 1661 di-GCN4-di presby 2792 SGE715 1675 di-GCN4-di wt 2795 SGE72 1675 di-GCN4=di resb + rov 2796 SGE 20 1675 di- C 4-di rov 2802 SGE720 1675 di-P53 presby 2948 SGE720 1675 di-P53 prov 2944 SGE720 .1675, di-COMP presby 2813 SGE720 1675 di-COMP prov 3083 pSGE720 2761 di-K158C presby 1 2 pSGE720 2761 di-K 1.58C presby+prov 3084 pSGE720 2761 di-K158C prov Table 5 Strain Plasmid rHb Host Descrip.
SGE1675 none none gyrA96(NaIR), laciQ', endA, hsdR17, relAl, supE44, recJ (a deriv. flf JM l 07) SGE1464 pSGE720 1.1 SGE1675 SGE1480 - SGE726 1.0 SGE1675 SGE1483 pSGE728 0.0 SGE1675 SGE270.6 pSGE733 0.1 SGE1675 SGE2761 none none SGE1675 + rpsL (strR) by Pl transduction SGE2782 SGE767 9+1.1 SGE1675 SGE2784 SGE768 9.1 SGEl675' SGE3083 pSGE1001 mt. SGE2761 with rHb di-SGE3084 pSGE1222 mut. SGE2761 with rHb di-ocK 158MK82D
SGE3138 pMON7124K none gyrA96(NalR), Iac1Q', endA, relAl, supE44, rec! (a deriv. of SGE1675) SGE3172 pSGE1237 mut. SGE2761 with rHb di-ocK15800N108K; K82D
SGE3261 pSGE768 9.1 SGE3138 Example 2 Construction of beta mutations pSGE728 was constructed by Xhol digestion and deletion from pSGE720 of one alpha subunit and the di-alpha glycine linker. The resulting plasmid, pSGE726, contains a single alpha gene rather than a di-alpha gene (rHbI.0). The Presbyterian mutation in beta was replaced by digestion with Bg1II and HindlII, and ligation to introduce wild-type beta and create pSGE728 (rHbO.0). The Presbyterian mutation in the beta gene of pSGE720 was replaced by digestion and ligation as for pSGE728, to introduce wild-type beta and created pSGE733 (di-alpha and wild type beta;
rHb0.1).
The Providence mutation (PLys82-+Asp) was introduced into the rHb 1.1 background to cneate rHb9+1.1. The Lys82--*Asp mutation was created by PCR
amplification of a portion of the P-globin gene using an oligonucleotide containing an Asp codon in placeof the Lys82. CBG124 (wild-type beta coding sequence 5' primer near BspEl site) and CBG119 (3' primer containing the PK82D mutatiop downstream of Asp718 site) were used to amplify a small DNA fragment from 'pSGE761 as template.
CBG 119 5'AGC GAA GGT ACC GTC CAG GTT (SEQ.ID.NO: 1) CBG124 5' CCT GAC TCC GGA AGA AAA ATC C (SEQ.ID.NO: 2) The PCR product and vector were digested with BspEI and Asp718 and ligated.
DNA sequencing of plasmid isolated from transformants was performed with Sequenase kit reagents and protocols (United States Biochemical, 33P
(Amersham, Inc.), and priniers synthesized on an Applied Biosystems 380B DNA synthesizer.
Sequencing confirmed the Providence and Presbyterian mutations and the plasmid (pSGE767) was transformed into SGE 1675 to produce SGE2782.
The Providence mutation was introduced into the rHbO.l background to cieate rHb9.l. A BamHUAsp7l8 fragment from pSGE767 was isolated and ligated into digested pSGE733 (rHbO.1). Sequencing confinmed the mutation and the plasmid (pSGE768) was transformed into SGE1675 to produce SGE2784, or SGE3138 to produce SGE3261. Similar steps were used to introduce Providence(,,) and the combination of Providence(.,) and Presbyterian in a plasmid with a. di-alpha Lysl58-+Cys mutation (Table 5: SGE3083, SGE3084 and SGE3172).

Example 3 A. Bacterial growth and lysis procedures Each isolate tested was grown ovemight at 37 C as a patch on an LB plate supplemented with tetracycline. Inoculums were transferred via sterile toothpicks to 0.75 mt aliquots of fresh. DM-1 media as described in Looker et 'al., (1992), with 15 g/ml tetracycline, 0.1 M IPTG (Sigma) and 0.05 mg/n-d hemin (Ameresco) in sterile 12 nun (i.d.) borosilicate tubes. The cultures were incubated at 30 C
for 18 hours with 350 rpm shaking in a New. Brunswick Series 25 Incubator Shaker.
TheØ2 ml aliquots were transfenred to a 96 well flat bottom microtite,r plate (Immunlon 4 from Dynatech). 'Jbe optical density of each well was recorded at 650 nm using the Spectral Dynamics microplate reader and Softmax software package. The cells were pelleted to 5* the bottom of the microplates at 3000 rpm at 4 C . for 10 minutes using the Beckman~
RK6 centrifuge, 3.7 rotor and microplate adapter buckets. The spent media was removed and the cells were resuspended in 0.1 ml 25 mM Borax with light vortexing of .
microplate. The microplates were covered with parafilm and stored at -80 C
overnight and then thawed at 30 C in a water incubator until just thawed. Lysozyme (Ameresco ] 0 Ultra pure grade) was added to 0.17 mg/ml final volume from a I mghnl stock containing 0.05 M NaCI. The samples were mixed by light vortexing of microplate, covered and incubated at room temperature for 30 minutes. DNAase I
(Boehinger Mannheim grade II from bovine pancreas) was added to 0.02 mghril from 0.1 mghril DNAase stock containing 7.5 mM CaC12 and 75 mM MgClz. The samples were mixed 15 by light vortexing of microplate, covered and.incubated at room temperatune for 15 minutes. The microplates were covered with parafilm and stored at -80 C for 90 nun.
to 20 hours and then placed at 30 C in a water incubator until just thawed.
The cell debris were pelleted to thc bottom of the microplates at 3000 rpm at 4 C for 10 nainutes using the Beckman RK6 centrifuge, 3.7 rotor and microplate adapter buckets.
The 20 cleared lysate was transferned to wells of a fresh microplate and stored at 4 C for approximately 12 hours.

B. EMA
Cleared lysates were diluted 1:800 or, 1:1600 into PBS with 0.1% (w/v) casein 25 and 1.08% (w/v) Tweeri 80. Recombinant di-alpha hemoglobin (rHb) standards were diluted serially into this buffer and used in the procedure in order to produce a standard curve. For the following procedure, all reagents were added in 0.1 zril aliquots unless otherwise indicated. Aliquots of 5 ghril goat affinity purified anti-rHbl.l antibodies in Borate Buffered Saline were used to coat wells of Immulon 4 mieroplates (Dynatech) 30 for 18 hours at 4 C. Tbe wells were then completely filled with casein blocker (Pierce) for a short time to block nonspecific binding. After washing the plates thrice with ELISA wash buffer, diluted samples and standards were then added and the plates were incubated at 37 C for 60 minutes. The plates were washed as above and biotin Iabeled Goat anti-rHbl.1 antibody at 50 ng/ml in PBS with l% casein were added to each well.

The plates were covered and incubated at 37 C for 60 minutes and were washed as above. A 1:10,000 dilution of Streptavidin-Horse Radish Peroxidase (Southern Biotechnology Associates) into 0.1% casein in PBS was prepared and distributed and the plates were covered and incubated at 37 C for 20 minutes and were washed as above. Working TMB Peroxidase Substrate Solution B was prepared according to manufacturer's instructions (Kiekegaard) and quickly added. After 10 nunutes, I M
phosphoric acid was added to each well to quench the rcaction. The absorbance at M50-650 nm was read for each plate. The values were then normalized against the standard curve and adjusted for dilution and for the original culture optical density at 650 nm.
Example 4 Fermentations Fermentations were performed a defined medium in 15L Biolaffite fermentors, generally as described in Looker et al. "Expression of Recombinant Human Hemoglobin in Escherichia coli." Meth. Enzvmol. 231:364-374.(1994) using DM59(60) medium under Glucose excess (BAR) conditions with induction for 16 hours at 28 C, except.where noted. In order to minimize the contribution of day-to-day and fermentor-to-fermentor variability to the yield outcomes, some experiments distributed strains among the fermentors and sets of fermentations, such that each set of fermentations included side-by=side controls and strains were not assigned the same fermentor more than once, when possible. Induction of expression was achieved by addition of IPTG between 10 and 200pM on attaining a cell density of an O.D.600 of approximately 30. Incubation was continued for 16 hours post induction, and hemin was added in five shots of 10, 13, 17, 17, and 17 ml at 0, 3, 6, 9, and 12 hours post induction respectively, delivering a total concentration of 0.37g/L of hemin.
One ml fermentation samples were withdrawn at 4, 8, 12, 14 and 16 hours post induction and assayed for soluble rHbl.l. In other studies, hemin was added in five shots at 25 ml each and collected at 12, 14 and 16 hours.
'30 Example S

Detemzination Of Percent Soluble And Insoluble rHb The accumulation of soluble and insoluble rHb was measured from at least 2 and usually at least 3 independent fermentations per condition examined. One ml samples were withdrawn into 1.7 ml Eppendorf tubes to appropriate times past induction. These one ml samples were centrifuged in an Eppendorf centrifuge for three minutes and the supernatants were removed. Pellets were stored at 80 C until assayed.
Soluble and insoluble rHb was determined as described (Weickert et 'al., AOL
Environ. Micro., 63:4313-4320 (1997)) except that after SDS-PAGE
electrophoresis, the rHb was detected by either silver staining or western blotting (10). Gels were silver stained using the reagents and protocol of Dafichi Pure Chemicals Co., Ltd.
(Tokyo, Japan).

