DE2306071A1 - METHOD OF OBTAINING ORGOTEIN FROM RED BLOOD CELLS - Google Patents

Description

Dr. Joachim Rasper

Patent attorney DIA 4

62 Wiesbaden ======

iftrsiodttr Hihe 22 Tel. 5ί 284!

DIAGNOSTIC DATA, TEG.
Mountain View, CaI., V.St.A.

Process for obtaining orgotein from red blood cells

The invention relates to a new method for obtaining a Orgotein-rich protein concentrate from red blood cells.

Orgotein is a family of relatives Protein compounds that are a characteristic combination of physical, have chemical and pharmacological properties. Each of these related connections, hereinafter referred to as "fellow species" can be referred to physically as the isolated, largely pure form of a globular, soluble in buffer solution and water Define a protein that has a very compact, natural conformation and that is heat-labile, but against warming for several minutes at 65 C in water or a buffer solution, which is a salt of a divalent metal with an ionic radius of 0.60 contains up to 1.00 A-3 units, is stable, and which in gel electrophoresis at pH 8.45 in 0.01 m trisglycine buffer is a characteristic

309886/1059

stic multi-band pattern results. This protein is chemically through it characterized in that it contains all - (or all but one) essential amino acids, a small amount of carbohydrate, no lipids, 0.1 to 1.0 moles - ^ - metals in the form of about 3 to about 5 g-atoms / mole one or more chelated, divalent metals having an ionic radius of 0.60 to 1.00 Α units, and essentially none contains chelated monovalent metals or cell toxins in the molecule. Pharmacodynamically, each of these species can be considered a. non-toxic, immunologically well-tolerated, injectable protein be defined, among the pharmacological activities of which its anti-inflammatory effect is of particular interest, as well like its compact conformation, with its. Chelated content divalent metals. The immunological similarity the orgotein conspecific is so strong that yours in a rabbit or another suitable animal produced antibodies from one or several other Orgotein conspecifics are recognized as antigen and / or by one or more antibodies that other Orgotein · * Belonging to conspecifics, recognized as an antigen, are, as is the case with the Gel immunoelectrophoresis and / or gel immunodiffusion. Although some of the physical and chemical properties as well as the nature and strength of the pharmacodynamic effectiveness of conspecifics vary to conspecifics, all orgotein conspecifics have the same as above mentioned combination of properties.

Comes from the recently published literature show that to the Orgotein family of metalloproteins includes the proteins ,. "which have already been previously isolated in different purities _s and which one the names hepatocuprein, Mann & Keilin, Proc. Royal. Soc "for Biol. Sci., 126 303 (1939); cerebrocuprein, Porter & Ainsworth, J. Neuroehem., 1_, 260 (1957)" erythrocuprein, Liarkowitz et al "J. Biol. Chem., 234 " 40 ( 1959)> and cytocuprein; Carrico & Deutsch, J. Bio. Chem. 244 1 6087 (1969). Other publications on this subject are: Mohamed & Greenberg, J. Gen. Physiol. 3_J_, 433 (1954 ) 5 Porter- & Folch, Arch. Neurol. Psychiat. 7_7_, 8 (1957) ϊ Porter & Ainsworth. J. Neuroehem., 5_, 91 (1959) i Krimmel et al., J. Biöl. Chem., 23J; , 46; V / yman, Biochem. Biophys ", Acta 4_5_, 387 (1960) 1 Shields et al-,

3 0 9 8 8 6/1059

J. Clin. Inv., 4_ü_, 2007 (I96I) j Markowitz et al., 'Anal. Chem., 3/3, 1594 (1961); Porter et al., Arch. Biochem. Bioph. , £ 1, 319 (1964); Stansell 5 Deutsch, J. Biol. Chem., 24J3, 4299 (1965); ibid , 240 , 4306 (1965); Stansell & Deutech, Clin. Chem. Acta, J ^, 598 (1966); KcCord & Fridovich, J. Biol. Cheai., 24JL, 5753 (1969); Hartz & Deutsch, J. Biol. Chem., 244 , 4565 (1969); . McCord & Fridovich, J. Biol Chem, 244 6056 t (1969). Carrico Sc Deutsch, ibid , 245 »723 (197O). These metalloproteins are reported to have very high superoxide dismutase (sodase) activity (McCord & Fridovich, J. Biol. Chem., 244 , 6-49 (1969)} Keele, McCord and Fridovich, J. Biol. Chem. , 245 , 6176 (1970); ibid, 24JL, 2875 (1971)

