Isolation of superoxide dismutase

A method for isolating superoxide dismutase. The method involves contacting red blood cells containing proteinaceous impurities with a water-miscible organic solvent at a pH in the range of 5 to 8 and a temperature of from 15.degree. to 50.degree. C., removing the impurities and obtaining purified superoxide dismutase.

BACKGROHND OF THE INVENTION 
1. Field of the Invention 
In 1939 a copper-containing protein was isolated from erythrocytes and 
found to be one of a family of water-soluble metalloprotein congeners. 
These proteins have been assigned the non-proprietary name "orgotein" by 
the U.S. Adopted Name Council. It was later determined that these proteins 
possessed enzymatic activity which catalyzed the reaction 
EQU O.sub.2.sup.- +O.sub.2.sup.- +2H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2 
An alternate name, superoxide dismutase, was proposed for these proteins, 
because they dismuted superoxide radicals to peroxide and molecular 
oxygen. It is theorized that superoxide dismutase may perform a role in 
protecting cells from the toxic effects of physiologically produced 
superoxide radicals, and against the deleterious effects of ultraviolet 
radiation. 
2. Description of the Prior Art 
U.S. Pat. No. 3,579,495 describes a method for isolating orgotein from red 
blood cells. The method disclosed therein involves first removing 
hemoglobin from the lysed red blood cells by precipitating the hemoglobin 
with an organic solvent at a temperature of 5.degree. to -15.degree. C. 
and a pH of from 5 to 8. The organic solvent is a water-immiscible 
solvent. The patentees indicate that halogenated solvents, e.g., methylene 
chloride and chloroform are preferred, and that a small amount of a 
water-miscible solvent can be added to increase the contact between the 
hemoglobin and the precipitating solvent. After precipitation of the 
hemoglobin, the hemoglobin is removed. The hemoglobin-free supernatant is 
then heated at 60.degree.to 80.degree. C. in the presence of a divalent 
metal cation such as Mg.sub.++, Cu.sup.++, or Mn.sup.++, to remove 
proteinaceous impurities. The divalent metal cations are then removed and 
the isolated orgotein recovered from the supernatant. 
U.S. Pat. No. 3,813,289 also describes a method for isolating orgotein from 
red blood cells. The method involves heating hemoglobin-containing red 
blood cells at a temperature of from 60.degree. to 80.degree. C. and a pH 
of from 5 to 8, to precipitate the hemoglobin. The precipitated hemoglobin 
is removed and the isolated orgotein removed from the supernatant. The 
patentees indicate that the advantage of this claimed process over the 
process claimed in U.S. Pat. No. 3,579,495 is that hemoglobin, which 
constitutes 99 percent of the proteins in red blood cells, is removed 
without the use of an organic solvent. 
The method described in U.S. Pat. No. 3,579,495 and in U.S. Pat. No. 
3,813,289 present technical difficulties in removing the proteinaceous 
impurities. 
In the method described in U.S. Pat. No. 3,579,495, when the suspension is 
centrifuged, a three-phase system is produced. The upper phase is an 
aqueous phase containing orgotein; the middle phase contains the 
proteinaceous impurities as a gelatinous material; and the lower phase is 
an organic phase. 
In the method described in U.S. Pat. No. 3,813,289, when the suspension is 
centrifuged, a liquid phase containing the orgotein and an insoluble phase 
containing proteinaceous impurities are produced. 
In both processes, the impurities are difficult to compact and difficult to 
remove. 
Neither of the above references suggest or disclose that superoxide 
dismutase can be isolated from red blood cells by rapidly denaturing the 
hemoglobin and other proteinaceous impurities with a water-miscible 
organic compound wherein the proteinaceous impurities are compactible and 
easy to remove. 
SUMMARY OF THE INVENTION 
The present invention is directed to a method for isolating superoxide 
dismutase from red blood cells which contain proteinaceous impurities. The 
method involves contacting the red blood cells with a water-miscible 
organic solvent for a time sufficient to cause denaturing of the 
proteinaceous impurities. The pH is maintained in the range 5 to 8, and 
the temperature in the range 15.degree. and 50.degree. C. After the 
proteins are denatured, purified superoxide dismutase is recovered from 
the red blood cells. 