Example 6 Hemin Concentration Estimation Hemin concentration in the fermentation broth was estimated as follows:
samples of fermentation broth were centrifuged and an aliquot of the supematant was added to a solution of 300pM Human Serum Albumin (HSA, Baxter) in 50mM tris pH

7.5 such that the concentration of hemin did not exceed 50 M. In the presence of excess HSA, hemin fornis a 1:1 complex with a well defined absorbance spectrum as dexribed in Beaven et al., Eur. J. Biochem., 41:539-456 (1974), and the hemin concentration was estimated from the absorbance at 625nm. By subtracting the.
new hemzn concentration from that of the previous sample, the quantity of hemin bound by or taken up by the cells was calculated during the entine time course of rHb accumulation.

Example 7 .
Comparison of,Pnesbyterian Beta and Wild-Type Beta Relative to Hb containing wild-type beta, expression of hemoglobin containing Pmsbyterian beta only produces about half the amount of soluble hemoglobin in E.coli.
This differcncx must be due to the prcsence of the Prrsbyterian mutation in beta.
Recombinant hemoglobin expression improvements may therefore be achieved by improvements in the beta globin. The lower expression with beta Pnes. may be due to a disruption of the molecular folding pathway by the asn108--0ys change of the Presbyterian mutation, or general destabilization of the beta globin, as indicated by in vitro experiences. Table 6 shows the results of the comparison between Presyterian beta and wild-type beta.

Alpha Beta Expression N
MONO PRESBYTERIAN 0.46 3 MONO WII..D-TYPE 1.0 3 DI - PRESBYTERIAN 0.58 3 DI WILD-TYPE 1.3 3 Soluble expression normalized to expression of mono-alpha with wild-type beta globin.
Example 8 Naturally-occurring Beta Variants Eleven naturally occurring beta variants were selected due to their reported lower.Oxygen affinity as compared to wild-type beta globin, and were cloned into the rHb expression plasmid to be co-expressed with di-alpha globin. It was believed that some of these variants =possess the desired oxygen affinity without compromising soluble yield. Strains were fermented and expression was measured. The results are reported in Table 7.

Relative Expression of rHb Containing Beta Globin Variants (15L Fermentations) Variant Substitution ExFression*
Wild-type None 1.0 Cheverly F45S 0.5 Okaloosa IABR _e,0.05 Korle-Bu D73N 0.85 Vancouver D73Y. 1.1 Mobile D73V 0.35 T'ilburg D73G 0.40 Kansas N 102T 1.2 Beth Israel N102S 0.67 Saint Mande N 102Y 0.44 ProvidencemP K82D 1.0 Presby+erian N 108K 0.44 * Soluble expression normalized to expression of di-alpha with wild-type beta globin.

Recombinant hemoglobins Kansas, Vancouver, and Providence have soluble expression equal to or gneater than that of wild-type beta globin. Recombinant hemoglobins Okaloosa and Cheverly, which are reported to be slightly unstable in red 5; blood cells (Carver & Kutlow, 'International Hemoglobin Information Center Variant List. Hemoglobin 19:37-149 (1995)) had lower expression. Isolated Presbyterian beta subunits has been observed to precipitate in vitro and are considered to bo less stable compared to other beta subunits.
The effect of the Presbyterian mutation in beta suggests dw alternative mutations, with less profound' effects on solubility, would. be useful. The Providence(Asp) mutation, PLys82--+Asp, reduces the oxygen affinity of hemoglobin (Bonaventura, J., et al., 1976. J. Biol Chem. 251:7563-7571) but has no reported effect on protein stability. As a test 'of whether other stable naturally occurring mutations can provide equivalent funcdonal properties to Presbyterian Beta (reduced oxygen affinity), without the. expression consequences, a version of rHbl.1 that uses Providence(Asp), not 'Presbyterian beta was created. In accord with the previous variant nomenclature, this molecule was designated rHb9.1. An additional beta variant molecule was also created, that combined the Providence(Asp) and Presbyterian mutations. Expression of these molecules was compared to expression of rHb 1.1 and rHb0.1 in 15L fermentations, and these betas were then combined with many other rHbs.

Example 9 Effect of Beta Mutations on Di-Alpha Constructs The expression of many different hemoglobins containing genetically fused di-alphas or with domains that niight serve to oligomerize the di-alpha rHbs were examined with Providence beta globin and/or with Presbyterian beta. Hemoglobin expression levels were measured for cells obtained from 15L fermentations.
The presence of Presbyterian (N108K) beta reduced the soluble rHb yield by greater than a 2-fold for both mono and di-alpha hemoglobins (Table 6). This reduction is apparent throughout the fenmentation as soluble rHb . Alternative beta subunits had a profound effect on the soluble di-alpha rHb yields (Table 4).
Providence(Asp) beta (K82D), a mutation known to lower oxygen affinity, resulted in much higher soluble rHb yields than did beta containing the Presbyterian mutation (Table 8).

Strain rHb alpha beta Ave.Peak S. D. N
Express.
SGE2706 rHb0.1 wt 1.0 0,33 9-SGE1464 rHbl.1 di Presby. 0.43 0.3,1 10 S 8 r b9.1 Provid. 1= 7 t0.51 1 SGE2782 rHb9+1.1 (h ov+ res = 0.46 7 Peak averages are normalized to rHb0.1.
~
The combination of Presbyterian and Providence (Asp) mutations resulted in -2-fold higher soluble rHb accumulation than with the Presbyterian mutation alone, ] 0 indicating that Providence(Asp) at least partially "rescues" the instability associated with the Presbyterian mutation. The effect on soluble Hb accumulation is apparent throughout the fermentation as soluble rHb accumulates as shown in Table 9.
'Table 9 reports the data relating to the accumulation of soluble rHb during fermentations of several mutant beta globins with di-alpha. The rHb containing Providence(Asp) beta globin accumulated to the highest level. The combination of Providence(Asp) with Presbyterian in beta partially "rescued" the expression of the rHb containing Presbyterian beta, whose soluble accumulation was the lowest of all the molecules tested.

Time Post SGE1464 SGE2783 SGE2706 SGE2784 Induction (Hrs) Presby. Prov + Presby wt beta Prov.
rHb 1.1; N=10 rHb9+I.1; N=7 rHb0.1; N=9 rHb9.1; N=10 4 0.30 0.28 0.86 8 0.70 1.09 1.79 1.67 12 1.02 1.97 2.32 2.56 14 1.01 2.21 2.69- 2.91 16 1.22 2.43 2.77 3.07 When co-expressed with di-oe(rHb9.1), the Providence mutation improved the soluble expression of rHb by 47% to 25.3t5.4% of the soluble cell protein, and the total globin expression increased to 44.4f12.4% (Table 10).

Effect of beta globin mutations on soluble accumulation of rHb*

Strain 1464 2782 2706 2784 rHb 1.1 9+1.1 0.1 9.1 beta Asn l 08-4Lys Asn ] 08-+Lys+ Lys82--+Asp wt Lys82-4Asp g/L sol 1.0t0.1 2.6 0.5 . 2.1t0.5 3.1 0.7 ) ave. % sol 8.1 t 1.4 17.5 4.0 17.2 2.2 25.3t5.4 ave. % tot. 20.6f1.6 . 35.7 7.5 37.8t5.1 44.4t12.4 solubilityt 44.1t4.0 52.4f10.2 50.8 3.7 58.2 5.1 N* 3 5 3 3 K82D incr. +116% +47%
*N=number of ermentations used for determining the average % soluble and total expression.
t solubility = the % of the total globin present in soluble form. It was determined independently for each fermentation sample and the averages are shown. Note:
this average is not necessarily equivalent to dividing the % Sol. rHbl.1 by the %
total globin since fermentations with higher globin accumulation would have greater "weight" in an average value.
incr. = the % increase in soluble expression with the Lys82-+Asp mutation in beta globin relative to soluble expression with the identical beta globin without Lys82-~Asp.