In Be-PS 667,828 and CB-PS 1,160,151 a multi-stage process for isolating orgotein from animal tissue, such as, for example, beef liver, is described. US Pat. No. 5,687,927 describes an improved process in which some process steps are omitted and the yield is increased considerably. US Pat. No. 3,579,495 claims a multi-stage process for the isolation of orgotein from red blood cells, which comprises a solvent pre-cleaning and a heating stage, with a total yield, based on packed red cells ^ of about 0.01 . fo is achieved.

In some of the methods described in the above literature, a chromatographic purification step on diethylaminoethyl cellulose has been used as part of a multi-step process for the isolation of orgotein from red blood cells. In the simplest of these procedures, the red blood cells were freed of hemoglobin by solvent precipitation and then using K ₂ HPO. and acetone fractionated. A portion of the fractionated soluble proteins from hemolyzed red bovine blood cells was chromatographed at pH 7.4, 190 mg of orgotein with a superoxide dismutase activity of 3 × 3ΟΟ Sinneiten / mg, corresponding to a total yield of 0.006 <p and a yield of from 3 liters of packed cells Orgotein of 60 / O. Although this process is sufficient for the laboratory, it is too uneconomical for technical production. If you wanted to apply this process to a larger scale without changing it, you would need more than 7,500 liters of chloroform, 4,500 liters of ethanol to obtain 1 kg of orgotein,

309886/1059

7,000 kg of K-HPO and 5,000 l of acetone.

In the patent application P 22 59 4Ο5 · 4 a solvent-free process is described with which largely pure orgotein is obtained directly from hemolyzed red blood cells in a single process step by ion exchange chromatography. While that method avoids the difficulties associated with the use of a mixed solvent and ammonium sulfate as the precipitating agent, it has the disadvantage that large volumes of liquids have to be chromatographed, so that chromatographic columns of the appropriate size are required. Außerden are more than 99 8 j i ° of the proteins which are chromatographed, foreign proteins, living the

1 -

the duration of the column and the speed of the chromatography.

The procedure requires dialysis of the entire hemolysate, in order to reduce its ionic strength to the necessary, low level. The dialyzed lysate is not stable, and so is the ion exchange column becomes clogged by precipitating proteins, if not the lysate during the giving up of the samples is clarified. The flow velocity in the column is relatively low, and the adsorptive capacity of the column for orgotein is decreased due to the adsorption of large amounts of proteinaceous impurities. Also is the selectivity of the adsorption subsequent elution for some Orgotein conspecifics, E.g. those from the horse-d, worse than those for others. In short: that The method described in the co-pending patent application represents represents a significant advance over older methods, but has some disadvantages of its own.

It has now been found that these disadvantages of the patent application P 22 59 405 * 4 described method can be overcome, his However, simplicity and economy are retained if the red blood cells are pretreated so that a solution is formed which Although it contains orgotein, it is largely free of hemoglobin and Is carboxylic acid anhydrase. Such a solution can be chromatographed using a much smaller chromatography column than is required when using Hemolyzed Red Blood Cells. That

3 0 9 8 8 6/1059

is especially true when the starting solution is concentrated prior to chromatography, which is not easy to do with hemolyzed red blood cells. In addition, since less than 1 -ρ of the ^D roteins that pass the chromatography column of the method of patent application P 22 59 4Ο5 · 4 must be chromatographed in the "method according to the present invention in order to isolate the same amount of orgotein, the in the column useful operating time of the resin used much longer, the flow rates much higher and the selectivity of the resin much, so that a less precise controlled elution satisfies better. the inventive method has compared to the said patent application the apparent advantage _r that the carbonic anhydrase and the hemoglobin, which makes up about 99 / ° of the red blood cell protein, can be removed simultaneously without the use of organic solvents.

The aim of the invention is therefore a method for isolating orgotein from red blood cells, which is simple and economical and a Orgotein concentrate freed from hemoglobin and carboxylic acid anhydrase supplies, from which pure orgotein can be obtained easily and economically on an industrial scale.

