The Effect of Hydrolysis of Proteins on their Ultraviolet - Absorption Spectra near 210 m , u

Proteins show a region of intense absorption at wavelengths shorter than 230 m,u that was first extensively investigated by Goldfarb, Saidel & Mosovitch (1951), who concluded that a large part of the absorption was due to the peptide bond. The only other major contribution came from the aromatic amino acid residues. Since proteins generally have similar peptidebond contents, it is to be expected that near 2 10 m IL they will all have similar specific absorptions, provided that the aromatic amino acid content is not unusually high or low (Tombs, Cooke, Souter & Maclagan, 1960). Use has been made of this similarity in methods for the assay of mixtures of proteins, at either 215 and 225 mu (Waddell, 1956; Bendixen, 1957; Murphy & Kies, 1960) or 210 mgu (Tombs, Souter & Maclagan, 1959). The specific absorption of proteins at 210 m,e is approximately 200, and this great sensitivity is useful, e.g. for analysis of the effluent in protein chromatography, since as little as 1 mg. can be satisfactorily fractionated (Tombs, Cooke, Burston & Maclagan, 1961). Since during proteolysis peptide bonds are split, a decrease in the absorption at 210 m,u is to be expected, and it might be possible to devise a very sensitive procedure for the determination of the number of bonds split. This approach has been successful in following the splitting of acetylmethionine at 238 m,u (Mitz & Schlueter, 1958) and of simple peptides near 230 m, (Schmitt & Siebert, 1961). On the other hand, the action of pepsin has been reported to produce a small rise in the absorption at 210 mp (W. H. Taylor, personal communication). This is at first sight contrary to expectation and requires some explanation. We have therefore investigated the change in ultraviolet absorption produced by acid and enzymic hydrolysis, in an attempt to reconcile the slight rise reported in peptide hydrolysis with the decrease expected as a result of peptide-bond splitting.