This improvement was apparent throughout the entire induction period in the fermentation (Table 11), resulting in an average soluble yield of 3.1t0.7 g/L.
Soluble and total accumulation of rHb9.1 was significantly better than soluble and total accumulation of rHb0.1 under identical conditions (P<0.05; Multiple Range tests) (Table 11). Table 11 reports the data relating to soluble accumulation of four variants for up to 16 hours after induction. Heme supplementation was at 0, 3, 6, 9 and hours post induction to a final concentration of 0.63mM.

Time post Soluble Soluble Soluble Soluble induction (hrs) rHb 1.1 rHb9+1.1 rHbU. l rHb9. i (% cell protein) (% cell protein) (%cell protein) (%cell protein) 4 6.2 ,.1 .9 5.2 8.4 10.7 14.6 12 7.5 12.2 15.9 18.5 14 7.8 14.9 17.9 22.4 16 8.1 17. 17.2 25.3 The addition of the Providence mutation to the Presbyterian 0-globin subunit (rHb9+1.1) resulted in a 116% increase -in soluble accumulation (Tables 10 and 11) compared to that with the Presbyterian subunit alone (P<0.02; T-test of the means).
This rescued rHbl.1 soluble expression to a level statistically indistinguishable from wild type rHbO.l (Table 10). Molecules that accumulated to higher levels did so in part because a gneater proportion of the protein accumulating. within the cell remained soluble (Table 12). In Table 12, total globin was measured by Western blot from samples identified in Table .4 1. The rHbs with higher soluble expression wer cornelated with higher total globin (R-squared = 0.99) and with a higher percentage of the total globin present as soluble rHb. (R-squared = 0.97).

Soluble Expression Total Globin Soluble Globin (% cell protein) (% cell rotein) (% total protein) 9.0 20.6 44.1.
18.8 35.7 52.4 19.0 37.8 50.8 25.3 44. 58.2 The presence of these beta globin mutations had similar effects on soluble accumulation of a recombinant hemoglobin having a Lys 158-4Cys mutation in di-alpha. The presence of Providence,õ,) improved soluble expression from a maximum of 11.3% with the Presbyterian beta to 24.4 Jb (Figure 1). The combination of Providence(,0 and Presbyterian mutations in beta resulted in an intermediate level of expression with a maximum of 16.4% soluble globin accumulation (Figure 1).
Table 13 shows the average peak rHb yields of various di-alpha molecules co-expressed with different beta subunits. For di-alpha and three other modified alpha molecules tested, co-expression with the Providence (Asp) subunit resulted in the highest soluble accumulation. Wild-type beta was always second best and Presbyterian beta the worst.

Oligomer Presby wild type Presby+Prov Prov Molecule di-a 1.26 2.91 2.45 4.39 di-di-al 0.30 1.22 3.34 dia-GCN4-dia 0.10 0.40 0.34 1.70 dia-K158C 1.08 - 2.13 3.36 Table 14 shows the effect of Providence(Asp) beta on expression of many other recombinant hemoglobin molecules. The soluble accumulation of these alpha-based oligomers Nvas substantially improved by co-expression with Providence(Asp) beta.

Oligomer Molecule Presby Prov di-a 1.26 4.39 di-di-al 0.30 3.34 di-di-a2 0.20 4.24 di-di-ot3 0.20 4.07 dia-GCN4-dia 0.10 1.70 dia-P53 0.09 1.58 dia-COMP BLOD 1.47 dia-K158C 1 =08 3.36 *BLOD = below limit of detection Table 15 shows the soluble accumulation of additional alpha-based oligomers co-expressed with Providence(Asp) beta. The use of Providence(Asp) beta resulted in substantial accumulation of additional recombinant hemoglobin molecules, including tri- and tetra-di-alpha.

Oligomer Molecule Relative Soluble Yield diaGCN4 3.35 dai-Mnt 3.16 dia-tetraZIP 1.56 tria 1.04 tetraa 0.79 Example 10 Comparison of Relative Expression in ELISA and Fermentors Based on the promising results obtained with a few naturally occurring variants at beta D73, K83 and N 102, three low-complexity libraries with all 20 amino acids at each of these three positions were created. The libraries were pre-screened by the ELISA of Example 3 and expression was subsequently verified in . 15 Liter fermentations The expression results are shown in Table 16. Other variants at these 3 positions were reported by JEI.ISA to have low expression and were not fermented.
The fermentation results geinerally matched those predicted by ELISA in 10 out of 13 variants examined. Relative standard deviation (RSD) of expression in fermentors is generally less that 20%, while the RSD of the ELISA is about 40%.

Variant ELISAI Fermentor2 Wild-type 1.0 :1.0 N102A 1.3 0.9 N102V 0.5 0.7 D73I 1.3 1.2 D73M 1.3 1.1 D73K 1.3 0.7 D73E 1.5 1.2 D73L 1.4 0.8 D73T 1.0 1.0 K82E 2.1 1.3 K82G 1.6 1.3 K82H 1.6 0.9 K82P 2.0 N1D
K82Q 1.7 1.0 K82S 1.0 1.0 K82N 1.0 N/D
~ Expression was normalized to OD600 of cultures and to the soluble level measured for rHb with di-alpha and wild-type beta. Value shown is the mean of at least 2 assays.
2 Expression was normalized to OD600 of cultures and to the soluble level measured for rHb with di-alpha and wild-type beta. Values shown are the mean of at least two fermentations and are relative to wild-type controls run at the same time.

Example 11 Improving Soluble rHbO.0 Accumulation by Hemin Addition In 30 C fermentations with 15 M IPTG to induce rHb expression, rHbO.0 consistently accumulated to higher soluble levels than rHbl.1 to an average of 10.0 1.1% of the soluble cell protein versus 6.5 1.6% for rHbl.1 in 10 hours.
At 28 C, with 100 M IPTG, soluble rHbO.0 accumulated to an average of 14.1 0.4%, while rHbl.1 increased by a similar magnitude, to 9.2 2.8%. Extending the induction period from 10 to 16 hours improved the soluble accumulation of rHbl.1 to 10.9f1.9% of the soluble cell protein, but resulted in the soluble yield of rHbO.0 declining from 16.5t0.3% at 8 hours post induction to 11.0 0.3% at 14 hours post induction. Total globin remained relatively constant between 23.9 5.1% at 8 hours and 22.5f4.0% at 14 hours.

The possibility that additional henie supplementation could support continued soluble rHbO.0 accumulation was tested by including two more hemin additions, at 9 and 12 hours post induction, increasing the final concentration 85%, from 0.34 to 0.63mM. These additions prevented the soluble rHbO.0 decline observed late in the previous fermentations. Soluble rHbO.0 accumulated to an average of 19.2 2.1 k of the soluble cell protein and total globin accumulated to 28.4 8.5% of the soluble oell protein. This 75% increase in soluble hemoglobin accumulation at 16 hours post induction was highly significant (P=0.0005; T-test of the means).

Expression Comparisons of Four rHbs Using Higher Hemin Supplementation Using higher henuin concentrations (0.63mM) established for rHbO.0 in 15L
fermentations at 28 C, the soluble expression of four different recombinant hemoglobins was examined in parallel fermentations (Table 17). All four strains were isogenic with identical high copy plasmids except for two globin gene differences: (1) the covalent linkage to form di-a and/or (2) the presence of the A-Presbyterian (Asn l 08--~Lys) mutation.
A significant increase in soluble expression was observed for the two strains expressing a wild type A-globin subunit rather than P-Presbyterian (P<0.05;
Multiple Range tests). The average increase in soluble rHb was about 2-fold from about 10%
of the soluble cell protein to about 20% or greater (Table 17). A slight increase (about 7-17%) in soluble accumulation was correlated with the expression of the di-a subunit with both wild type and A-Presbyterian subunits.

Table 17 Effect of di-alpha and beta subunits on accumulation of soluble rHb Strairi 1480 1464 1483 2706 rHb 1.0 1.1 0.0 0.1 alpha mono di mono di beta Asn 108--~Lys Asn l 08-+Lys wt wt ' /L sol 1.1 0.2 1.4t0.4 2.4t0.6 3. l t0.5 ave. % sol 9.6t0.5 11.2t4.5 19.8t1.4 21.2t3.4 ave. % tot 21.8 1.5 21.8t0.7 32.4 8.5 54.0 15.0 N* 3 3 3 3 di-a incr. t +17% +7%
wt P incr. $ +106% +89 j Fermentations at 28 C, induced for 16 hours with 100 M IPTG and supplemented with 5 aliquots of hemin to a final concentration of 0.63mM. Soluble yields are from samples 16 hours-post induction.
*N = number of fermentations used for determining the average % soluble and total expression.
t di-a incr. = the % increasein soluble expression with the di-alpha linker relative to the soluble expressioq with the unlinked alpha globin.
$ wt 0 incr. = the % increase in soluble expression with the wild type beta globin relative to the soluble expression with the Asn108---)Lys beta globin.

