DE3410159A1 - Process for obtaining Cu2Zn2superoxide dismutase from red blood cells from vertebrates - Google Patents

Abstract

The invention relates to a simple, low-cost and rapid process for obtaining superoxide dismutase of extremely high purity from red blood cells from vertebrates, especially mammals, preferably large animals, wherein, in the first stage of the process, haemolysed red blood corpuscles are heated without any further addition of chemicals, and in the second stage of the process after removal of the precipitate by chromatography of the resulting solution the superoxide dismutase is isolated.

Description

Process for the production of Cu Zn superoxide dismutase from red blood

9 2 vertebrate cells.

The invention relates to a method for isolating Cu2Zn2 superoxide dismutase from red blood cells of vertebrates, especially mammals.

Superoxide dismutases are enzymes which disproportionate the radical superoxide anion ° 2 in the presence of protons according to the following equation (Journal of Biological Chemistry, Volume 244, pp. 6049-5055, 1969).

Superoxide and its secondary products can occur in the physical process have a potentially toxic effect by, for example, the synovial fluid or also attack the genetic material.

There are superoxide dismutases that are either iron or manganese or copper and contain zinc. The latter enzyme occurs in the cells of higher living things and is the most common. Superoxide dismutase containing copper and zinc (Cu2Zn2 superoxide dismutase) from bovine red blood cells has a molecular weight of 31 300 and consists of two identical sub-units, each with a copper and a zinc. The sequence of the amino acids and their spatial arrangement is known.

Cu2Zn2 superoxide dismutase is one of the most resistant enzymes to denaturation by heat (Hoppe-Seyler's Zeitschrift für Physiologische Chemie, Vol. 353, pp. 1059-1068, 1972).

Nor is it destroyed by protein-degrading enzymes.

Purified bovine blood superoxide dismutase comes in two charge isomers with isoelectric points of pH 5.2 and pH 4.9.

Cu2Zn2 superoxide dismutase (hereinafter referred to as superoxide dismutase for short) is currently found called) in two areas.

On the one hand, it is used in analytical enzyme laboratories as a test protein used and on the other hand it is used pharmacologically against diseases that are summarized under the umbrella term rheumatism. For example, it is used in arthritis discussed a mechanism by which O2 radicals are formed. These in turn lead to decompose the synovial fluid and thereby aggravate the condition. Superoxide dismutase can intercept the 02 radicals and thereby prevent the breakdown of the synovial fluid.

So far the remedy is "Orgotein" (purified superoxide dismutase ) has mainly been used for joint inflammation in horses.

Meanwhile, bovine superoxide dismutase is also available for use on humans under the trade name "Peroxinorm" on the market.

The main difficulty in isolating superoxide dismutase from Blood is the separation of hemoglobin. The first isolation of superoxide dismutase succeeded as early as 1938 through complex chromatography processes. In the fifties and sixties of our century, the enzyme was hemolyzed by treating red blood cells isolated with heavy metal salts (e.g. lead acetate).

In 1969 an isolation method was developed based on the Separation of the hemoglobin with organic chlorinated solvents is based (Journal of Biological Chemistry, supra). This method was later modified and improved (Biochimica et Biophysica Acta, Vol. 243, pp. 203-213, 1971). This isolation method gives very pure superoxide dismutase, but has the disadvantage that it is very expensive is (duration approx. 7 days), has a high consumption of chemicals and occupational health Considerable aspects because of the use of chlorinated organic compounds Concerns exist. The total yield of pure superoxide dismutase is only 20c: from the enzyme found in red blood cells.

Also in 1969 a purely aqueous isolation method was developed, which could be increased by modifications up to 50% overall yield ( FEBS Letters, Vol. 155, pp. 15-18, 1983). This insulation that is on the bond of superoxide dismutase based on weakly basic ion exchangers also provides a very pure enzyme. However, it is very complex and at best for the laboratory scale suitable.

Furthermore, an isolation method is known, which is a connection the earlier heavy metal method with heat treatment (JA-OS 53-31 206). Here, hemolyzed red blood cells become divalent in the presence of them Metal salts are heated and hemoglobin is precipitated as a result. This isolation method supplies only impure superoxide di-smutase of comparatively low activity.

