Raman difference spectroscopy of tertiary and quaternary structure changes in methaemoglobins.

There have been several resonance Raman scattering investigations of the effect of inositol hexaphosphate (IHP) on methaemoglobins. In those studies the sensitivity for detecting frequency differences was limited to 1-2 cm-1, and consequently frequency differences were not detected although spectral intensity differences due to changes in spin equilibria were. Shelnutt et al. recently reported on the observation of frequency differences induced by changes in the quaternary structure of chemically modified deoxyhaemoglobins. An improved Raman difference spectroscopic technique with 0.1 cm-1 sensitivity allowed the detection of these differences. We report here the application of this technique to a series of methaemoglobins with and without the addition of IHP. In addition to the intensity changes resulting from changes in the spin equilibria, we have observed frequency differences. In all liganded methaemo-globins that we examined a decrease in frequency of the mode in the 1,370 cm-1 region was observed on addition of IHP. In those in which a quaternary structure change is known to occur the frequency difference is greater than 0.5 cm-1. In those in which no quaternary structure change occurs [metHbA(CN-) and methHbA(N-3)] the frequency difference is smaller (approximately 0.15 cm-1).