Spectrophotometric titration and ultraviolet difference spectra of myosin and the meromyosins.

Spectrophotometric titration studies and ultraviolet difference spectra are two techniques which have been used in recent years for determining what role, if any, the tyrosine residues in a protein play in maintaining the secondary and tertiary structure of proteins. Crammer and Neuberger (2) utilized a spectrophotometric procedure to investigate the ionization of phenolic groups in ovalbumin and insulin. Similar investigations were carried out with plasma albumin (3, 4), ribonuclease (5, 6), and two derivatives of ribonuclease (7). From these studies it was found that some of the tyrosyl residues do not ionize freely and it was suggested that intramolecular hydrogen bonds between phenolic hydrogens and side chain carboxylate groups could exist in proteins (2). Ultraviolet difference spectra have been used for studying the anomalous tyrosyl residues and in the case of insulin and ribonuclease results which were interpreted as substantiating this hypothesis were obtained (8, 9). Wetlaufer et al. (10) working with the model compounds 0-methyltyrosine and glycyl0-methyltyrosine showed that other explanations must be considered. These authors concluded that the small spectral shifts observed when the model compounds were titrated with acid or base could be attributed to a change in the state of ionization of the molecules. Blumenfeld and Perlmann (11) used this as a partial explanation for the spectral shifts observed during the autolysis of pepsin. Bigelow and Ottesen (7), with the use of two derivatives of ribonuclease, suggested that two of the anomalous tyrosyl residues of ribonuclease might be hydrogen bonded to nonionizing acceptors. As part of a general program relating to some of the chemical and physical properties of the muscle proteins, it was felt that it would be of some interest to carry out such studies on myosin and two fragments produced by the limited tryptic digestion of myosin, L-meromyosin and H-meromyosin (12). These results have shown the existence of abnormal tyrosyl residues in myosin and L-meromyosin whereas the tyrosyl residues in H-meromyosin appear to be normal.