This goal is achieved according to the invention in that at least the Red blood cells - percentage of blood at a pH of 5 to 8, at which the hemoglobin precipitates, heated to a temperature of about 60 to 80 C, the heated mixture is cooled, the precipitated protein is separated from the aqueous solution of the heated mixture and the orgotein is isolated from the supernatant solution.

It is surprising that both hemoglobin and carboxylic acid anhydrase can be selectively and essentially completely precipitated while the orgotein remains in solution, in view of the large proportion of the starting proteins, namely at least 95; - ■> those precipitated will. In addition, hemoglobin is a relatively heat-resistant protein. It is also used outside the scope of the invention pH range not or only partially made insoluble.

309886/1059

According to a preferred embodiment, the invention comprises Procedure the following stages:

(1) Dilute the whole blood with up to about 3 »e.g. * 0.5 Ms. 1.5 volumes of water; ·

(2) Warming the whole blood to about 65 to 75 G for the duration of about 1 "to 8 hours;

(3) Cool the heated mixture, preferably to at least 40 G and especially at tree temperature or below;

(4) Separation of the precipitated proteins, preferably by centrifugation, and

(5) Separating the crude orgotein from the supernatant solution, preferably by means of ion exchange chromatography.

According to other embodiments of the invention, one or several of the following variations can be introduced into the process: '

(a) "In step (1) serum-free blood cells, preferably those in 1 to 3 volumes of water are suspended as starting material for. the heating level used;

(b) The heating is at a lower temperature for a longer period Duration, e.g. at 60 C for 3 or more hours, or at one higher temperature for shorter duration, e.g. at 6C G for 30 minutes carried out up to an hour;

(c) The heating is carried out at a pH of about 7;

(d) Stage (3) is carried out after or during stage (4)!

(e) The orgotein is made from the excess in step (5-) by precipitation insulated with acetone or other water-miscible organic solvents; *. -

(f) The orgotein is obtained from the supernatant in step (5) by ultrafiltration or isolated by freeze drying;

3 0 9888 / 10S9

(g) Some of the water is passed through before the isolation of the orgotein Distillation under vacuum, e.g. at 20 to 60 G removed.

The heating stage can be carried out on the whole blood, and this is preferred because the mechanical process steps of the Removal of serum and ashing of packed red cells eliminated will. The warming level can also be performed on serum-free red blood cells be performed. Purer orgotein is obtained, but this benefit is offset by the cost associated with the There is no need to separate and wash the red blood cells before heating. It can also take the extra protein with little contamination Effort can be separated by means of a proteolytic enzyme, as described below. Therefore it is preferred from the whole Ran out of blood.

Although the warming level can be applied to undiluted blood or serum-free, packed red blood cells, but problems with mixing and heat exchange arise. That's why all blood and especially the serum-free red blood cells are preferably mixed with water, with up to 3, preferably about 1 'to 2 volumes before heating. The water

if desired, a buffering agent and / or a divalent metal ion, such as Cu and / or Zn, the supplying material contained in relatively low concentrations, e.g. below 0.05 m. When using whole Blood can contain trisodium citrate or another coagulation inhibitor may be added to prevent coagulation prior to hemolysis of the hemoglobin.

The heating stage is preferably near the iso! Electric Point of the hemoglobin of the red blood cells, namely at about pH 7 » carried out. The isoelectric pH of various hemoglobins is in the range of 6.5 to 7 »5, usually between 6.7 and 7» 4 · When it is low pH value, e.g. below 5 », the hemoglobin is denatured, but only partially precipitated. Hemoglobin of various origins, e.g. from Cattle or horses, will precipitate at all pH values within the range of 5 to 8. Other types of hemoglobin only fall near theirs isoelectric point.

309886 / 1O5S

The pH ¥ ert of the red blood cells can be adjusted with any suitable acid or Base can be adjusted. - · '.

The heating step is carried out at about 60 to 80 ° C., preferably about 70 to 75 θ ' , until all or almost all of the hemoglobin has precipitated. The exact heating temperature and duration is not critical if only the hemoglobin is largely precipitated. Higher. Temperatures and longer times are at the expense of the total Orgotein yield. Lower temperatures and shorter times deliver a

^y ο

less pure orgotein, around 1 to 2 hours at around 75 C is sufficient. Heating for 1 hour or more at 60 to 75 ^C, the final yield of pure orgotein not influenced in any significant way, but an hour is 1/4 to 3/4 of the destroyed Orgoteins by heating at 80 C for the duration. The heating step can be carried out batchwise or continuously using long heating and cooling zones.

