Modification of hemoglobin with site-directed bifunctional reagents.

Abstract

Reaction of deoxy-Hb with the periodate-oxidized derivatives of nicotinamide adenine dinucleotide (o-NAD), phosphoribosyl pyrophosphate (o-PRPP), adenosine triphosphate (o-ATP), glucose-1-phosphate (o-glc-1-P) and nicotinamide adenine dinucleotide phosphate (o-NADP) led to formation of cross-link adducts in varying yields as determined by SDS-polyacrylamide gel electrophoresis. Oxygen equilibrium studies were performed on Hb's cross-linked with o-NAD, o-PRPP and o-ATP. These derivatives were found to have increased oxygen affinity and were cross-linked between the beta chains. Inositol hexaphosphate (IHP) blocked modification by these reagents, suggesting that modification was occurring in the organic phosphate binding site. In addition, it was found that the bifunctional reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) also led to formation of Hb cross-linked between the beta chains, but resulted in a derivative with a dramatically decreased oxygen affinity, properties making it a potential candidate as an Hb-based cell-free blood substitute.