Example 13 Improving Soluble rHb9.1 Accumulation By Hemin Addition A 69% increase in the heniin supplementation, to 1.07mM, significantly increased the rate of soluble rHb9.1 accumulation (Figure 2A; P=0.001) and an average of 35.6 2.5% of the soluble cell protein, was soluble rHb9.1. These fermentations achieved an average soluble yield of 6.0t0.3 g/L rHb9.1, almost twice the 3.1f0.3 g/L
achieved with the lower heme concentration (Table 18). Total rHb9.1 globin accumulation averaged 55.6t6.9% of the soluble cell protein, and total globin yield from 16 hour fermentations was 9.4 g/L. A maximum soluble accumulation of rHb9. l to 39% of the soluble cell protein and a maximum total globin accumulation to 65%
were observed from an individual fermentation. Silver stained gels of the soluble and insoluble fractions of cell lysates from fermentations reveal prominent di-a and OProvidence bands constituting the majority of the protein in each of these lysate fractions.

Time Post lnduction Soluble rHb9.1 (hours) ( ) 3.45 12 4.79 14 4.95 16 5.77 18 5.95 20 5.77 22 6.28 24 6.42 Extending the induction period to 24 hours, and continuing the hemin additions every three hours, resulted in a marginal increase in the average soluble rHb9.1 yield, from 6.0f0.3 to 6.4 0.2 g/L, but did not result in any increase in the %
soluble or total rHb9.1 (Table 18). However, it resulted in the highest yield, 6.8 g/L, of soluble rHb9.1 from an individual fermentation. The effect on soluble rHb9.1 accumulation of adding at induction, a single bolus of hemin equal to the sum of hemin additions used in a typical fermentation was tested. There was no significant difference in the soluble rHb9.1 accumulation with this strategy.

Example 14 Heme To rHb9.1 Stoichiometry The molar concentration of hemin added was compared to the molar accumulation of soluble rHb9.1 on a per heme basis. The molar concentration of soluble rHb9.1 was calculated from the soluble yields. The molar concentration of hemin supplied was calculated from the known mass of hemin added. Each mole of hemoglobin contains 4 moles of globin subunits (2a + 20 per rHb) which each contain one heme group, therefore each mole of hemoglobin contains 4 moles of heme.
The accumulation of soluble rHb9.1 was strongly coirelated with the amount of hemin supplied with an apparently linear irelationship up to about 1nzM hemin (Figure 3A).
Addition of hemin to a concentration>ImM did not improve soluble rHb9.1 accumulation, indicating that it was not limiting under these conditions (Figure 3A).
The correlation of accumulation of soluble rHb9.l subunits (mM), with the mM
hemin supplied was very strong over this initial concentration range (Figure 3A;_R-squared =
0.90; 50 samples). The comparison indicated that an approximately three-fold niolar excess of hemin was required for soluble accumulation of rHb9.1 to a range of concentrations (heavy line in Figure 2). There was not a single sample in 70 examined that was accompanied by less than a 2.5-fold molar excess of hemin. This was therefore taken as the. lower limit of hemin excess required for maximal soluble rHb accumulation.
To determine the relative importance of time of accumulation and hemin concentration, two sets of data were compared from different hemin supplementation strategies which resulted in nearly identical concentrations of hemin at different times during accumulation. One resulted in the addition of a total of 0.64mM hemin by 6 houts post induction, and the second msulted in the addition of 0.63mM hemin by 12 hourspost induction. The concentration of soluble rHb9.1 was compared on a per heme basis at 8 hours post induction for the first condition, and 14 and 16 hours post induction for the second co2idition. Regardless of the time since induction, the soluble rHb9.l -accumulation was indistinguishable (0.22t0.02 mM and 0.19t0.04 mM
respectively), suggesting that under these conditions, hemin was more important than time of accumulation in achieving high soluble yields.
The loss of heme from the media was examined in l5L fermentations, grown at 28 C, in which rHbl.l expression was induced. by the addition of l00 M IPTG, and correlated with the accumulation of soluble rHb. An approximately stoichiometric relationship was observed, indicating that only the heme required to support soluble rHb accumulation disappeared from the media (Figure 3).

Example 15 Soluble Accumulation Of rHbO. I In Heme-Free Fermentations A study was conducted to test whether rHbO.1 would accumulate to higher soluble levels than rHbl.l in fermentations without heniin supplementation.
Two fermentations at 28 C induced with 100 M IPTG resulted in an average soluble rHb0.1 yield of just 2.5f0.2% of the soluble cell protein, similar to that seen with rHbl.l in Verderber et al., J. Bacteriol., 179:4583-4590 (1997). Using 10 M
IPTG
for induction, an average of 1.6t0.3% of the soluble cell protein was soluble rHb0.1.
Dividing the soluble rHb0.1 accumulation in the absence of hemin by the accumulation in the presence of hemin an estimate was made that a maximum of 12-15% of the accumulation in the presence of hemin can be due to E. coli heme biosynthesis.

Example 16 Functionality Of rHbs Samples of six variants of recombinant hemoglobins were purified. Two parameters of functionality, oxygen binding affinity (Po and cooperativity (nMu) were measured (Table 19). As expected, the hemoglobin mutations affected the oxygen binding in the recombinant molecules to approximately the same degree they did in the native hemoglobin molecules. The con-elation between the two functional parameters, and between each and the soluble expression levels, were examined. The two functional parameters were not correlated with each other (R2 = 0.04; P =
0.69). In spite of this, both were equally correlated with the percent soluble expression R2 =
0.046 for P50 (P = 0.14), and R2 = 0.47 for nMu (P = 0.13). Since the P values were greater than 0.1, the correlation of low P., and/or nM,. with higher expression was not statistically significant. In addition, a sample of soluble rHb9.1 from a fermentation yielding 3.7 g/L, was examined for norvaline substitution, which had been previously observed in rHbl.1 (19, 20). The level of norvaline was below the liniit of -25 quantitation, indicating that high level soluble expression did not necessarily increase the level of this amino acid misincorporation.

Effect of beta globin mutations on rHb functionality ,g)~ 1Hh beta mutation EQ nm di- 0:l wt 10 2.2 9.1 Lys82--+Asp 15 2.0 1.1 Asn l 08-+Lys' 31 '2.4 9+1.1 AsnlO8-4Lys+ 44 2.2 Lys82-4Asp mono 0.0 wt 15 2.6 1.0 Asn 108--4Lys 36 2.6 5..
Example 17 Genetie Construction of.NO Mutants Mutations were introduced into cloned human alpha and beta genes via site directed PCR-based mutage'nesis as described in general by Innis et al., PCR
Protocols:
A Guide to Methods and Annlications (1990). In general, the desired. mutations were introduced into synthetic DNA oligonucleotides which were synthesized according to the manufacturer's instructions on an Applied Biosystems 392 DNA
synthesizer. Following standard deblocking procedure, oligonucleotides were, dried by vacuum-centrifugation, resuspended in the desired buffer and diluted to, 10-50 pmoV l in sterile water.
These oligonucleotides were used as primers in PCR reactions whcre a recombinant plasmid carrying cloned wild type alpha and beta genes, such as pSGE728 (Fig. 4), was used as template DNA.
These mutagenic oligonucleotide primers were chosen to span the site at which the mutation(s) was to be introduced and a nearby restriction endonuclease recognition site to facilitat,e cloning of resulting PCR products carrying mutadons of interest. A
second oligonucleotide primer was also required in the PCR rraction to allow DNA
amplification. This primer also could be designed to contain globin gene mutation(s) or alternatively could consist of wild-type globin gene seguencx from a neighboring rsgib-nzsf-th-c-alpha-or-beta g}obin-gene.- This-second primer-was-also-chosen-to- contain a rr,striction tndonuclease recognition site so that the resulting PCR product could be cloned into appropriately digested pSGE728 for subsequent expression of the mutated alpha or beta globin. Partial restriction maps of the alpha and. beta genes -from pSGE728 are shown in Fgures 5 and 6.
The lengths of the mutagenic oligonucleotides were determ,ined by the distance between the site to be mutated and the closest trstriction site that could be incorporated into the mutagenic oligonucleotide at a position between tht 5-prime end of the oligo and the site to be mutated. The mutagenic oligos were typically 30 to 45 nucleotides in length and contained mutations affecting one or two codons although more positions l0 potentially could also be altered if desired. It was generally desirable to place the mutated DNA sequences as far as feasible from the 3-prime end of the oligo so as to avoid or minimize potential problems during the primer annealing step of the PCR
reaction. The mutated nucleotides were generally placed 5-10 nucleotides upstream of the 3-prime end of the mutagenic primer. The globin gene restriction site incorporated near the 5-prime end of the mutagenic oligonucieotide was generally placed 5-nucleotides downstream of the 5-prime end to facilitate subsequent digestion of PCR
products. Oligonucleotides which were employed solely as primers in PCR (i. e.
did not contain mutations) weretypically 24-36 nucleotides in length and contained globin gene restriction sites generally located 6-12 nucleotides downstream of the 5-prime end of the oligonucleotide.
PCR reactions were generally performed in an Applied Biosystems GeneAmp 9600. PCR reaction conditions were empirically detenmined: denaturation was typically at 959 C for 15-60 seconds, generally annealing temperaturts ranged from 45-60 C for 15-30 seconds with many reactions being run in 50-55 C'range for annealing, and extensions were done at 72 C for 15-120 seconds.
In some instances the annealing temperature was raised during the course of the reaction: e.g. a reaction might consist of 5 rounds with an annealing temperature of 45 C followed by 20 rounds at an annealing temperatuie of 60 C. Typically Teactions consisted of a total of 25-30 cycles. The reactions were typically performed in 10 mM
Tris-HCl (pH 8.3), 50 mM KCI, 1.5 mM MgClz 0.2 mM dNTPs (Phannacia)'and 0.00196 gelatin. Oligonucleotide primers were added at concentrations usually 0.5-1.0 pM: Purified plasmdd DNA (about 0.1-10 ng per rr,action) such as pSGE728 was generally used as template. AmpliTaq DNA Polymerase (Perkin Elmer) was typically used at 1-10 units per rcaction and Teaction volumes.tanged from 20-100 ul.

