CA2035810A1 - Enrichment of delta-bilipeptides solutions - Google Patents

Abstract

ABSTRACT

A method for removing a substantial quantity of contaminating proteins or peptides, such as albumin from a delta-bilirubin or bilipeptide solution is described, thus providing an enriched solution of delta-bilipeptide. The method involves the use of a chromatographic gel of solid material to which either albumin, an albumin-resembling peptide or delta-bilipeptide has been covalently attached, addition of the delta-bilipeptide-containing solution thereto and elution of the delta-bilipeptide therefrom as an enriched solution.

Description

The present invention relates to delta-bilirubin and compounds resembling delta-bilirubin. More particularly, the present invention relates to processes for enriching delta-bilirubin and the like in solution.

Bilirubins are breakdown products of heme, found naturally in human serum. Four main types of bilirubin are understood to exist, namely, unconjugated bilirubin (Bu), mono-sugar-conjugated bilirubin, di-sugar-conjugated bilirubin and delta-bilirubin.

Delta-bilirubin comprises bilirubin covalently linked to albumin via a peptide bond between a propionic acid side chain of the tetrapyrrole group of bilirubin, and the epsilon amino group of a lysine residue in albumin. This lysine residue is located between amino acid residues 97 and 224 in the N-terminal half of the albumin protein. Delta-bilirubin is thus often referred to as biliproteinl BP.

Delta-bilirubin has been tested for its ability to protect various mammalian cell types against oxy-radical damage.
The results indicate a biomedical utility for delta-bilirubin.
Further, research indicates that the compound has utility in retarding oxidative degradation of food products, drug formulations, cosmetics and perfumes.

The isolation of delta-bilirubin from sera is described by Lauff et al., in "Clinical Chemistry", 28: 629-637, 1982. This method involves a high pressure liquid S chromatographic separation of delta-bilirubin from the other bilirubin fractions existing in sera, specifically, unconjugated bilirubin, mono-conjugated bilirubin and di-conjugated bilirubin.

K~saka et al., "Japanese Journal of Clin. Lab.
Automation", 11:88 92, 1986, describe a modified method of extracting delta-bilirubin from icteric (i.e. jaundiced) sera.

These extraction methods provide a means for isolating delta-bilirubin from all other types of bilirubin; however, lS they do not provide a means for obtaining a relatively purified solution of delta-bilirubin. The isolated delta-bilirubin fraction is substantially free from other bilirubin compounds; however, unbound albumin protein from the serum sample remains present as a contaminant.
McDona~h et al. (J. Clin. Invest. 74:L763-770, 1984) describe the biosynthetic formation of a compound similar to delta-bilirubin by incubating synthesized conjugated bilirubin with albumin. Again, the product is obtained as a mixture with albumin.

It is desirable to reduce the amount of peptide or protein contamination of delta-bilirubin and similar compounds as much as possible, in order to provide a biochemical material of predictable, controlled constitution and properties and to minimize the risk of undesirable biochemical side effects on use thereof.
However, many different methods for separation of delta-bilirubin from albumin and other peptide compounds have been attempted, but without great success.

Accordingly, it is an object of the present invention to provide a novel and more efficient method for separating delta-bilirubin and similar compounds from free albumin and peptides present in the reaction solution in which the delta-bilirubin or similar compound has been produced, thus providing an enriched solution thereof.

The present invention provides a process whereby very substantial quantities of albumin and other peptide compounds can be removed from liquid mixtures thereof containing delta-bilirubin or similar compounds (collectively referred to herein as delta-bilipeptides as discussed below), to provide a solution of delta-bilipeptide sufficiently enriched in delta-bilipeptide so as to be biochemically useful and of substantially constant and predictable composition.

