SOLVENT PERTURBATION STUDIES OF HEME PROTEINS AND OTHER COLORED PROTEINS .I.

The solvent perturbation technique of difference spectroscopy has been applied and extended to the study of the location of the aromatic tryptophyl and tyrosyl residues in the heme proteins. Based on experiments on unfolded hemoglobin and myoglobin and their apoproteins in 8 m urea and acid solutions, and their model compound analogs, corrections for the heme-contribution to the difference spectra in the 270-295-mμ aromatic region have been suggested, and their validity and limitations have been described and tested. These corrections are based on extrapolations dictated by the difference spectra of the nonaromatic, neighboring bands, or the γ-and δ-bands in the case of the heme proteins. The location of the tryptophyl residues in horse heart cytochrome c, beef liver catalase, sperm whale myoglobin, and horse hemoglobin, in the ferri state have been studied, using 20% ethylene glycol, glycerol, sucrose, and 90% deuterium oxide as perturbants. The data obtained on cytochrome c suggest that the single tryptophyl residue in this protein is about 55 to 68% exposed. In the case of beef liver catalase, a much larger subunit protein, a substantially greater fraction of the approximately 40 tryptophyls appear to be buried. In this protein 35 to 40% of the tryptophyl seem to be affected by the perturbing influence of solvent. Comparison of the hemoglobin and myoglobin data indicate a somewhat greater degree of burial of the tryptophyls in hemoglobin than in myoglobin (in myoglobin there are two tryptophyls whereas in hemoglobin there are six such groups). A more detailed discussion and analysis of the horse hemoglobin data based on studies with six perturbants, as well as the perturbation data for five other mammalian hemoglobins obtained with 20% ethylene glucol, is presented in the accompanying paper. © 1969.