Following the PCR reaction the reaction products were purified using the QlAquick PCR Purification Kit (QIAGEN Inc. Santa Clarita, CA). The purified products were then subjected to restriction endonuclease digestion with the appropriate enzymes to generate DNA fragments suitable for cloning into similarly cut pSGE728.
Restriction digests were performed according to vendor protocols.
The digested PCR fragments could be cloned directly or first subjected to agarose gel electrophoresis and purified out of :the agarose gels. Gel composition and electrophoresis conditions were chosen based on the DNA fragment size. Many fragments were about 60-250 base pairs in length and for these fragments resolution in gel electrophoresis is optimal with gels such as 3% NuSeive agarose or 4%
Metaphor agarose, both obtained from FMC BioProducts (Rockland, ME) and used according to vendor protocols. Following elect-ophoresis, DNA fragments wr.re purified out of agarose gel slices using the QIAEX II Gel Extraction Kit (QIAGEN~Inc. Santa Clarita, CA) according to the vendor protocols. The vector pSGE728 was also digested with the enzymes appropriate for cloning the mutagenized PCR fragment(s) of interest and similarly gel-purified following more conventional electrophoresis.
Digested and purified mutagenized PCR fragments were ligated with digested and purified pSGE728 veetor fragment using T4 DNA ligase (New England BioLabs.
Beverly, MA) according to the vendor protocols and ligation products were used to transform E coli. E coli strain JM l 09 obtained as competent cells from Promega (Madison, WI) was often used for this purpose although other strains of E coli could also be employed. as well as other various methods for preparation of competent cells (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Springs Harbor, 1989)). Transformants were selected for tetracycline-resistance and could subsequently be sequenced to identify the mutations of intetrst and confirm the sequence of the mutagenized cloned PCR segment. Plasmid templates were prepared . using the QIAprep Spin Miniprep Kit (QIAGEN) and sequenced using the AmpliCycleTm Sequencing Kit (Perkin Elmer, Foster City, CA) according to the vendor protocol.
Some sequencing was performed manually using Sequenase .(United States Biochemical, Cleveland, OH) but, the prefenred method is automated sequencing using the Amp1iC,ycleT"' Sequencing Kit (Perkin Elmer, Foster City, CA) to perfonm sequence neactions according to the vendor protocol and analysis on an ABI
Prisnri 377 DNA Sequencer (Applied Biosystems Division of Perkin Elmer, Foster City, CA).
Variations of this procedure were sometimes used if a site to be mutated was not located sufficiently close to a restriction site that was unique in pSGE728.
In that case a so-called "helper" DNA fragment might be employed to facilitate cloning steps. For example in the alpha gene (Fig. 5) the codon for the V62 position of that protein is somewhat distant from unique restriction sites so that the Mae IlI site shown in that figure, which is not unique to the plasmid but is unique to the segment from the Bam HI site to the V62 codon. Therefore, the V62 codon was mutated on a PCR
fragment spanning the Mae III through Mlu I segment of the alpha gene and this 'fragment following Mae III digestion was ligated to a gel-purified Bam H I - MaeIII
ffagment of pSGE728. This ligation product was digested with Bam HI and MIuI ( both of which are unique cutters within pSGE728) and the Bam HI-Mlu I fragment was gel purified and ligated to Bam HI- Mlu I- and gel-purified vector fragment of pSGE728.
Alternatively V62 mutations could have been incorporated into longer oligonucleotides that spanned unique sites such as Mlu I.
For construction of large libraries of mutant hemoglobins in which two to four amino acids were simultaneously mutated essentially randomly, similar procedures were followed for mutagenic oligonucleotide design, PCR reactions, and cloning steps.
However, it was sometimes desirable to omit restriction sites from certain mutagenic oligos and instead to incorporate the restriction sites necessary for cloning into pSGE728 via a subsequent, PCR amplification with a primer that pardally overlapped this particular mutagenic " oligo and spanned a useful restriction site. These amplifications needed to be performed under conditions designed to rigorously exclude environmental contaminants of wild type globin sequences such as pSGE728 and other recombinant plasmids carrying globin sequences. These sorts of environmental contaminants could potentially be preferentially primed in this latter type of amplification because they can anneal to the full length of these oligo primers whereas the PCR fragments which are the target templates only anneal to a smaller portion of such primers.
In some instances two or more mutagenized PCR segments were ligated together to create segments containing as many as four mutagenized sites prior to cloning into appropriately digested pSGE728 vector. The appropriately sized ligation products were identified and subsequently purified by agarose gel electrophoresis. In some instances the purification step was preceded or followed by PCR
amplification using primers that would specifically amplify the ligation product of intenrst.
To generate a wide spectrum of amino acid substitutions at positions of interest, mutagenic oligos were synthesized with degeneracies at positions of interest.
For "randomization" of a given position two degenerate oligos were synthesized, one of which contained the sequence N(T/A/C)T at the codon to be randomized while the other contained the sequence (A!I')(T/G)(T/G) at the same position. These two oligos could be pooled prior to PCR but more usually two independent PCR reactions were used with such pairs and the PCR products roughly quantitated (using an AlphaImagerT"'2000 Documentation & Analysis System from Alpha Innotech Corp San Leandro, CA) by visualization following gel electrophoresis. Once quantitated, roughly equivalent quantities of each fragment could be. pooled for subsequent cloning steps. This "randomization" results in 20 different codons generating 16 different amino acid substitutions: there are..two codons each for F, I, L, and S; I
codon for each of D. R. N. A, T. P. C, V, M, H, W, and Y. The amino acids E. K, Q, and G
are absent from "randomized" positions in these libraries.
Following ligation into pSGE728 vector and transformation into E coli, a numbr,r, ,(typically, 24-28) of independent Transformants were picked and the mutagenized PCR segment cloned in each was sequenced.. Plasmid templates were prepared using the QIAprep Spin Miniprep Kit (QIAGEN) and sequenced using the AmpiiCycle'm Sequencing Kit (Perkin Elmer, Foster City, CA) according to the vendor protocol. Sequences were run and analyzed on an ABI Prism 377 DNA Sequencer (Applied Biosystems Division of Perkin Elmer, Foster City, CA). Sequences were analyzed to assess the distn'bution of amino acid substitutions within a given library and the frequencies of PCR-induced and synthetic oligo-induced errors in DNA
sequence.
Subsequently clones from librarieswere picked and analyzed as described below.
Example 18 Construction of a and P Mutants (Di-alphaand Di-di-alpha) For -some studies it was desirable to produce recombinant hemoglobins that were mutated in both the alpha and beta subunits. Toward that end, alpha and beta .
mutations can be combined in derivatives of pSGE728. Typically such combinations can be achieved by cutting mutant derivatives of pSGE728 with appropriate restriction endonucleases that sepaiate the alpha and beta sequences, gel-purifying the zestriction fragment containing the beta gene of the mutant beta derivative of pSGE728, gel-purifying the mstriction fragment containing the alpha gene of the mutant alpha derivative, ligating these two fragments together, transforming E coli and analyzing the resulting transformants to verify the presence of both alpha and beta mutations.

