Spectral demonstration of semihemoglobin formation during CN-hemin incorporation into human apohemoglobins.
The incorporation of CN-hemin into three human adult apohemoglobin species (apohemoglobin, alpha-apohemoglobin, and apohemoglobin modified at its beta93 sulfhydryl with p-hydroxymercuribenzoate) has been monitored at micromolar concentrations in 0.05 M potassium phosphate buffer, pH 7.0, at 10 degrees C. In all cases, Soret spectral blue shifts accompanied CN-protohemoglobin but not CN-deuterohemoglobin formation. This finding in conjunction with isofocusing studies provided evidence of a CN-protosemi-alpha-hemoglobin intermediate, the formation of which appeared to be a direct consequence of CN-protohemin-alpha heme pocket interactions. The kinetics of full reconstitution of CN-protohemoglobin and CN-deuterohemoglobin revealed four distinct phases that apparently correlated with heme insertion (Phase I), local structural rearrangement (Phase II), global conformational response (Phase III), and irreversible histidine iron bond formation (Phase IV). These phases exhibited rates of 7.8-22 x 10(7) M(-1) s(-1), 0.19-0.23 s(-1), 0.085-0.12 s(-1), and 0.008-0.012 s(-1), respectively. Partial (50%) reconstitution with CN-protohemin, in contrast, revealed only three kinetic phases (with Phase III missing) of heme incorporation into native and p-hydroxymercuribenzoate-modified apohemoglobin. Furthermore, the absence of 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, the consequence of which may be that intermolecular heme transfer is encouraged under conditions of heme deficiency in vivo.
Vasudevan
G
G
Department of Chemistry, College of Arts and Sciences, University of Massachusetts, Lowell 01854, USA.
McDonald
M J
MJ
D000328:Adult; D001059:Apoproteins / Q000737:chemistry*; D003486:Cyanides / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D006427:Hemin / Q000031:analogs & derivatives* / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D007526:Isoelectric Point; D013057:Spectrum Analysis
D001059:Apoproteins; D003486:Cyanides; D006420:Hemeproteins; D006454:Hemoglobins; C023478:apohemoglobin; D006427:Hemin