The method is based upon the discovery that albumin and similar proteins have chemical affinity for the bilirubin nucleus to a sufficient extent that they will selectively covalently bind delta-bilipeptide so as to extract it preferen~ially from a mixture of delta-bilipeptide with albumin and albumin-like peptides. Thus, the delta-bilipeptide can be attached to a chromatographic column gel to extract covalently the albumin peptide from the mixture whilst the delta-bilipeptide is merely held to the column material more weakly, perhaps on an ion S exchange, electrostatic attraction basis, or through other conceivable forces such as weak affinity based interactions.
Thus, the delta-bilipeptide can be preferentially eluted from the column to obtain an aqueous solution substantiall~ enriched in delta-bilipeptide and substantially free from albumin peptides.
Conversely, an albumin peptide can be attached to the column material to bond the delta-bilipeptide covalently whilst the albumin peptide is held only by electrostatic attraction, so that the albumin peptide can be preferentially eluted, and a solution enriched in delta-bilipeptide can be obtained by subsequent elution.

The discovery that albumin peptides and delta-bilipeptides can be selectively separated in this way is surprising, and not to be predicted from a consideration of the prior art or the chemical nature of the substances involved. The molecular size of the bilirubin nucleus is so small compared with that of human serum albumin (molecular weight 546 v 60,000) that one would not expect it to be sufficiently clearly located and identified within a mixture of the two for affinity chromatographic separation. This is especially so when, as in delta-bilirubin, the bilirubin nucleus is already bound to a molecule of albumin. One would expect it to be effectively hidden in the enfolded protein chains, and effectively protected from chemical bonding thereby.
Surprisingly, this is not the case. There appears to be some ?

unusual feature in the stereochemistry of delta-bilirubin and other delta-peptides which leaves the bilirubin nucleus available for covalent a~finity binding to another albumin molecule or albumin peptide.

The process of the present invention is thus useful in separating natural delta-bilirubin from serum, to remove therefrom substantially all of the serum proteins such as HSA
which would normally contaminate it. In addition, however, it is useful in separating synthetically produced delta-bilirubin and other delta-bilipeptides from albumin and other peptides which may be used as reagents in synthetic preparation of delta-bilipeptides and which may be present in delta-bilipeptide solutions as unreacted reagents or reaction by-products.
The term "delta-bilipeptide" as used herein means a compound which is either delta-bilirubin itself or which resembles delta-bilirubin and has the general conformational structure of delta-bilirubin, but in which the albumin portion is either truncated or is replaced with a shorter amino acid sequence of at least 6 amino acids but including the sequence -Lys-Glx-Arg- at the point of covalent attachment to a propionic acid group of the bilirubin nucleus, via the aforesaid -Lys- residue. In the above sequence, Lys represents lysine, Glx represents glutamine or glutamic acid and Arg represents arginine.

The term "albumin-like peptide" as used herein means a peptide having an amino acid sequence corresponding to that of human serum albumin, comprising at least about ~0 amino acid units.

Synthetic methods for preparing such bilipeptides are described and claimed in my co-pending U.S. patent application serial number , filed on even date herewith, and the disclosure of which is incorporated herein by reference.
The methods involve the use or production of peptides as reagents or reaction products. Accordingly, the process of the present invention is useful in connection therewith.

Moreover, a synthetic method for preparing delta-bilirubin itself is based on site-specific reaction of unconjugated bilirubin, pre-activated by reaction with Woodward's reagent K, with human serum albumin. The method of the present invention is useful in connection with extraction of the product from that process also.

A preferred embodiment of the invention is exemplified in the following drawing in which;

Figure 1 illustrates the chromatographic elution pattern resulting from the enrichment of a delta-bilirubin sample as descnbed in Example 3.
When albumin or a peptide having an albumin-like amino acid sequence is covalently linked to the column material, there is a marked preference for the binding of the delta-bilipeptides in the li~uid mixture applied to the column, by chemical affinity forming covalent bonds. The free propionic acid group on the bilirubin nucleus is believed to react with an amine group on the albumin or similar peptide linked to the column. The other proteinaceous materials in the solution are held to the column by weaker forces including but not limited to electrostatic attraction. Consequently, initial elution of the column, e.g. with water, produces a solution rich in albumin and peptide material and substantially free from any delta-bilipeptides.
This elution is continued until the eluent water contains no proteinaceous material, then the elution medium can be changed to an ionic hydrolysis medium, whereupon the delta-bilipeptide is eluted from the column. Suitable such ionic elutants include aqueous sodium phosphate or potassium phosphate buffered to a basic pH. Other suitable such buffers are described by Good, "Biochemistry", vol. 5, page 467, 1966. Very weak, basic buffered ionic eluents are preferred, e.g. 0.05 molar or less, in dissolved salt, to avoid complications of subsequent salt emoval from the product, but stronger buffers are also effective. The solut;on so obtained is substantially enriched in delta-bilipeptide and almost but not completely free from residual albumin or other contaminating proteins or peptides.

