STABILITY OF THE HEME-GLOBIN LINKAGE IN ALPHA-BETA DIMERS AND ISOLATED CHAINS OF HUMAN HEMOGLOBIN - A STUDY OF THE HEME TRANSFER-REACTION FROM THE IMMOBILIZED PROTEINS TO ALBUMIN

The stability of the heme-globin linkage in alpha beta dimers and in t he isolated chains of human hemoglobin has been probed by studying the transfer of heme from the proteins immobilized onto CNBr-activated Se pharose 4B to human albumin. The kinetic and equilibrium features of t he reaction have been measured spectrophotometrically given the stabil ity of the heme donors and the ease with which heme donor and acceptor can be separated. Isolated alpha and beta chains transfer heme to alb umin at similar rates (1-6 x 10(-2) s(-1) at pH 9.0 and 20 degrees C) in the ferrous CO-bound and in the ferric state. In alpha beta dimers the heme-globin linkage is strengthened considerably, albeit to a diff erent extent in the ferrous CO-bound and ferric met-aquo derivatives. Only in the latter heme is lost at a measurable rate, 0.065 +/- 0.011 x 10(-2) s(-1) for alpha heme and 2.8 +/- 0.6 x 10(-2) s(-1) for beta heme at pH 9.0 and 20 degrees C, which is very close to the rate measu red with soluble met-aquo-hemoglobin at micromolar concentrations. The se results indicate that in human hemoglobin the heme-globin linkage i n the alpha chains is stabilized by interactions between unlike chains at the alpha(1) beta(2) interface, whereas heme binding to the beta c hains is stabilized by interactions at the alpha(1) beta(2) interface. These long range factors have to be taken into account in addition to the local factors at the heme pocket when evaluating the effect of po int mutation and chemical modification.