Magnetic circular dichroism analysis of the IHP effect on spin equilibria in human ferric hemoglobins

Magnetic circular dichroism (MCD) spectroscopy has been to explore the connection between optical spectra and the high spin population of several hemoglobins under various conditions. It is found that the effectiveness of IHP in inducing spectral changes can be markedly affected by solvent. For example, the IHP-induced spectral changes in the visible region for nitritomethemoglobin-A in mixed buffer solvent systems (glycerol or polyethylene glycol (PEG), mw 190-210) are more than double those observed in aqueous buffers. We estimate that IHP induces a mix of R/T forms in bis-tris phosphate buffers, for NO-2metHb that is only about 50%. While PEG and glycerol both lead to enhanced IHP-induced spectral differences, they behave differently in two aspects. PEG shifts the visible MCD and absorption spectra of F-metHb-A, supposedly already biased towards the T form by ligand, in the same direction that IHP does. PEG also maximizes the spin state changes with IHP for three R from hemoglobins and N-3metHb-A, and so appears to stabilize the T form in all cases. Glycerol does not. In addition, the apparent binding constant for NO-2 to H2OmetHb-A differs between these two solvents. Comparison of the data from several hemoglobins leads to the conclusion that the changes in spin state distributions induced by IHP correlate well with quaternary structure for a given hemoglobin. An analogous correlation amongst various proteins between initial spin state distribution (IHP) absent) and quaternary structure is not found.