For alpha and beta mutations at residues B 10, E l 1, G8 and E7 such combinations can be made by digesting the mutant derivatives of pSGE728 with Bsp HI which cuts within the tetracycline-resistance gene and SacII which. cuts within the beta gene, about 28 base pairs from the start of the beta coding sequence.
Digestion with SacII and BspHl (New England BioLabs, Beverly MA) according to the vendor protocols results in two DNA fragments: one 937 bp in length containing a portion of the gene for tetracycline-resistance and nearly all of the beta gene and including the codons for amino acid residues B 10, El 1, G8 and E7, and the second -2318 bp in length which contains a portion of the gene for tetracycline-resistance and all of the alpha gene. These digestion products can be readily separated by electrophoresis on agarose gels of (0.6-1.0) k using SeaKem GTG agarose (FMC BioProducts, Rockland, ME) according to the vendor protocols. Subsequently the 937 bp fragment derived from the beta mutant derivative of pSGE728 can be excised out of the agarose gel and purified using the QIAEX II Gel Extraction Kit (QIAGEN Inc. Santa Clarita, CA) according to the vendor protocols.
Similarly the 2318 bp fragment from the pSGE728 derivative carrying the alpha mutation can also be excised from the gel and purified. These two purified fragments -can be ligated together using T4 DNA ligase (New England BioLabs Beverly, MA) according to the vendor protocols and ligation products were used to transform E coli.
E coli strain JM 109 obtained as competent cells from Promega (Madison, WI) can be used for this purpose although other strains of E coli could also be employed as well as other various methods for preparation of competent cells (Sambrook et al., u ra ..
Selection for tetracycline resistant transformants selects for reconstitution of the tetracycline-resistance gene and this is nearly always associated with reconstitution of the beta gene at the SacII site. When individual transformants thus obtained are analyzed by determining DNA sequence for alpha and beta genes and gross plasmid structure, more than 90% are found to be the desired recombinants which have both the alpha and beta mutations. For sequence analysis plasmid templates can be prepared using the QlAprep Spin Miniprep Kit (QIAGEN) and sequenced using the AinpliCycle'*' Sequencing Kit (Perkin Elmer, Foster City, CA) according to the vendor protocol. Sequences were run and analyzed on an ABI Prism 377 DNA Sequencer (Applied Biosystems Division of Perkin Elmer, foster City, CA).
For some studies it is desirable to produce recombinant hemoglobins carrying amino acid substitutions in both alpha and beta and in which the alpha subunits are genetically fused by a glycine (or some other amino acid) linker. Methods have been described in Looker et al., Nature 356:258-260 (1992) for the construction of such "di-alpha" fusions and these methods could be applied to construct di-alpha versions of any mutant alpha gene. Such di-alpha mutants could readily be eombined with any beta mutant of interest as described above.
For some studies it is desirable to produce recombinant hemoglobins carrying amino acid substitutions in both di-alpha and beta and. in which the di-alpha subunits are genetically fused by a peptide linker so that a"di-hemoglobin" or ."di-di-alpha"
molecule is produced. Methods are described in WO 96/40920, for the construction of such "di-di alpha" fusions to produce di-hemoglobins and these methods could be applied to contruct di-di alpha versions of any mutant alpha gene. Such di-di alpha mutants could readily be combined with any beta mutant of interests as described above.

Example 19 Small-scale production for in vitro kinetic assays For kinetic mcasu'mments on large numbers of hemoglobin variants E coli stTains containing rzcombinant plasmids, such as derivatives of pSGE728, which encode variant hemoglobins were typically grown in shake flasks usually at volumes of about 50 ml. Generally defined media supplemented with about 0.2% yeast extract were used for cell growth and tetracycline was added, generally at 15 g/ml to select for maintenance of the recombinant plasmid. Expression of the hemoglobin genes was induced by additioa of IPTG, usually at a concentration of lUO M and hemin was added to a final concentration of 50 g/ml generally at the time of induction.
Cells ware ---generally grown and induced at 28 C. Cells grown to stationaiy phase, such as typical saturated overnight cultures, could be dircetly inoculated (generally at a dilution ranging from 1/50 to l/l 000) into media containing IPTG and hemin or such cultuncs could bc inoculated into media lacking IPTG and hemin, grown to log phase, e.g. 0.4-0.7 CD
0 A6W and then induced by the addition of IPTG with hemin typically being added to the cultures at the time of induction. Culturrs were generally grown under inducing conditions ovemight (-1420 hours) although shorter times, e.g. about 6 hours could also be employed. At the end of this time, cells were pelletcd by centrifugation and the cell pelleted wene either frozen and stored at -80 C or procesnd immediately.

Recombinant hemoglobins 'were purified by small-scale column chromatography using Fast Flow Zn-Chelating Sepharose (Pharmacia). During the purification, cells, lysates, all buffers and eluted hemoglobins were kept cold on ice as much as possible. Typically, a pellet of a 50 ml cultune was resuspended with 1.0 ml ice-cold 25 mM sodium tetraborate and transfcrred to a 1.7 ml eppendorf tube.
Cells were usually lysed by sonication, although enzymatic lysis by lsysozyme could also be employed. Sonicated lysates were clarified by centrifugation (generally about 14,000 x g for 15-20 minutes at 4 C) following addition of 20 ul of 20 mM ZnAcetate.
Supernatants were loaded onto a -150-200 ul column that had previously 'been equilibrated as follows:
2-10 column volumes 0.5 M NaOH
6-10_column volumes 0.5 M NaCI, 20.mM Tris-HCI pH 8.1 @ 0 C
3-10 column volumes 20 mM ZnAcetate 6-10 column volumes 0.5 M NaCI, 20 mM Tris-HCl pH 8.1 @ 0 C
Following the loading, the column was washed with at least 9 column volumes 0.5 M NaC1, 20 mM Tris-HCI pH 8.1 at 0 C followed by at least 3 column volumes 0.05 M NaCI, 20 mM Tris-HC1 pH 8.1 at 0 C and then eluted with -1.0 ml of the desired buffer (e.g. 0.1 M Na phosphate pH 7.0) containing 30 mM EDTA.
Hemoglobin was typically recovered in a volume of -200-400 ul. These samples were used in kinetic assays such as NO oxidation and 02 dissociation. If not used immediately, samples were frotien and stored --80 C.

. Example 20 Purification of heme pocket mutants All molecules, monomeric and dimeric were first captured by immobilized mental affinity chromatography (IMAC) and further processed as described in WO 96/15151. The hemoglobin solution was then diafiltered into the appropriate load buffer for further purification. For monomeric hemoglobins, the appropriate load buffer was 20mM Tris (pH 9.0) for loading onto an anion exchange column (Q
sepharose FF, Pharmacia, Uppsala, Sweden). The protein which was loaded onto the column at 15gm/L is washed with three column volumes of 12.5mM Tris =
(pH7.8). The protein is then eluted in two to three volumes of 12.3mM Tris (pH7.6) or if the pI of the protein is below 7.5 it is eluted in a Bis-Tris buffer at the appropriate pH. pI was used to determine proper wash and elution conditions for each protein, both monomeric and dimeric. Certain mutations on the surface of some of the heme pocket mutants were found to effect this value. For dimeric hemoglobins, the appropriate load buffer was 10 mM KPi (pH 7.0) for loading onto a ceramic hydroxyapatite (CHT) column (BioRad) or 20 mM Tris (pH 8.0) for loading onto a hydrophobic interaction chromatography (HIC) column (BioRad). The protein is loaded onto the CH'T column at 20gm/L the column is then washed with eight column volumes of 30-40mM KPi (7.0). Five column volumes of 85-90mM Kpi (pH7.0) are used to elute the protein from the coiumn. When the HIC column is.used protein is 1.0 loaded at 15gm/L, and the column is then washed with five column volumes of 1.2M
Ammonium Sulfate/20mNI Tris (pH 8.0). The protein is eluted using 3 column volumes of 1M Ammonium Sulfate/20mM Tris (pH 8.0). The wash steps for both CHT and HIC columns were developed to allow the monomeric hemoglobin to be eluted while leaving the di-hemoglobins and larger molecules bound to the column.
Pools from either column are diafiltered to prepare for loading onto .an anion exchange column. The anion exchange step is designed to wash away remaining monomeric hemoglobin from the di-hemoglobin yielding a di-hemoglobin pool that is 98%
pure on a size basis. The anion exchange column is a Super Q 650M (TosoHass). The column is equilibrated with 20mM Tris (pH9.0) and 15gm/L of protein is loaded on the column. The column is then washed with three column volumes of 10-15mM Tris (pH
7.6-7.8) and the protein is eluted in thnx column volumes of 15-30mM Tris (pH
7.6-7.8) or if pH is between 7.3-7.6 then a Bis-Tris buffer is used. T'he protein from this point on is handled as described in WO 96/15151.

