CONFORMATIONAL DIFFERENCES IN CO DERIVATIVES OF HBA, HBC (E-BETA-6K) AND HBS (E-BETA-6V) IN THE PRESENCE AND ABSENCE OF INOSITOL HEXAPHOSPHATE DETECTED USING ULTRAVIOLET RESONANCE RAMAN-SPECTROSCOPY

The 229 nm excited ultraviolet resonance Raman spectra of the carbon m onoxide saturated derivatives of adult human hemoglobin (HbA), HbC (E beta 6K) and HbS (E beta 6V) in the presence and absence of inositol h exaphosphate (IHP) at pH 6.35 reveal a distinct pattern of local and g lobal conformational differences. In the absence of IHP, the protein s pecific spectral differences are all in the form of intensity changes. The intensity differences in the W3 band indicate that, compared with WbA, the mutant Hbs have the beta-chain A helix more tightly packed a gainst the E helix, with HbS having the largest change. Intensity diff erences in other Raman bands indicate that the local perturbation asso ciated with the beta 6 substitution propagates to other regions of the globin, The IHP-induced changes are consistent with two types of effe cts, a further tightening of the packing of the A helix against the E helix and the appearance of T state features indicative of a strained or weakened R quaternary conformation. The protein specificity of thes e effects can be accounted for by a model in which the linkage of the A and H helices of the B-subunits through the Glu beta 7-Lys beta 132 (H10) salt bridge is modulated by the beta 6-sensitive packing of the A helix against the E helix. This mechanism also accounts for the prot ein specificity of the IHP effects since Lys beta 132 is linked to two of the residues that form the central cavity IHP binding site. (C) 19 98 John Wiley & Sons, Ltd.