The reaction of 4-( p-trimethylammoniumphenylazo)-2-hydroxymercuriphenol with the thiol groups of proteins

Abstract

1. 1. A new water-soluble azomercurial, 4-( p-trimethylammoniumphenylazo)-2-hydroxymercuriphenol (TAMP), has been prepared and tested as a thiol probe. 2. 2. As for 4-( p-sulfophenylazo)-2-mercuriphenol (SAMP), the p K of TAMP undergoes a shift when the reagent forms a mercaptide with the thiol groups of native proteins, enabling spectrophotometric titrations to be carried out at 440 nm. 3. 3. TAMP gives the same results as SAMP with β-lactoglobulin. It does not, however, adsorb on bovine serum albumin. A TAMP-ovalbumin derivative also showed two p K values. Thiol titrations on ovalbumin are subject to deviations caused by the differences in reactivity of the thiols. 4. 4. In 8 M urea and 5 M guanidinium chloride the p K's of the TAMP-protein derivatives are nearly identical and equal to those of the derivatives with small mercaptans. Spectrophotometric titrations that are independent of the environment of the thiol group are possible in 8 M urea. 5. 5. Addition of EDTA raises the p K of the free reagent. This enlarges the p K differences with the mercaptides of small mercaptans and denatured proteins to an extent that these titrations become also highly sensitive.