Modification of hemoglobin a with dimethyl adipimidate: Contribution of individual reacted subunits to changes in oxygen affinity

Abstract

The effect of dimethyl adipimidate, a bifunctional imidoester, on the oxygen affinity of hemoglobin A has been studied. Treatment of human oxyhemoglobin with 5 mM dimethyl adipimidate at pH 8.5, room temperature is accompanied by an increase in oxygen affinity in the presence and absence of 2,3-diphosphoglyceric acid. Circular dichroism measurements in the ultraviolet region indicate that dimethyl adipimidate-treated hemoglobin exhibits a reduced conformational change upon deoxygenation. In order to study the contribution of reacted individual subunits, α and β subunits of dimethyl adipimidate-treated and untreated hemoglobin have been separated and reconstituted to form hybrid tetramers containing either the α-treated ( α t β c ) or the β-treated subunits ( α c β t). Electrophoresis on sodium dodecyl sulfate polyacrylamide gels of isolated α and β globin subunits as well as hybrid tetramers from dimethyl adipimidate-treated hemoglobin reveals that 20% of the globin subunits are cross-linked. In the absence of 2,3-diphosphoglyceric acid, modification of α subunits increases the oxygen affinity and reduces the conformational change of the tetramer upon deoxygenation whereas modification of β subunits has no effect. However, treatment of β subunits decreases the effect of 2,3-diphosphoglyceric acid on the oxygen affinity of the hybrids and reduces the 2,3-diphosphoglyceric acid-induced spectral changes in oxyhemoglobin. Therefore the interaction of dimethyl adipimidate with both the α and β subunits contributes to regulating the oxygen affinity of human hemoglobin.