Ultraviolet D I F F E R E N C E Spectra of Pepsin*,$ the Journal of General Physiology

A shift of pH of pepsin solutions from 4.6 to 1.0 gives rise to spectral displacemerits in the ultraviolet. If represented as difference spectra three peaks with maxima at 2770, 2850, and 2930 ,~mgstr~ms are present which can be attributed to the tyrosine and tryptophan residues in the protein. On mild autolysis of pepsin at pH 2.0 the absorbancy in the ultraviolet further decreases. Although some of these effects can be ascribed to the occurrence of hydrogen bonding between the aromatic residues and a carboxylate ion, those observed on autolysis are caused by charge effects of newly formed polar groups in the vicinity of a chromophore. No direct relation between the optical properties described here and enzymic activity of pepsin has been observed. The ultraviolet absorption bands of protein solutions are displaced to shorter wave lengths on titration of a protein with acid (1), upon denaturation with urea or guanidine salts (2-4), and on mild proteolysis (2, 3, 5, 6). Recently, Laskowski, Scheraga, and coworkers have studied the spectral shifts that occur if the pH of solutions of insulin and ribonuclease is altered from neutrality to pH 1.0 (6, 7). On the basis of their results these investigators proposed the hypothesis, at present widely accepted, that these small spectral displacements reflect changes in the hydrogen bonding between the tyrosyl hydroxyls and neighboring basic groups of the protein; e.g., carboxylates. An alternative explanation, however, follows from the work of Wetlaufer, Edsall, and Hollingworth, who observed spectral changes of the same order of magnitude if solutions of tyrosine, O-methyltyrosine, and glycyl O-methyltyrosine of pH 7.0 are compared with those of pH 1.0 (8). Since the occurrence, in these materials, of intramolecular hydrogen bonds can be excluded these * This work was supported in part by Grant B-930 of the United States Public Health Service, National Institutes of Health. A preliminary report of this work was presented at the Meeting of the American Society of Biological Chemists, Philadelphia, 1958. § Holder of a Postdoctoral Fellowship in Medical Sciences, 1957-59, administered by the National Research Council and the National Science Foundation.