Example 21 Measurement of Reaction Between oxyHb and NO

Nitric oxide gas was passed through a column of NaOH pellets and- used to thoroughly flush a tonometer. Anaerobic buffer (0.1 M sodium phosphate, pH
7.4) was injected into the tononieter and equilibrated with the NO to make a stock solution.
Dilutions of the stock solution were made in glass syringes containing anaerobic buffer.
Time courses of the reaction of oxyhemoglobin with NO were collected at 420 and 402 nm using an Applied Photophysics stopped-flow device. Temperature was 20 C.
Data were collected and analyzed using the software program !SX.17MV supplied by Applied Photophysics.

Substituted alpha and beta subunits having approximately equal values of k'NO, ax were combined into tetrameric hemoglobin constructs for use in animal models.
These "paired-mutant" rHbs (e.g. rHb2, rHb3, and rHb4; Table 20) were constructed with a genetically fused, mutated di-alpha subunit to prevent dissociation of the hemoglobins into alpha/beta dimers as described above. In Table 2, the mutations are designated, for example, as ON 108K, which means the native human beta globin sequence residue number 108; with wildtype Asn (N) replaced by Leu (K).

rHb description (mm AMAP H ) ( M~S ,) (mms Hg) (h~ )~
rHbl.l rHb0.1 +ONl08K 25.8 2Ø 58 32 0.051 rHb0.1 wild-type + dialpha 28.4 f 5.7 58 10 0.042 fusion -rHb rHb0.1 +(3Y 145H 24.1 2.7 56 2.7 0.041 Bethesda rHb2 rHB0.1 + 19.3 3.4 24 4.0 0.089 aV62F/ V67F/ L106I
rHb3 rHb0.1 + 13.0 1.6 15 3.4 0.10 aV62L/PVb7F
rHb4 rHb0.1 + 7.6 1.0 2 5.2 0.072 aL29F/RV67W

The resulting recombinant oxyhemoglobins have monophasic reactions with nitric oxide (Figure 7), simplifying interpretation of in vivo experiments by having only one rate constant to consider. The values of k'NO, ox for rHb2, rHb3, and rHb4 are 24, 15, and 2 M''s'', respectively. Recombinant hemoglobins rHbl.1, rHbO.1 (des val, di-a), rHbO.0 (des val, wildtype type), and rHb Bethesda have a wide range of values (32, 10, 15, and 2.7 mm Hg, respectively) due to different tendencies to form the low-affinity T-state quaternary structure. However, the latter four proteins have wild-type amino acids in the heme pockets and identical high values of the rate of NO-induced oxidation (-60 M's''). The data suggest that, regardless of the position of the allosteric (RrT) equilibrium, only substitutions within the heme pocket affect the NO
neactivity of oxyhemoglobin.

All three of the paired-mutant hemoglobins have oxygen dissociation curves that are significantly left-shifted compared to rHbl.1 (Table 20). The low values of P50 simplify interpretation of blood-pressure experiments by minimizing the formation of deoxyhemoglobin. In experiments with top-load administration and a normal complenient of red blood cells, these high-affinity hemoglobins will remain nearly 100 ,b oxygen-saturated throughout the circulation. Therefore, only the reactions of oxyhemoglobin with NO need be considered For rHb 1:1, the rate of NO binding to deoxyhemoglobin is similar to the rate of NO reaction with oxyhemoglobin, so the two forms of rHbl.1 have similar ability to scavenge NO. Generally, the rate of NO-induced oxidation is very similar to that for NO binding to the deoxy forms of myoglobins and hemoglobins, since the rate-limiting step in both processes is ligand entry into the distal pockets (Eich et aL, suFra.l .

Tables 21 and 22 provide additional.NO binding data for various mutant hemoglobins.

Strain Description k'Nq.o kozI Pso ~x (j.iM' s' ) (s' (mmHg) SGE3004 diaV62IJOV67F 15 2/6 4.5 1.85 SGE3005 3004 + St. Mande 15 6/43 8.6 1.38 SGE3263 3004 + Prov. + Presby. 15 13.4 1.64 SGE3173 di-di 3004 13 SGE2821 diaL29F, H58Q/PL106W 12 12/30 42 1.7 SGE3006 2821 + Prov + St. Mande 13 -100 1.83 SGE 3007 2821 + Prov + Pres 13 8/25/80 -100 1.7 SGE2822 di-di 2821 11 13/32 SGE1738 2822 + Pres SGE3001 diaL29F/(3V67W 1.5/4 0.3/4 6 1.63 SGE1728 3001 + Pres 1.2/5.5 0.3/2/21 SGE1729 3001 + Prov 1/4 0.3/2/10 SGE3011 diaL29W,H58Q/Pv67W 1.5/5 4/15 46 1.3 SGE3010 3011 + Prov 1.5/5 6/20 52 1.43 SGE2966 3011 + Pres SGE2968 3011 + Prov + Pres 1.6/5.5 1 l4 1.89 SGE2967 3011 + St. Mande 2.5/10 75 1.60 SGE2963 di-di 3011 37 1.13 SGE2971 di-di 3011 + Prov 1.5/5 6/20 42.8 1.23 SGE2969 di-di 3011 + Pres SGE2972 di-di 3011 + Prov + Pres SGE2970 di-di 3011 + St. Mande SGF3012 diaL29W. H580/(3L106W 2/9 87 1.57 In Table 21, the values for k'~,_( M"'s') and k02 (s'') are in some cases expressed as two values (e.g., 2/6), which reflects the range of results that were sometimes obtained during analysis of these mutant combinations.