DETAILED DESCRIPTION OF THE INVENTION 
The red blood cells which are used as the starting material in the claimed 
method can be obtained from animals such as cattle, horses, goats, sheep, 
pigs, rabbits, chickens and humans. Bovine red blood cells are preferred, 
as bovine blood is readily available from slaughter houses. Fresh whole 
blood or whole blod stabilized with an anticoagulant to prevent clotting, 
can be used. The whole blood can be centrifuged to separate the red blood 
cells from the plasma. Such centrifugation may lyse the cells. Subsequent 
dilution with water and water-miscible solvent will achieve complete 
lysis. 
The whole blood can be used as collected, or can be diluted with water to 
facilitate ease of handling. For example, addition of 3 volumes of water 
provides a red blood cell suspension which is easy to process. 
Red blood cells contain only a small amount of superoxide dismutase. It is 
estimated that less than 0.1 percent (wt/wt basis) superoxide dismutase is 
present; the remaining 99.9 percent consists of unwanted proteinaceous 
components. Hemoglobin constitutes a major amount of the impurities. 
A water-miscible organic solvent such as methanol, ethanol, propanol, 
isopropanol, or acetone, is then added to the red blood cells. From about 
5 percent to 60 percent of solvent (v/v), based on the amount of diluted 
red blood cells present can be used. Because of its favorable cost and 
availability, solvents such as isopropanol are preferred. The pH is 
maintained in the range of 5 to 8. A preferred pH range is 5.5 to 6.5. 
Bovine red blood cells normally have a slightly basic pH; the pH can be 
adjusted to the range described above by addition of a mineral acid, e.g., 
HCl, H.sub.2 SO.sub.4, HNO.sub.3 ; HCL is preferred. 
The red blood cells in the water-miscible organic solvent are maintained at 
temperature of from 15.degree. to 50.degree. C., for a time sufficient to 
cause denaturation of proteinaceous impurities present. Denaturation of 
the proteinaceous impurities is a time-temperature dependent phenomenon, 
and is also dependent upon the amount of red blood cells being processed. 
It has been determined that the denaturation can be achieved by 
maintaining the temperature at 50.degree. C. for about 15 minutes to 1 
hour, or can be achieved in a longer period of time by maintaining the 
temperature between about 15.degree. C. to 35.degree. C., for a time 
period of up to 48 hours. 
Prior to denaturation, the red blood cell-water miscible organic solvent 
mixture has a deep red opaque appearance. As denaturation occurs, the 
mixture begins to separate into a solid and a liquid phase. The solid 
phase contains proteinaceous impurities; the liquid supernatant which 
contains superoxide dismutase in solution becomes a light gold color. 
It has been determined that preferred conditions for isolating superoxide 
dismutase by the claimed method involve using a pH of 5.5 to 6.5 and a 
temperature of about 50.degree. C. With the amount of red blood cells 
processed in Example 1, acceptable clarity was achieved in 15 minutes; 
maximum clarity of the supernatant was achieved in about 1 hour. 
The resulting supernatant-solid phase which is produced during denaturation 
can be separated by conventional liquid-solid separation techniques, e.g., 
filtration or centrifugation. The proteinaceous impurities are easily 
compactible and can be readily removed by these techniques. 
After separation, the superoxide dismutase is present in a small amount in 
a large volume of supernatant and it is necessary to concentrate the 
supernatant. Conventional techniques can be used, e.g., ultrafiltration, 
affinity chromatography, ion-exchange chromatography, or by precipitation 
techniques, for example by addition of ammonium sulfate or organic 
solvents such as acetone, or a combination of such techniques. A preferred 
method is ultrafiltration and precipitation. 
Subsequent to concentration of the superoxide dismutase, the superoxide 
dismutase can be further purified by conventional techniques such as 
affinity chromatography, ion-exchange chromatography and gel filtration. 