In the preferred method of operating according to the invention, an intermediate elution step is used, after the water elution to remove the albumin and albumin residues. This intermediate elution suitably uses methanol, ethanol proponal or mixtures therof as eluent, and serves to extract further proteinaceous material, especially denatured proteins, from the column, without disturbing the covalently bonded delta-bilipeptide. In this way, delta-bilipeptide contaminated with even smaller quantities of residual proteins is finally obtained.

When delta-bilipeptide e.g. delta-bilirubin is S covalently linked to the column material, the reverse situation occurs, namely, there is afflnity covalent binding of the albumin and/or other peptide constituents of the mixture to the column, utilizing the free acid groups of the bilirubin attached to the column. The delta-bilipeptide constituent of the solution is held merely by forces. Then, initial elution with water yields an enriched delta-bilirubin solution, and subsequent elution with alkaline buffered sodium phosphate solution will remove the proteinaceous material from the column subsequently. This however, is a less preferred procedure, since it is likely to lead to a solution containing larger quantities of contaminating albumin and peptide residues than the former situation, where substantially all such residues are removed prior to elution of the delta-bilipeptide from the column.

The process of the present invention provides delta-bilipeptides which are sufficiently free from albumin and peptide residues, no matter how the delta-bilipeptide has been previously obtained or synthesiæd, that it can be put to beneficial biochemical use. The residual albumin and peptides are present in such very small amounts that the delta-bilirubin is for practical purposes biochemically useful, for administration to patients as a cytoprotective agent, for use as a food or cosmetic antioxidant, etc. It is also sufficiently pure that the results of so using it are consistent, predictable and reproducible. Moreover, it is sut`ficiently pure to permit its ùse as a starting matenal for further chemical modification, e.g. enzymatic cleavage of delta-bilirubin to form a delta-bilipeptide.

The nature of the chromatographic gel material is not critical, provided that it is solid, inert, biochemically acceptable and capable of ready derivatization to bond the albumin peptide or delta-bilipeptide thereto. Any of the commercially available chromatographic gels commonly used in protein and peptide separations can be used. Commonly they are crosslinked polysaccharides. Methods of attaching peptides to them covalently are well known to those skilled in the art. Use of carbodiimide as a coupling agent is one suitable and preferred such method.
The present invention will now be described with reference to the following specific, non-limiting examples.

Synthesis of Delta-Bilirubin usin~ Woodward's Rea~ent The first step in synthesizing delta-bilirubin using Woodward's Reagent is the formation of the Bilirubin-Woodward's Reagent K Complex (BW). Based on the method of Kuenzle et al. (J. Biol. Chem. 251:801-807, 1976), 116.8 mg or 0.2 mmol of unconjugated bilirubin (Sigma) were combined with 151.8 mg (0.6 mmol) of Woodward's Reagent K (Sigma), 20 ml of acetonitrile (Aldrich) and 0 3 ml of tIiethylamine (Aldrich).
This mixture was stilTed fo- 45 minutes at 20C followed by an ,S 1l~
evaporation step conducted at 30C. To the evaporated mixture was added 5 ml of distilled water.

Bilirubin-reagent conjugates were formed which were isolated using a Sephadex G-25 column (2.5 X 7.5 cm).
The fractions were eluted with water at a flow rate of 50 ml/hr.