Subunit Name k'Noox kos ( lVl-'s" at 20 0(s'' at 20 0 awild type 1483 (rHbO.0) 60 10 a-(B I0)L29F A51 3 0.2 aL29F/aH58Q 1733 (3341 di*) 14 9 a(B l 0)L29W A57 3 -2 aL29W/aH58Q (3350 dia) 2.7 8 a (B 10)L29A 3159 -w.t. 8 a(E11)V62F aEl l#10 26 4 a(E11)V62W . aEl 1#6 22 4 a(E] 1)V62L aEl 1 #7 16 4 a(G8)L101I
a(G8)L101F HB9, HB12 w.t.
a(G8)LIOIW HB1, HB5 w.t.
wild type 1483 (rHbO.0) 60 35 p(B l0)L28F AS4 w.t.
(3(B l 0)L28W AS6 w.t.
P(E11)V67F 17,25 (JD16ap) 12 3 P(E1I)V67W 3313 (NS2) 6-5 3 PV67W/PH63Q(PK82D) 1735 .3/3 2/20 PV67W/PH63A(PK82D) 1734 3(E11)V67L 1724 (JD 15 sm) 16 24 P(G8)L 106I 1727 (JD 18 sm) 60 45 P(G8)L106F(13N108K) 1723 (JD4 sm) 30 60 P(G8)L106W 3267 ) 30 60 OV67"L106M JDI 12 PV67F/pL106I JD9 24 3 PV67F/PL106W JD16 w.t. -5 PV67F/pL106M EVI 25,5 2 BV67M/6L106F 0.8.3 40.1.4 "w.t.." indicates that the mutant was not distinguishable from rHb0.0 Example 22 Hemodynamic Data Hemodynamic responses to hemoglobin administtation were obtained in conscious, unrestrained rats. Male Sprague-Dawley rats were chmnically instrumented with indwelling arterial and venous catheters at least 48 hours prior to experimentation.
Top-load doses of 350 mg/kg of the rHb solutions (5 g/dI) were administered to separate groups of rats via intravenous infusion at a rate of 0.5 mUmin. Human serum albumin (HSA, 5 g/dl) was administered to another group of rats as a volume control.
Arterial pressure was monitored continuously for 30 minutes prior to and 90 minutes following administration. All data are shown as mean standard error.
Statistical comparisons between rHbI.1 and the other hemoglobins or HSA were made by repeated measures.analysis of variance, p-values <_ 0.05 were considered significant.
The mean arterial pressure (MAP) responses elicited by rHbl .l and each of the three paired-mutant rHbs were determined in conscious rats (Figure 8). The magnitude of the pressor response decreased as the rat,e constant for NO scavenging was decreased. At the lowest rate of NO oxidation, the pressor response was nearly as low as that observed following administration of an equivalent volume of 5% human serum albumin (HSA). These effects were not due simply to the low Pso values for the distal pocket mutants, as shown in Figure 9. The pressor effect of rHbl .l (P.50 = 32 mm Hg) was identical to that of rHb Bethesda, which had a P50 value (2.7 mm Hg) lower than any of the other hemoglobins in this study.
These results (Table 20, Figures 8 and 9) indicate that Oz delivery to arteriolar smooth muscle or parenchyma tissue is unlikely to be the mechanism of the observed pressor effect. The Pso of rHb Bethesda is low enough that this hemoglobin is essentially incapable of delivering oxygen to normal tissues, in top-load experiments where a normal complement of erythrocyte hemoglobin is present and maintains normal tissue pOz levels. However, rHb Bethesda elicits a pressor response identical to that of rHb1.l, which has a high Pio and is capable of oxygen delivery. rHbO.1 has a similar pressor effect and an interrnediate P50 (Table 20). The results indicate that the mechanism of the pressor response is depletion of nitric oxide, not excessive oxygen delivery to arterioles. The changes in blood pressure correlate with the rates of NO-induced oxidation but not with oxygen affinity (Table 20).
The autooxidation rate of each hemoglobin was detenmined to examine the hypothesis that NO scavenging is mediated by methemoglobin (Alayash et al., Mol.
Med. Todax 1: 122-127 (1995); Alayash et al., Arch. Biochem. BioRhvs 303: 332-(1993)). There is no cornelation between autooxidation rates and pressor responses (Table 20), making it unlikely that the production of inethemoglobin (or the concomitant generation of superoxide radical) is responsible for the increase in blood pressure. All of the autooxidation rates fall within a narrow range, with the largest value only about 2-fold greater than the smallest. rHb4 has the least effect on blood pressuTe but has an intermediate rate of autooxidation. Furthermore, the process of autooxidation is quite slow, with in vitro half-times greater than 6 hours at 37 C, while the pressor response reaches a maximum in only 5-10 minutes.
For each timecourse in Figure 8, the post-infusion blood-pressure data were averaged, and the results were plotted against the value of NO-induced oxidation. As shown in Figure 10, there is a direct relationship between the magnitude of pressor response and the rate of nitric oxide oxidation. The graph indicates that nitric oxide scavenging activity can account for nearly all of the pressor response of extracellular hemoglobin. If the value obtAined for the albumin volume control is taken as the lower limit, there is 'very little pressor response remaining when the rate of NO-induced oxidation is extrapolated to zero. These results suggest strongly that the fundamental mechanism of the pressor response is reaction of nitric oxide in the distal heme pockets of oxy- and deoxyhemoglobin.
Therefore, it has been shown that the rate of reaction of hemoglobin with nitric oxide can be lowered significantly by replacing native distal heme pocket residues with larger, hydrophobic amino acids. The reactivity of NO toward both alpha and beta subunits can be controlled by this strategy. In contrast, amino acid substitutions outside the region of the heme pockets, some of which affect oxygen affinity, appear to have no effect on the oxidative reaction with NO. Similarly, fusion of the alpha subunits to stabilize the tetrameric form of hemoglobin has no effect on NO
reactivity, as the value of NO-induced oxidation for rHbO.0 is the same as those for rHbO.l and rHb1.1.
The foregoing description of the invention is exemplary for purposes of illustration and explanation. It will be apparent to those skilled in the art that changes and modifications are possible without departing from the spirit and scope of the DEMANDES OU BREVETS VOLUMINEUX
LA PRtSENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.

CECI EST LE TOME DE _2 NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.

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Claims (12)

1. A hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the alpha subunit:
E11(Val.-- > Leu) and E7(His.-- > Gln);
E11(Val.-- > Phe or Trp) and E7(His.-- > Gln);
E11(Val.-- > Phe or Trp or Leu) and E7(His.-- > Gln) and G8(Leu.-- > Phe or Trp);
B10(Leu.-- > Phe) and E4(Val.-- > Leu);
B10(Leu.-- > Trp) and E4(Val.-- > Leu);
B10(Leu.-- > Trp) and E7(His.-- > Gln);
B10(Leu.-- > Trp) and E11(Val.-- > Phe);
B10(Leu.-- > Trp) and E11(Val.-- > Trp);
B10(Leu.-- > Trp) and E11(Val.-- > Leu) and G8(Leu.-- > Trp);
B10(Leu.-- > Trp) and E11(Val.-- > Leu) and G8(Leu.-- > Phe);
B10(Leu.-- > Trp) and E11(Val.-- > Phe) and G8(Leu.-- > Trp);
B10(Leu.-- > Trp) and E11(Val.-- > Phe) and G8(Leu.-- > IIc);
B10(Leu.-- > Trp) and E7(His.-- > Gln) and E11(Val.-- > Leu) and G8(Leu.-- >
Trp);
B10(Leu.-- > Trp) and E11(Val.-- > Trp) and G8(Leu.-- > Trp);
E11(Val.-- > Leu) and G8(Leu.-- > Phe);
E11(Val.-- > Leu) and G8(Leu.-- > Trp);
B13(Met.-- > Phe or Trp);
G12(Leu.-- > Phe or Trp); or B14(Phe.-- > Trp).

2. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are:
B10(Leu.-- > Trp) and E7(His.-- > Gln).

3. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are:
B10(Leu.-- > Phe), E7(His.-- > Gln) and G8(Leu.-- > Trp).

4. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu-- > Trp), E7(His-- > Gln), E11(Val-- > Leu) and G8(Leu-- > Trp).

5. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu-- > Trp) and E11(Val-- > Phe).

6. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu-- > Trp) and E11(Val-- > Trp).

7. The hemoglobin mutant of claim 1, wherein mutations in the alpha subunit are B10(Leu-- > Trp), E11(Val-- > Phe) and G8(Leu-- > Ile).

8. A hemoglobin mutant with reduced rate of reaction with nitric oxide selected from the following mutation in the beta subunit:
B13(Leu-- > Phe or Trp);
G12(Leu-- > Phe or Trp);
B10(Leu-- > Phe) and E4(Val-- > Leu);
B10(Leu-- > Trp) and E4(Val-- > Leu);
B14(Leu-- > Phe or Trp);
G8(Leu-- > Phe) and G12(Leu-- > Trp);
E11(Val-- > Leu) and G8(Leu-- > Trp);
E11(Val-- > Trp) and G8(Leu-- > Met);
E11(Val-- > Leu) and G8(Leu-- > Phe);
E11(Val-- > Leu) and G8(Leu-- > Met);
E11(Val-- > Phe) and G8(Leu-- > Ile);
E11(Val-- > Phe) and G8(Leu-- > Phe);
E11(Val-- > Phe) and G8(Leu-- > Trp);
E11(Val-- > Phe) and G8(Leu-- > Met);
E11(Val-- > Met) and G8(Leu-- > Trp);
E11(Val-- > Met) and G8(Leu-- > Trp) and E7(His-- > Gln);
E11(Val-- > Trp) and G8(Leu-- > Ile);
E7(His-- > Gln) and E11(Val-- > Trp);

E7(His-- > Gln) and E11(Val-- > Leu);
E7(His-- > Gln) and E11(Val-- > Phe);
E7(His-- > Gln) and E11(Val-- > Phe) and G8(Leu-- > Phe or Trp);
E7(His-- > Gln) and E11(Val-- > Leu or Trp) and G8(Leu-- > Phe or Trp);
E11(Val-- > Trp or Phe) and G12(Leu-- > Trp or Met);
E11(Val-- > Trp or Phe) and B13(Leu-- > Trp or Met);
B10(Leu-- > Trp) and B13(Leu-- > Trp or Met);
B10(Leu-- > Phe) and B13(Leu-- > Trp);
B10(Leu-- > Trp or Phe) and G12(Leu-- > Trp);
B10(Leu-- > Phe) and G12(Leu-- > Met);
G8(Leu-- > Trp) and G12(Leu-- > Trp or Met); or G8(Leu-- > Trp) and B13(Leu-- > Trp or Met).

9. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val-- > Met) and G8(Leu-- > Trp).

10. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val-- > Met), G8(Leu-- > Trp) and E7(His-- > Gln).

11. The hemoglobin mutant of claim 8, wherein mutations in the beta subunit are E11(Val-- > Leu) and G8(Leu-- > Trp).

12. A hemoglobin mutant having the following mutations: alphaB10(Leu-- > Trp), alphaE7(His-- > Gln) and beta E11(Val-- > Trp); or alpha B10(Leu-- > Phe), alpha E7(His-- > Gln) and beta G8(Leu-- > Trp).