Heat-labile proteins, including enzymes, are made from red blood cells by heat denaturation together with the hemoglobin. Carboxylic acid anhydrase is a well-known form of heat-labile substances Proteins. The red blood cells are heated until the carboxylic acid anhydrase is inactivated. In terms of the heat sensitivity of the Carboxylic acid anhydrase is published in "The Enzymes", Volume 5, (19 ^ 1) »ITew York, referenced. ·

When the hemoglobin is precipitated, so are the carboxylic acid anhydrases and all or almost all other heat-labile enzymes and non-enzymatic

The proteins in the supernatant solution have precipitated because the hemoglobin is more stable. At 60 ° C, heating for more than 20 minutes is usually required to remove the carboxylic acid anhydrase and the others To inactivate heat-labile proteins., and at 65 C 10 to 15 are sufficient Minutes. .

The completeness of the hemoglobin precipitation can easily be seen from the color of the protruding part can be recognized, which should be at least pale pink.

Occasionally it can happen? that not when using older blood all hemoglobin of the red blood cells fails when the

3098.86 / tO69

is carried out with an acidic ρΗ-ΐ / ert. In such a Before or after removing the hemoglobin precipitated by the warming, find the best color by adjusting the pH to 7 or above, preferably to 7 to 7.5, precipitated before the Orgotein is precipitated, e.g. before or after the separation of the precipitated hemoglobin, for example as part of the pre-precipitation of Foreign proteins using acetone. The precipitated red color can only come on removed in the same way as the hemoglobin precipitated in the heating step.

After heating, the heated red blood cells will be on under about 50 C, preferably cooled to room temperature or below, e.g. by passing them through a heat exchanger.

The hemoglobin and other proteins precipitated in the heating stage v / ground then removed, preferably by centrifugation, and v / ground discarded. Settling or filtration, preferably vacuum filtration with filter aids such as diatomaceous earth, can also be used will.

After removing the precipitated hemoglobin, the orgotein is made out isolated from the supernatant, e.g. by adding acetone to the cooled solution, by lyophilization, by ultrafiltration or by Adsorption on an ion exchange resin column and subsequent elution.

The preferred method to separate the orgotein from the supernatant is ion exchange chromatography, for example using the technique described in US Pat. No. 3,579,495 is.

A particularly preferred method that is very easy on the extraction of Orgotein can be turned off in technical quantities, consists in replacing the supernatant obtained on heating with a diethylaminoethyl cellulose to direct. The supernatant, which has an ionic strength of less than 0.01 m, is a diethylaminoethyl cellulose or another weakly basic ion exchange = column passed,

309886/1059

- .10 -

wherein orgotein is adsorbed on the ion exchange resin, whereupon the column is washed with water or buffer solution having an ionic strength of less than about 0.01 m and the orgotein is eluted at a higher ionic strength, eg about 0.02 to about 0.03 m will. Depending on the conditions used, can raw, are eluted ie 50 Jo pure until high purity Orgotein from the column. ·.

Alternatively, the supernatant can be obtained by molecular sieve chromatography cleaned, e.g. by passing it through a column of 3pichlorohydrin cross-linked Dext'ran resin (Sephadex G-75 Superfine) will. In the case of a model system, the head of the column can be interchangeable so that when the top of the column has adsorbed orgotein to the point of exhaustion, this head is removed and with fresh resin can be replaced, leaving the rest of the column intact. ·

The supernatant can be added before the orgotein is separated off a proteolytic enzyme are prepurified, surprisingly Orgotein is compared to digestion by proteolytic enzymes practically inert. Therefore the orgotein in the protruding part can thereby cleaned so that the foreign proteins are selectively broken down, preferably using Strep. griseus proteinase, pancreatin, subtilisin, Papain or bromelain, in a weight ratio of enzyme to proteins in that. Projecting from about 1: 2 to 1: 1000, depending on the one selected Enzyme and the ratio of foreign proteins to orgotein in the mixture. His or largely pure orgotein can be obtained from the open-minded Mixtures can be isolated in a conventional manner, for example by ultrafiltration. The protein fragments from the enzymatic Digestion and excess ions can be conveniently removed from the digested Mixture can be removed by dialysis before separating the orgotein.