These techniques help to remove any residual amount of organic solvent 
associated with the superoxide dismutase. The superoxide dismutase 
recovered has a purity in the range from 80 to greater than 95 percent.

EXAMPLE I 
A 368 liter portion of fresh bovine red blood cells which had been 
separated from the plasma portion by centrifugation was collected at a 
slaughter house. The red blood cells were diluted with 1104 liters of 
water and mixed well. The pH of the red blood cells was about 7.1; 
approximately 31 liters of 1 N HCl were added to adjust the pH to 6.0. 
Total volume of the red blood cell mixture was 1503 liters. 
For each liter of diluted red blood cells, 33 ml of 99 percent pure 
isopropanol was added. A total of 502 liters was added. The red blood 
cell-isopropanol solution was heated to 50.degree. C. and held at that 
temperature for one hour, then cooled to 15.degree. C. The proteinaceous 
impurities settled to the bottom as a dark red precipitate, leaving a 
light gold colored supernatant. 
The precipitate was an easily compactible mass and was removed by 
filtration through cellulose filter media, using a plate and frame press. 
The volume of filtrate was 1202 liters and the precipitate, which was 
discarded, weighed 633 kg. 
The filtrate containing superoxide dismutase was concentrated by 
ultrafiltering the liquid using hollow fiber cartridges, commercially 
available from Rohm and Haas, Kansas City, Missouri, under the trade 
designation Romicon PM-10. 
Approximately 550 liters of concentrated filtrate was obtained. The 
filtrate was cooled to about 0 to 5.degree. C. and 1.5 volumes of 
-5.degree. acetone was added; total volume of acetone added was 825 
liters. Following addition of the acetone, a precipitate formed which was 
rich in superoxide dismutase. 
The acetone solution was mixed for about 3 hours and allowed to settle for 
48 hours. The solution containing the superoxide dismutase precipitate was 
centrifuged at a high speed, low flow (13,000 g, 1.5 to 1.0 
liters/minute). 
The precipitate collected was determined to weigh 228 grams (wet weight). 
The precipitate was dissolved in a buffer solution of 
tris(hydroxymethyl)aminomethane (pH 7.0) and centrifuged to remove 
insoluble impurities. The semi-purified dissolved superoxide dismutase was 
chromatographed on cross-linked dextran. A final yield of 9 grams of 
superoxide dismutase was obtained. 
The activity of the superoxide dismutase was determined by a method 
involving the aerobic reduction of nitro-blue tetrazolium by NADH in the 
presence of phenazine methosulfate. This reduction is inhibited by the 
superoxide dismutase enzyme. [See Biochemie, 57:657 (1975)]. One unit of 
superoxide dismutase is defined as that amount of enzyme which will cause 
a 50 percent inhibition in the rate of reduction of cytochrome C at 
25.degree. C. and a pH of 7.8[See I. J. Biol. Chem., 224:6049 (1969)]. 
Based on this method of assay, a purity of 87 percent was achieved. 
EXAMPLE II 
A 970 liter portion of fresh bovine red blood cells which has been 
separated from the plasma portion by centrifugation was collected at a 
slaughter house. The red blood cells were diluted with 2910 liters of 
water and mixed well. The pH of the red blood cells was about 7.0; 
approximately 47 liters of 1 N HCl were added to adjust the pH to 6.0. 
Total volume of the red blood cell mixture was 3927 liters. 
A total of 1318 liters of 99 percent pure isopropranol was added. The red 
blood cell-isopropanol solution was heated to 35.degree. C. for about 12 
hours, then cooled to 15.degree. C. The proteinaceous impurities formed a 
precipitate, leaving a light gold color supernatant, containing the 
superoxide dismutase. 
The precipitate was an easily compactible mass and was removed by 
filtration through cellulose filter media, using a plate and frame press. 
The precipitate was discarded and the supernatant was stored at -5.degree. 
C. for future use. 
One portion of the supernatant was concentrated to isolate superoxide 
dismutase by the procedure described in Example I. Based on the enzyme 
inhibition test described previously, the isolated superoxide dismutase 
was determined to have about 80 percent purity.