Vol. 66, No. 1, 1975 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CIRCULAR DICHROISMAND RAMAN STUDIES OF THE ALLOSTERIC TRANSITION IN METHEMOGLOBIN F. A. Ferrone~ Department of Physics, and W. C. Topp, Department of Chemistry Princeton University Princeton, New Jersey 08540 Received July 11,1975 SUMMARY. Circular Dichroism and resonant Raman spectra have been measured for methemoglobin with and without inositol hexaphosphate (IHP) between pH 5 and pH 9. The transition induced by IHP is accompanied by large changes in the rotational strength of the Soret band. Without IHP, the rotational strength of methemoglobin euqals that of oxyhemoglobin; with IHP it equals that of deoxyhemoglobin. The rotational strength of the 260 nm band shows no change with IHP; the Raman bands change in a manner indicative of a shift in spin state equilibrium. The most direct explanation of these results is that IHP changes quaternary structure with few tertiary changes. It is pro- posed that the Soret rotational strength is a quantitative marker for quater- nary structure. INTRODUCTION. A promising approach to the study of cooperativity in hemo- globin was opened by the demonstration that, upon addition of inositol hexa- phosphate (IHP), methemoglobin could e~hibit a transition similar to, if not identical with that observed in ferrous hemoglobin upon deoxygenation. (1,2) Subsequently, questions have been raised as to the degree to which the met- hemoglobin plus IHP system resembles the ferrous deoxy structure. (3,4) While awaiting the x-ray crystallography which will prove definitive, one can turn to optical probes of structure such as circular dichroism and resonant Raman scattering for useful insights. Empirical studies of the Soret circular dichroism show a sensitivity to events of a quaternary nature, including chain separation (5,6), C-terminal digestion (6) or cooperativity-lowering mutation (7). Calculations by Hsu and Woody account for the Soret circular dichroism as the result of coupled oscil- *Supported by NSF contract 40474 Cop.vrlght © 1975 by Academzc Pres~, lnc All rzghts of reproductton in any form reserved 444