QUATERNARY STRUCTURE REGULATES HEMIN DISSOCIATION FROM HUMAN HEMOGLOBIN

Rate constants for hemin dissociation from the alpha and beta subunits of native and recombinant human hemoglobins were measured as a functi on of protein concentration at pH 7.0, 37 degrees C, using H64Y/V68F a pomyoglobin as a hemin acceptor reagent, Hemin dissociation rates were also measured for native isolated alpha and beta chains and for recom binant hemoglobin tetramers stabilized by alpha subunit fusion, The ra te constant for hemin dissociation from beta subunits in native hemogl obin increases from 1.5 h(-1) in tetramers at high protein concentrati on to 15 h(-1) in dimers at low concentrations, The rate of hemin diss ociation from alpha subunits in native hemoglobin is significantly sma ller (0.3-0.6 h(-1)) and shows little dependence on protein concentrat ion, Recombinant hemoglobins containing a fused di-alpha subunit remai n tetrameric under all concentrations and show rates of hemin loss sim ilar to those observed for wild-type and native hemoglobin at high pro tein concentration, Rates of hemin dissociation from monomeric alpha a nd beta chains are much greater, 12 and 40 h(-1), respectively, at pH 7, 37 degrees C, Aggregation of monomers to form alpha(1) beta(1) dime rs greatly stabilizes bound hemin in cu chains, decreasing its rate of hemin loss similar to 20-fold. In contrast, dimer formation has littl e stabilizing effect on hemin binding to beta subunits, A significant reduction in the rate of hemin loss from beta subunits does occur afte r formation of the alpha(1) beta(2) interface in tetrameric hemoglobin , These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic gro up to be removed by serum albumin and apohemopexin.