SPECTRAL DEMONSTRATION OF SEMIHEMOGLOBIN FORMATION DURING CN-HEMIN INCORPORATION INTO HUMAN APOHEMOGLOBINS

The incorporation of CN-hemin into three human adult apohemoglobin spe cies (apohemoglobin, alpha-apohemoglobin, and apohemoglobin modified a t its beta 93 sulfhydryl with p-hydroxymercuribenzoate) has been monit ored at micromolar concentrations in 0.05 M potassium phosphate buffer , pH 7.0, at 10 degrees C, In all cases, Soret spectral blue shifts ac companied CN-protohemoglobin but not CN-deuterohemoglobin formation. T his finding in conjunction with isofocusing studies provided evidence of a CN-protosemi-alpha-hemoglobin intermediate, the formation of whic h appeared to be a direct consequence of CN-protohemin-alpha heme pock et interactions, The kinetics of full reconstitution of CN-protohemogl obin and CN-deuterohemoglobin revealed four distinct phases that appar ently correlated with heme insertion (Phase I), local structural rearr angement (Phase II), global conformational response (Phase III), and i rreversible histidine iron bond formation (Phase IV). These phases exh ibited rates of 7.8-22 x 10(7) M(-1) s(-1), 0.19-0.23 s(-1), 0.085-0.1 2 s(-1) and 0.008-0.012 s(-1), respectively. Partial (50%) reconstitut ion with CN-protohemin, in contrast, revealed only three kinetic phase s (with Phase III missing) of heme incorporation into native and p-hyd roxymercuribenzoate-modified apohemoglobin, Furthermore, the absence o f Phase III slowed the rate of proximal bond formation, These findings support the premise that irreversible assembly of CN-protosemi-alpha- hemoglobin is deterred by the presence of a heme-free beta partner, th e consequence of which may be that intermolecular heme transfer is enc ouraged under conditions of heme deficiency in vivo.