Circular Dichroism Studies of Cyanate-Induced Conformational Changes in Hemoglobins A and S.

Circular dichroism and difference spectroscopy have been used to study dilute aqueous solutions of oxygenated, deoxygenated, and carbamoylated deoxygenated hemoglobins A and S (HbA and HbS, respectively). The spectra of HbA and HbS, in comparable state of oxygenation or carbamoylation, are identical, strongly implying identical conformations about the heme groups of the respective proteins. The spectra of the oxygenated forms change little upon addition of KCNO, which is known to carbamoylate the NH2 terminals of the individual chains (Cerami and Manning, 1971). The spectra of the deoxygenated forms, on the other hand, are markedly altered. The decreased magnitude of the 430-nm extremum with increased cyanate concentration can be used to calculate an addition curve which becomes asymptotic at a cyanate:heme molar ratio of approximately 10(3). This conformational change occurs in the absence of O2 and has been predicted (Njikam et al.,1973); it can also be demonstrated by difference spectroscopy techniques, whereby a comparable addition curve can be constructed from changes in the 555-nm absorption, while the 541-nm absorption remains invariant. The change described corresponds to the formation of a new conformation, corresponding to carbamoyldeoxyhemoglobin, carrying one carbamoyl group per chain. In the presence of a small quantity of oxygen, however, the above reported changes in CD are accompanied by a concomitant rise in the 415-nm peak-corresponding to the formation of oxyhemoglobin-while those in the difference spectra reflect not only a change in the 555-nm band but also a parallel one at 541 nm, confirming the formation of oxyhemoglobin. Thus the conformation achieved upon carbamoylation of deoxyhemoglobin has the higher oxygen affinity predicted by Nigen et al. (1974) for carbamoyldeoxyhemoglobin. Cyanate has been used (Cerami and Manning, 1971) as an antisickling reagent in vivo and in vitro, but, although it has been shown that it binds covalently to the NH2-terminal residues of hemoglobin (Lee and Manning, 1973), its effect on hemoglobin conformation has not been previously shown nor has its mechanism of action been fully clarified. The results presented here show that the effect of cyanate on hemoglobin is the formation of a new conformation with heightened oxygen affinity. Since oxyHbS does not aggregate while deoxyHbS does, in a temperature-dependent fashion, the formation of carbamoyldeoxyHbS interferes with such aggregation in vitro in deoxygenated samples. In vivo, where there are generally low residual concentrations of O2, the formation of oxyHb is favored by the higher O2 affinity of carbamoyldeoxyHbS, and aggregation with concomitant red cell sickling is therefore disfavored.