Human serum albumin (HSA) was reacted with the bilirubin-reagent conjugates (BW). Conjugate 2.8~mol was combined with 3.0~mol of HSA in 10 ml of degassed phosphate bu~fer solution. EDTA was added to a final concentration of 1 mM and the pH was subsequently adjusted to 8.5 with imidazole (11.0 mmol). The solution was stirred under nitrogen at room temperature in the dark for 8 hours.
The obtained BW-delta-bilirubin monomer product was purified by adding the reaction mixture to a Sephacryl S 100 column, followed by elution with phosphate buffer solution at a flow rate of 50 ml/hr. The delta-bilirubin product was dialysed exhaustively against water, washed and condensed with ultra-filtration.

Finally, Woodward's Reagent was removed from the BW-delta-bilirubin product. The product was subjected to a phenyl Sepharose CL~B column for the purpose of removing Woodward's Reagent K to form isolated delh-bilirubin. This was accomplished by dissolving the lyophilized BW-delh-bilirubin monomer in 2M ammonium sul~ate (40 mg/ml) and then adding it to the phenyl Sepharose CL-4B column saturated in 2M

ammonium sulfate. Distilled water was used to elute delta-bilirubin which was then dialysed and lyophilized.

Enrichment of Synthesized Delta-bilirubin Chemically synthesized delta-bilirubin produced as described in Example 1 may be further purified following synthesis by application to a column of CH-Sepharose chromatographic gel to which human serum albumin (fatty acid free) is covalently linked with carbodiimide. The CH-Sepharose gel is prepared as follows:

(i) 10-15 g of dry CH-Sepharose (Pharmacia) is swelled in 1 liter of 0.5 M NaCl overnight.

(ii) The swollen gel is filtered and washed in 3-4 litres of 0.3 M NaCl. 0 (iii) The pH of a solution containing 3 g of 3X
crystallized human serum albumin in 60 mL of double-distilled water is adjusted to 4.5. This solution is added to the gel.
(iv) A solution of ethyl-3, 3-dimethyl aminopropyl carbodiimide HCl is prepared by adding 23 g of the solid to 60 mL doubled distilled water. The pH is adjusted to 4.5 with 0.5 M NaOH.

;` t ~!

(v) The carbodiimide solution is added dropwise to the gel/HSA solution over 20 minutes maintaining a pH of 4.5 for the 20 minute addition period as well as the subsequent hour.

(vi) The mixture is stirred for 24 hours at room temperature and then the gel is collected on a sintered glass funnel.

(vii) The gel is washed through 3 cycles, each cycle comprising 102 washes. The first wash is with 0.5 Iitres of 0.1 M
sodium acetate buffer, pH 4, plus 1 M NaC 1. The second wash is with 0.5 liter of 0.1 M sodium borate buffer, pH
8.0, plus 1 M NaCl.

15(viii) The gel is further washed with 4 litres of double-distilled water to remove any buffer salts and then the gel is suspended in 120mL of double-distilled water and stored at 04C until use.

Free, contaminating albumin was eluted with double-distilled water. Denatured serum proteins were eluted with 95% ethanol. Delta-bilirubin was eluted using 0.05 M to 0.1 M sodium phosphate buffer, pH 7.5 ~/-0.05.

~

Enrichment of Extracted Naturally Occurrin~ Delta-Bilirubin Delta-bilirubin isolated from pooled icteric sera according to Lauff et al. (Clin. Chem. 28, 1982, p. 636) was enriched using the column described in Example 2.

The delta-bilirubin samaple was added to the column and eluted as shown in Figure 1. Initially, material was eluted with double-distilled water and it was found to contain albumin for the most part. This was confirrned by immuno-electrophoresis and by the use of anti-HSA antibody. Once the absorbance of the eluent at 430 nm decreased to zero, the column was washed with 2-3 column volumes of 95% ethanol (B).
Turbid material containing some denatured serum proteins was washed off the column with these ethanol elutions. When the absorbance dwindled to zero, the column was eluted with a 0.05 M (0.1 mM) sodium phosphate buffer, pH 7.0-8.0 (C). The ratio of absorbance at 280 nm to 430 nm over the elution of this peak was 0.8: 0.9, and characterized the molar ratio of bilirubin:
albumin. The ratio of 0.8: 0.9 is substantially close to a ratio of 1:1. This is in contrast to the 0.05: 0.2 ratio obtained for serum-isolated delta-bilirubin without enrichment.