If the orgotein is to be separated from the supernatant in one step, it can be precipitated therefrom by means of acetone, about 1 to 1.5 volumes of acetone, calculated on the volume of the liquid containing the orgotein, are sufficient to precipitate all of the orgotein Orgotein of a higher unit, for example of 50 ^c / o and more, can be obtained in individual cases by the process according to the invention by, a

309886 / 1-059

Part of the dissolved proteins is pre-precipitated with acetone. V / enn Using less than an equal volume of acetone, such as 1/2 or 5/4 the volume, will result in little or no orgotein precipitated with the foreign proteins. After removing the precipitated foreign proteins, for example in the same way as above for the removal of the precipitated hemoglobin is described, the orgotein is mixed with the remaining amount of acetone, e.g. 3/4 to 1.5 volume, calculated on aqueous solution, precipitated. However, this solvent pre-precipitation brings about very little proteinaceous contamination Failure when the heating stage is at 75 to 80 C for 1 to 2 hours carried out at pH 7.0 because the precipitation is in the heating stage is so perfect.

Pre-precipitation of foreign proteins and orgotein is preferred Carried out in the cold, e.g. at 0 to 5 ° C, but any temperature, e.g. ambient temperature up to the boiling point of acetone, can be used will.

Acetone is used economically in the process according to the invention Considerations are used, but other, more expensive, water-miscible solvents can be used to precipitate the orgotein such as methyl ethyl ketone and tetrahydrofuran, which are thus equivalent to acetone according to the invention.

The precipitated orgotein should be removed from residual solvent and moisture as soon as possible, e.g. by freeze-drying and lyophilization, or by redissolving in an aqueous vehicle for the purpose of further purification in order to avoid loss of orgotein.

It is true that an orgotein-rich precipitate that is hemoglobin-free and carboxylic acid anhydrase, also obtained by the acetone precipitation technique be, however, the hemoglobin-free supernatant solution is extremely well suited for a chromatographic cleaning on a Molecular sieve or weakly basic ion exchanger, e.g. according to the method described in TJS-PS 3,579,495 or P 22 59 405-4. In those cases in which it seems inconvenient to chromatograph large amounts of liquid, the hemoglobin-free supernatant can be used before chromatography by ultrafiltration or freeze drying

309886/1058

be concentrated. Also a batch adsorption of orgotein can be used on a weakly basic ion exchanger.

The invention is illustrated in more detail by the following examples;

Example 1 _ "'" ..

Washed erythrocytes from bovine are in double the volume of water, the 1 / ag Gu ⁺ and Zn ⁺⁺ to 5 / ug estimated -Orgotein in the red

> / ug

Contains blood cells, hemolyzed and for 1 ms 2 hours at pH of about 7, i.e. the isoelectric point of hemoglobin Heated between 70 and 75 ° C. Then it is diluted 2 to 3 times and cooled and the resulting slurry centrifuged at 4 ° C. That Supernatant, which contains largely pure orgotein, is dialyzed and lyophilized. -

Example 2

Washed and serum-free, frozen, packed red blood cells from Horse are suspended in twice the volume of water. With acetic acid is adjusted to pH 5.5. The suspension is heated to 65 ° C. for 15 minutes warmed up. It is then cooled to ambient temperature or below and the precipitated protein removed by centrifugation. The pH is set to 7 * 5 to precipitate the red color and centrifuged. 3/4 of its volume becomes ice-cold Acetone is added, the mixture is then centrifuged and the precipitate is discarded. A second time, 3/4 volume of cold acetone is exposed, and the resulting precipitate is centrifuged off about 50% orgotein. The raw orgotein is freeze-dried for storage and lyophilized.

Largely pure orgotein can be made from this raw orgotein by molecular sieve gel filtration using cross-linked dextran (Sephadex G-75) or by ion exchange resin chromatography using Diethylaminoethyl cellulose can be isolated »

Example 5

The procedure of Examples 1 or 2 is followed using red Bovine, human, chicken, sheep or pig blood cells repeated, whereby the heating in the vicinity of the respective isoelectric point

"305886/1059

of hemoglobin, ζ.3. at pH 6.8 to 7.4, preferably at about 7.0 is carried out.