Another known method (JA-OS 49-50 195) subjects hemolyzed red blood cells from heat treatment with subsequent proteolytic decomposition of the upper class. Here too, low yields of relatively impure enzyme are obtained. In addition, the use of proteolytic enzymes on an industrial scale is poor controllable.

Finally, an isolation method is known (DE-PS No. 31 24 228), which is an improvement on the latter two methods. In two Process stages are hemolyzed red blood cells in the presence of an inorganic Neutral salt and a transition metal salt heated and the precipitate formed removed. With this method, clear ones are obtained after two heat treatments Solutions without interfering hemoglobin, however, the process engineering is by the two-fold Heating quite expensive. The use of neutral salts requires an additional one Dialysis step before chromatography.

According to the information in this patent specification, a protein is obtained which can form a complex compound with superoxide dismutase and hemoglobin.

This so-called affinity protein is said to be used in the event of heat denaturation or freezing or aging of red blood cells to form complexes with superoxide dismutase and thereby inactivate them. This affinity protein is said to be a lipoprotein act that through the Addition of the monovalent neutral salts followed by Heat treatment can be separated.

More recent studies on which the invention is based allow this Theory seem doubtful. It is known that in the purification of superoxide dismutase an accompanying protein occurs that has properties similar to superoxide dismutase has, however, it shows hardly any lipophilic properties. It can neither at high nor at at low ionic strengths are separated by highly lipophilic adsorption gels. In addition, the well-known isolation method is contradicting itself. Namely, would Aging the above complex of superoxide dismutase and the affinity protein arise, the yield of free superoxide dismutase from aged red ones would be Blood cells lower than in unaged. This is not the case.

It was also found that the heat treatment of hemolyzed red blood cells for superoxide dismutase at high salt concentrations is much more unfavorable than with lower salt concentrations. High salt concentrations stabilize Haemogl obi n, while lower salt concentrations the Superoxi ddi stabilize smutase. When using lower salt concentrations there is also the advantage that the dialysis of the crude extract for the subsequent chromatography not applicable.

The purified superoxide dismutase obtained according to the above Process purified has an average specific enzyme activity of 2200 U / mg protein. Highly purified superoxide dismutase obtained from previous processes but has a specific activity of 3300 U / mg protein (Journal of Biological Chemistry, supra).

This means that the former enzyme is only two-thirds enriched and still contains 30C. Other protein contamination.

Superoxide dismutase is extremely weakly immunologically active. Enzymes different animal species including humans are similar in sequence the amino acid residues. Therefore, the purified enzyme can also be extracted from bovine blood easy to use on humans. Basic requirement for it is, however, that the superoxide dismutase is absolutely free of foreign proteins. When present even small traces of foreign proteins can cause severe allergic reactions occur in patients who have the primary clinical picture (e.g. arthritis) by far surpass.

The superoxide dismutase produced by the process described above has been purified is only enriched by up to two thirds of the purity. From the above There are therefore considerable concerns about clinical application.

Accordingly, it is an object of the invention to provide a method for isolation to make superoxide dismutase available, also on an industrial scale is easy, quick and cheap and also an absolute purity of the end product enables.

This task is with the characterizing features of the claim 1 solved and developed with those of the subclaims. The inventive method works in its first stage in principle without chemical or biochemical additives. Fresh, frozen or preserved blood can be used. One partial separation of blood vessels; a is recommended, but this is preferable complete removal.

Stabilization of the blood is advantageous, but not necessary with acid citrate, so that a pH of 6.5-7.0 is achieved.

The inventive method begins with the hemolysis of the red Blood cells, with two to four times the volume of distilled or Tap water is diluted with vigorous stirring. The solution is then 1-60 minutes, preferably 10-20 minutes, e.g. 15 minutes 0 with vigorous stirring to temperatures from 60-90 C, preferably from 70-800C, e.g. heated at 750C. It will be 93-97 of hemoglobin and most non-hemoglobin proteins except superoxide dismutation failed. It is consciously accepted that small amounts of hemoglobin are still present remain in the supernatant of the superoxide dismutase solution, so the solution is still weak is red. The remaining hemoglobin can easily be removed by the subsequent chromatography be separated.

