Dye-sensitized photooxidation as a tool for determining the degree of exposure of amino acid residues in proteins. The methionyl residues in ribonuclease A.

Optical rotation measurements and absorption difference spectroscopy of ribonuclease A in different water-acetic acid mixtures showed that at least three conformational situations exist for the protein molecule at different levels of acetic acid concentration. Hematoporphyrin-sensitized photooxidation of the native as well as of the denatured proteins allowed us to detect three discrete states of reactivity for the 4 methionyl residues of ribonuclease A. On the basis of the photooxidation kinetics and of the conformational analyses, we concluded that methionine-29 is partly exposed in the native protein and methionine-13 is partially buried, whereas methionine-30 and methionine-79 are deeply buried. The importance of the single methionyl residues for the catalytic activity of the enzyme has been also resolved. The described procedure appears to be a reliable tool for examining the state of the amino acid residues in proteins.