Example 4

The procedure of Examples 1, 2 or 3 is repeated with the difference that that the red blood cells are heated in the absence of additional water or in the presence of an equal volume of water.

Example 5

The procedure of Example 2 is repeated except that 1.5 volumes of acetone are added at once. Orgotein of lower purity is obtained. By rubbing the precipitate with a minimal amount of water, the orgotein is selectively extracted from the precipitate. A solution is obtained by centrifugation of orgotein of about the same purity as that of the orgotein obtained in Example 2.

Example 6

The procedure of Examples 2 or 3 is repeated with the difference, that the first centrifugation is omitted.

Example 7

The procedure of Examples 2, 3 »4 or 6 is repeated with the difference, that none of the precipitates are separated until the first 3/4 volume of acetone has been added.

Example 8

The process of any one of Examples 1 to 7 is repeated with the difference that the heating stage is heated to 80 ^° C. for about 30 minutes.

Ex iel 9

The procedure of Example 2 is repeated except that the acetone precipitate becomes replaced by ultrafiltration to reduce the volume to be chromatographed.

30 9 8 86/1059

'- H-- 230607Ί

Example 10 '

The procedure of. Example 2 is repeated, but the acetone

Precipitation replaced by lyophilization. .

Example 11

The procedure of Example 1 is repeated with the difference that beef or human red blood cells, diluted 1: 2 with water, "at pH 7> 0 to be heated to 65 C for 18 hours.

Example 12

The procedure of Example 2 is repeated, whereby red blood cells from cattle, humans, chicken, sheep or pigs are used, but the acetone precipitation is replaced by an enzymatic digestion of the protein-containing impurities, for example by removing the supernatant solution from heating stage 1 be added to 3 mg pancreatin per ml of packed red blood cells and at about 37 C ^for f 15 to 48 hours incubated v / ill. Essentially all proteins, with the exception of orgotein, are broken down into small, dialyzable fragments. Largely pure orgotein can be isolated from the digested mixture by ultrafiltration.

Example 13

The procedure of Example 1 is repeated, except that the starting material is used whole blood used with Ztrium Citrate against coagulation is stabilized.

2 volumes of freshly collected bovine blood (85 ^ml; g total protein / ml; 20 µg orgotein / ml) are mixed with 1 volume 3 »perpetual aqueous trisodium citrate standard solution and 2 volumes containing 25 µg Cu / ml and 25 µg Zn / ml , whereby a whole blood solution is obtained, 4.Ie contains 1000 / µg Cu ⁺⁺ and Zn ⁺⁺ ions per 100 ml. This is heated to 75 ° C. and kept at this temperature for 2 hours. It is then cooled to about 10 ° C. by adding 150 ml of ice water. This dilution also makes a separate wash after centrifugation unnecessary. ^{The supernatant} (234 ffl l) is centrifuged. The filtrate contains a total of 1.6 mg protein and 12 to 17 mg orgotein / ml; this corresponds to at least 40 times the purification and a 7Oxigen yield of orgotein

30 98 88 / 1OS9

from the parent blood. This compares to a 90% yield of orgotein and at least a 50-fold purification achieved when using unwashed red blood cells (90 mg total protein / ml) instead originates from whole blood.

The supernatant becomes largely pure orgotein through lyophilization as described in Example 1, or by molecular sieve filtration or diethylaminoethyl cellulose chromatography, as described in Example 2, isolated.

To purify the orgotein prior to the above-described separation on, be 59 ^m l of the supernatant from the heat cleaning stage 12? / Contain up I Ug Orgotein / ml, with 50 to I50 mg pancreatin and 0.1 fo sodium azide (to prevent Bacterial growth), kept at 37 C for 15 hours and then cooled. The solution contains 1 mg total protein / ml and 12 11 g orgotein / ml.