Enzymatic Clea~a~e to Produce Albumin Peptides Peptide fragments of albumin containing the t~ipeptide sequence Lys-Glx-Arg (where Glx is either glutamine or glutamic acid) are obtained by the cyanogen bromide cleavage of human serum albumin in 70% formic acid under nitrogen.
Fragments containing residues 124-297 (from the N-terminus of albumin) are isolated by chromatographic methods.
s The isolated fragment is subjected to limited proteolysis. Such limited proteolysis comprises incubation with pepsin at a pH of between 2.0 and 3.0 for 1 hour at 37C. The peptides are resolved on a Waters CN-propyl column. Further proteolysis is effected with trypsin at pH 8 for 30 minutes. The fragments are re-chromatographed on the Waters CN-propyl column.

EX~MPLE S
Synthesis of Bilipeptides usin~ Woodward's Rea~ent The suitable peptide fragments obtained according to Example 4, which include the tripeptide sequence Lys-Glx-Arg, are reacted with the bilirubin-reagent conjugates prepared as described in Example 1. Thus 2.8~mol of conjugate is combined with 3.0~mol of peptide in 10 ml of degassed phosphate buffer solution. EDTA is added to a final concentration of 1 mM and the pH was subsequently adjusted to 8.5 with imidazole (11.0 mmol). The solution is stirred under nitrogen at room temperature in the dark for 8 hours.

The bilipeptide is purified by adding the reaction mixture to a Sephacryl S 100 column, followed by elution with phosphate buffer solution at a flow rate of 50 ml/hr. The ? ~

bilipeptide is dialysed exhaustively against water, washed and condensed with ultra-filtration.

Enrichment of Bilipeptides The bilipeptides synthesized as described inExample 5 above may also be enriched using the column described in Example 2.
Free, contaminating albumin is eluted from the column with double-distilled water. Denatured serum proteins are eluted with 95% ethanol. Bilipeptides are eluted using 0.05 M to 0.1 M sodium phosphate buffer, pH 7.5 +/-0.05.

Claims (10)

1. A process of treating an aqueous mixture containing, as a first component, a delta-bilipeptide, and, as a second component, an albumin peptide, to obtain an aqueous mixture enriched in one or other of said components, which comprises:

applying the aqueous mixture to a solid, inert, biochemically acceptable chromatographic gel which has covalently bonded thereto either an albumin peptide or a delta-bilipeptide, so that one of said first and second components of the aqueous mixture covalently binds to the gel whilst the other component remains more weakly associated therewith;

and eluting said other component from the gel with water to obtain an aqueous solution enriched in said other component.

2. The process of claim 1 including the subsequent step of eluting the gel with a buffered ionic aqueous solution to recover there from an aqueous solution enriched in said one component.

3. The process of claim 2 wherein an albumin peptide is covalently bonded to the gel, so that delta-bilipeptide from the aqueous mixture is selectively covalently bonded to the gel and is recovered in the subsequent elution step with buffered ionic aqueous solution.

4. The process of claim 3 wherein the buffered ionic aqueous solution is buffered to pH about 7-8.

5. The process of claim 4 wherein the aqueous solution has a molarity of 0.05 molar or less, with respect to dissolved ions.

6. The process of claim 4 wherein said buffered solution is sodium phosphate solution.

7. The process of claim 3 wherein the initial aqueous mixture comprises a mixture of naturally occurring delta-bilirubin and HSA.

8. The process of claim 3 wherein the initial aqueous mixture comprises a reaction mixture from the process of coupling HSA to bilirubin.

9. The process of claim 3 wherein the initial aqueous mixture comprises a reaction mixture from the coupling of albumin peptides produced by enzymatic cleavage, to bilirubin activated with Woodward's reagent K.

10. The process of claim 1 wherein a delta-bilipeptide is covalently bonded to the chromatographic gel, so that albumin peptides from the aqueous mixture are covalently bonded to the gel.