Process for recovering enzymes from blood

• US 4,341,867 A
• Filed: 05/13/1980
• Issued: 07/27/1982
• Est. Priority Date: 04/21/1980
• Status: Expired due to Term

• Current Assignee: De Forenede Bryggerier A/S
• Original Assignee: De Forenede Bryggerier A/S
• Inventors: Johansen, Jack T.
• Primary Examiner(s): Shapiro, Lionel M.
• Application Number:
  US06/149,393
• Time in Patent Office: 805 Days
• Field of Search: 435/189, 435/192, 435/232, 435/259, 435/183, 435/814, 435/815, 435/816
• US Class Current: 435/189
• CPC Class Codes: C12N 9/00 Enzymes; Proenzymes; Compositions thereof preparations containing enzymes for cleaning teeth A61K8/66, A61Q11/00; medicinal preparations containing enzymes or proenzymes A61K38/43; enzyme containing detergent compositions C11D; enzymes with nucleic acid structure, e.g. ribozymes, C12N15/113; Processes for preparing, activating, inhibiting, separating or purifying enzymes preparation of malt C12C1/00
• CPC Class Codes: C12N 9/0065 acting on hydrogen peroxide as acceptor (1.11)
• CPC Class Codes: C12N 9/0089 acting on superoxide as acceptor (1.15)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: Y10S 435/816 by solubility

Abstract

Various enzymes, in particular Cu,Zn-superoxide dismutase (SOD), catalase and carbonic acid anhydrase, are recovered from blood by admixing wholly or partly isolated blood cells with ethanol or a homologous alcohol until a concentration of 10 to 70% by volume and allowing them to stand for hemolysis of the blood cells and denaturation of the hemoglobin, and then adding water up to the double volume or more, and removing the precipitate of cell residuals, hemoglobin and other denatured proteins from the suspension. Then the desired enzymes are isolated from the solution obtained.

In particular, SOD and catalase can both be isolated by chromatography of the solution at a pH of 4.7 to 5.5 on a cation exchange resin of the same polarity as SOD in the pH range used and elution of the resin with a buffer solution which has a pH in the range 4.7 to 7.5 and an ionic strength in the range 0.01 to 1.0 M, SOD being eluted at the lowest pH and/or the lowest ionic strength.

The process allows both the hemolysis of the blood cells and the precipitation of the hemoglobin to be carried out in one step while retaining the activity of the desired enzymes, so that they can be obtained in a high yield in a manner which lends itself to use on an industrial scale. Further, SOD and catalase may be isolated and purified in a single chromatography step.

Claims

1. A process for recovery one or more of the enzymes Cu,Zn-superoxide dismutase (SOD), catalase and carbonic acid anhydrase from blood by lysing the blood cells and isolating the desired enzymes from the solution obtained, characterized by admixing wholly or partly isolated blood cells with an alkanol having 2 to 4 carbon atoms until a concentration of 10 to 70% by volume and allowing them to stand for hemolysis of the blood cells and denaturation of the hemoglobin and then adding water in an amount so as at least approximately to double the volume of the mixture, removing the precipitate of cell residuals, hemoglobin and other denatured proteins from the suspension, and finally isolating the desired enzymes from the solution obtained.

2. A process according to claim 1, characteristized by isolating one or more of the enzymes Cu,Zn-superoxide dismutase (SOD), catalase and carbonic acid anhydrase from the solution.

3. A process according to claim 2, characterized by the fact that SOD and catalase are isolated from the solution by subjecting said solution at a pH of about 4.8 to chromatography on a cation exchange resin of the same polarity as SOD in the pH range used, eluting SOD and catalase from the resin with a buffer solution which has a pH in the range 4.7 to 7.5 and an ionic strength in the range 0.01 to 1.0 M, SOD being eluted at the lowest pH and/or lowest ionic strength.

4. A process according to claim 3, characterized by eluting SOD from the cation exchange resin with a buffer solution having a pH of 4.7 to 5.5 and an ionic strength of 0.02 to 0.2 M and eluting the catalase with a buffer solution having a pH of 6.5 to 7.5 and an ionic strength of 0.1 to 1.0 M.

5. A process according to claim 3 or 4, characterized by using as cation exchange resin carboxymethyl celluloses or cross-linked dextrans substituted with carboxymethyl groups or sulfopropyl groups or cross-linked agaroses substituted with carboxymethyl groups.

6. A process according to any one of claims 1, 2, 3 and 4 wherein said alkanol is ethanol.