Performing the methods described above can involve a lot of effort and complexity of the process to some extent against the desired Purity of the orgotein weighed out that from the after removal of the precipitated frozen particles obtained from the red blood cells is isolated. Acetone precipitation provides a purer orgotein, however, it has the disadvantage that an organic solvent is required. Column chromatography on a diethylaminoethyl cellulose ion exchange resin provides orgotein of good purity, but has the disadvantage that dialysis is required to maintain the ionic strength to bring it to a sufficiently low ¥ / ¥ value, and also the ITaehteil the chromatography of large amounts of liquid. The latter disadvantage can, however, be avoided by pre-concentrating the supernatant by freeze-drying or low-temperature evaporation of only part of the supernatant. A batch adsorption by slurry of the supernatant with an ion exchange resin is more convenient than column chromatography, but less effective. A lyophilization, unless preceded by dialysis, it does not in itself do any purification, but it is very convenient, the orgotein from the large volume to be separated from the protruding.

309886/1059

«16 -

Purified orgotein

The crude orgotein obtained by the process of any of the preceding examples can be isolated in pure form by molecular sieve gel chromatography of up to 2 to 5 g of crude orgotein on a column (3.2 × 86.5 cm; total layer volume: 711.5 ml; void volume: 265.5 Jal) of Sephadex G-75, a dextran resin crosslinked with epchlorhydrin, the eluate being collected in 5.4 ml fractions and their A- ^ ₁ - and A _{? Rn} -absorptions be measured. Starting around fraction 50, these absorptions rise steeply and reach a maximum value around fraction 68. Fractions 66 to 75 contain most of the entire orgotein in an ultra-pure state. With fraction 98, these absorptions fall to approximately up to fraction 116 on full. The only significant contaminants appear in tubes 45-60 and 118-150.

But it can also be an aqueous solution of up to 10 g of crude orgotein, as obtained according to Example 1 on a column (2.5 cm χ JO; layer volume 147 ^m l) τοπ diethylaminoethyl-cellulose (Fibrous DEAE-23) given are the rarde equilibrated with 0.005 ω ₉ phosphate buffer pH 6.0 "the column is developed at a flow rate of 240 ml / h with 0.005 m · Pliosphatpuffer. Orgotein is eluted with 4 liters of the same buffer, which has an increasing FaCl concentration from 0 to O ₉ O _- S m. The _; Cu and Zn contents and the -fi-pgc * ^u && Ap _fi0 absorptions of the eluate are measured as described, for example, in P .22 59 405 · 4 »There are three larger peaks, each with a fine structure, namely starting at 575 ⁰ ^ and 1425 ml of the eluate. The orgotein (2nd peak) is contained in the part of the eluate in which the ratio of Äpg ₍ - / ip _RO ~ A'absorptions = 1.27 and the Cu and Zn contents are greatest * The 750 ml- up to 1050 ml (0.025 to 0.055 m) of the eluate contains essentially all of the orgotein in a highly pure state »

The preceding examples can be repeated with equal success if the reagents used and process conditions are different from each other Examples are exchanged with each other.

3098-86 / TO-S9;

Claims (11)

Claims Process for obtaining a protein concentrate rich in orgotein, which is essentially free of hemoglobin and carboxylic acid anhydrase, from red blood cells, characterized in that at least the red blood cell portion of whole blood at a pH from 5 to 8, at which hemoglobin precipitates, to a temperature heated from about 60 to 80 C, cooled the heated mixture, separated the precipitated protein from the heated mixture and the Orgotein is isolated from the supernatant. 2. The method according to claim 1, characterized in that red blood cells can be used by Hin.d. 3. The method according to claim 1, characterized in that red blood cells used by humans. 4. The method according to any one of claims 1 to 5> characterized in that that whole blood is used. 5. The method according to any one of claims 1 to 3 »characterized in that that serum-free red blood cells are used. 6. The method according to any one of claims 1 to 4, characterized in that the starting material is diluted with up to ^ ~ iaGh. & Xi of its volume of water before heating. 7. The method according to any one of claims 1 to 5, characterized in that that the orgotein from the supernatant by lyophilization or Ultracentrifugation is separated. 8. The method according to any one of claims 1 to 6, characterized in that that the red blood cells are heated at a pH of about 6.8 to 7.4. 309886/1059 - .18 - 9. Method according to one of Claims 1 to 7s, characterized in that that the heating stage is carried out at about 65 to 7.5 C. 10. The method according to claim 8, characterized in that for about 1 to 2 hours
is heated. up to 2 hours at a pH of about 6.8 to 7s4 to about 75 11. The method according to claim 1, characterized in that the orgotein from the supernatant by precipitation with acetone at about 0 to 5 ° C is isolated.