Time-dependent ultraviolet absorption changes in proteins at high pH.
The effect of alkali on the ultraviolet absorption of several proteins was examined by difference spectrophotometry. In addition to the well known generation of phenolate ions from tyrosine, time-dependent changes occurred. These were relatively slow in water, but arose more quickly and to a greater degree in 6 M guanidine hydrochloride. These time-dependent changes were attributed to modification of the sulfur-containing amino acids, cystine and cysteine. The magnitude of the changes depended on the number and accessibility to solvent of the cystine, cysteine or derivatized species present. The increase in absorption at 295 nm typically reached a maximum value for disulfide containing proteins after ca. 1 h exposure to pH greater than 12 in 6 M guanidine hydrochloride; thereafter the changes were at least partially reversible. Taken in conjunction with amino acid analysis data, the results lend support to the beta-elimination mode of action of alkali on proteins. However the reaction mechanism appears to be complex and more than one chromophore, arising from more than one reaction pathway, seems to be involved in the alkaline degradation of the sulfur-containing amino acids. Particularly for proteins containing large amounts of cystine or cysteine, caution must be exercised when performing tyrosine titration experiments in order to recognize and minimize potential interference from other non-tyrosine related chromophores.