Production of alpha-alpha cross-linked hemoglobins in high yield

Abstract

A high yield method of production of a cross-linked hemoglobin derivative suitable for use as a blood substitute and plasma expander which is cross-linked between Lys 99 Alpha 1 and Lys 99 Alpha 2 in high percentage by adding the cross-linker in the presence of an added polyanion which blocks competing reactions at other sites of the protein.

What is claimed is: 
     
       1. A method of producing in commercially significant yields, a modified hemoglobin which is capable of functioning as a blood substitute and blood plasma expander, comprising: isolating unmodified hemoglobin or preparing a crude red cell lysate;   deoxygenating said hemoglobin;   .Iadd.selectively .Iaddend.cross-linking said deoxygenated hemoglobin intramolecularly between Lys 99 Alpha 1  and Lys 99 Alpha 2  in the presence of .[.an.]. .Iadd.from about a 1.5 molar up to about a 20 molar excess .Iaddend.added polyanion selected from the group consisting of 2,3-diphosphoglycerate, inositol hexaphosphate, inositol hexasulfate; and   purifying said alpha-alpha cross-linked hemoglobin.   
     
     
       2. The method of claim 1 wherein said polyanion is 2,3-diphosphoglycerate. 
     
     
       3. The method of claim 1 wherein said polyanion is inositol hexasulfate. 
     
     
       4. The method of claim 1 wherein said polyanion is inositol hexaphosphate. 
     
     
       5. The method of claim 1 wherein the reaction is conducted at a temperature of from about 0° C. to about 40° C. 
     
     
       6. The method of claim 5 wherein the reaction is conducted at a temperature of from about 35° C. to about 40° C. 
     
     
       7. The method of claim 6 wherein the reaction is conducted at a temperature of about 37° C. 
     
     
       8. The method of claim 1 wherein the cross-linking between Lys 99 Alpha 1  and Lys 99 Alpha 2  is with a cross-linking agent which is amino group-specific. 
     
     
       9. The method of claim 8 wherein said cross-linking agent is a phenyl ester cross-linking agent. 
     
     
       10. The method of claim 9 wherein said cross-linking agent is bis(3,5-dibromosalicyl)fumarate. 
     
     
       11. The method of claim 8 wherein the amount of cross-linking agent employed is up to a three fold molar excess of the amount of hemoglobin. 
     
     
       12. The method of claim 11 wherein the amount of cross-linking agent is from about 1.3 times to about 2.0 times the molar amount of said hemoglobin. .[.13. The method of claim 11 wherein the amount of the polyanion is from an amount equimolar to the amount of hemoglobin up to a 
     
     
        twenty fold excess of the equimolar amount..]. 14. The method of claim .[.13.]. .Iadd.1 .Iaddend.wherein the amount of the polyanion is from 
     
     
        about 5 to about 10 times the molar amount of said hemoglobin. .Iadd.15. A method of producing in commercially significant yields, a modified hemoglobin which is capable of functioning as a blood substitute and blood plasma expander, comprising: isolating unmodified hemoglobin or preparing a crude red cell lysate;   deoxygenating said hemoglobin;   selectively cross-linking said deoxygenated hemoglobin intramolecularly between Lys 99 Alpha 1  and Lys 99 Alpha 2  in the presence of from about a 1.5 molar up to about a 20 molar excess of added polyanion which binds electrostatically at the 2,3-diphosphoglycerate binding site located between the beta chains of said deoxyhemoglobin; and   purifying said alpha-alpha cross-linked hemoglobin. .Iaddend.