After the solution has cooled, it is centrifuged or filtered or pressed through several layers of textile fabric. The clear supernatant is diluted with Acetic acid adjusted to pH 6.4 to 6.8 and on diethylaminoethyl ion exchangers, preferably DEAE cellulose (particle size 40-160 cm, ether-bridged diethylaminoethyl glucose and crosslinking the cellulose with epichlorohydrin; under the trade name of Pharmacia "DEAE-Sephacel") chromatographed.

Under these conditions the remaining hemoglobin becomes complete severed. The eluate obtained is lyophilized. This is what is described above Completely denatured accompanying protein of superoxide dismutase. Alternatively, it can Volume can also be reduced by ultrafiltration. The remaining dissolved Superoxide dismutase is then subjected to gel filtration in distilled water subject.

A salt-free, highly pure product is obtained in this way (gel filtration over epichlorohydrin crosslinked dextran; Pharmacia trade name "Sephadex" G-75 ").

The entire isolation process can be carried out without affecting the yield and purity in distilled or tap water and at temperatures of 0-400C, preferably be carried out from 0-200C, e.g. at 40C. The following are three Examples of isolation methods are given according to the above sample. Superoxide dismutase is the only copper protein in red blood cells (FEBS Letters, Volume 155, Pp. 15-18. 190X3) Thus, the copper content of the individual solutions can be directly related to the Content of superoxide dismutase can be correlated. One liter of red blood cells from Beef contain around 500 grams of copper. This corresponds to approx. 120 mg superoxide di smutase.

Purity criteria for the enzyme were UV spectroscopy and activity determinations with the cytochrome c reductase test (Journal of Biological Chemistry, 1963, supra) or the formazan test (FERS Letters, vol. 61, pp. 209-212, 1976).

Example 1 375 ml of bovine red blood cells were washed and frozen for three months. The copper content was 185 cig (- 45 mg superoxide dismutase ). After thawing, it was dialyzed against tap water for 12 hours, with 750 ml Diluted water and heated to 750C for 15 minutes.

The supernatant contained 125 pg copper (-31 mg superoxide dismutase ) corresponding to 68% yield. It was colored pale red. In ion exchange chromatography the red hemoglobin ran through the column on a diethylaminoethyl cellulose column through while the superoxide dismutase was completely bound. The latter was eluted with a linear NaCl gradient from 0-200 mM. It has been lyophilized and dissolved in 12 ml of tap water. The remaining precipitate on dissolution contained no copper. After a molecular sieve filtration with distilled water, lyophilization was carried out. The yield was 27 mg with a specific activity of 3,300 U / mg. The duration isolation was 4 days.

2. ~ Example 250 ml human red blood cells that last six weeks were preserved, were dialyzed against tap water for 12 hours and then with diluted two volumes of water. The copper content was 120 9 (-30 mg superoxide dismutase). Heating at 74-760C for 15 minutes and separating the precipitate gave one Supernatant of 550 ml with a copper content of 78 9 (- 20 mg superoxide dismutase).

Ion exchange and gel filtration chromatography of the supernatant was carried out as in the 1st example. The superoxide dismutase yield was 17 mg with a specific activity of 3100 U / mg.

Example 3 320 ml of fresh red blood cells were once with 0.7 % NaCl and then diluted with 1,000 ml of water. The copper content was 165 pg (- 40 mg superoxide dismutase). The heating conditions were as in the example 1. In the supernatant of the heated hemolysate there were 110 po copper (- 27 mg superoxide dismutase. The further purification of the superoxide dismutase was carried out as in Examples 1 and 2. The yield of superoxide dismutase was 23 mg with a specific activity of 3,200 U / mg.

Claims (2)

Claims 1) Process for the isolation of Cu2Zn2 superoxide dismutase from red blood cells of vertebrates, especially mammals, preferably of large animals, characterized in that one hemolyzed in the first process stage red blood cells are heated and formed without any further addition of chemicals Separate precipitate from the remaining aqueous solution. 2) Method according to claim 1, characterized in that from that obtained after the precipitate has been separated off in the first process stage Solution isolated by chromatography the Cu2Zn2 superoxide dismutase.