The polarographic method of Tadao Hata has been studied as a means of estimating both primary and secondary binding of organic mercurials on proteins. p-Chloromercuric benzoate and ethyl mercuric hydroxide were found to bind strongly with native crystalline egg albumin in a molar ratio of 2 : 1 while phenylmercuric hydroxide was found to exhibit weak sulfhydryl binding and a large amount of secondary binding. Urea, 3 M , eliminated the secondary binding of the PMOH and increased the primary binding to the level of the PCMB and EMOH. Stronger denaturants such as 6 M urea and guanidine increased the sulfhydryl binding of the PCMB but decreased the binding of the PMOH. The results are interpreted in terms of two mechanisms of binding, whole molecule binding and dissociation, with whole molecule binding playing the primary role in mercaptide formation and dissociation accounting for the secondary binding of the PMOH.
HE ESTIMATION of the binding <Jf organic Tmercurials on proteins has been done primarily to ascertain the presence of reactive sulfhydryl groups. The methods employed include chemical and amperometric titrations and also spectographic estimations, with p-chloroniercuribenzoate selected, for the most part, as the test reagent. These methods, while successful in the study of the primary binding or sulfhydryl binding of the mercurial, do not lend themselves to the study of the total binding sites, including those referred to as secondary. The specificities of these methods either exclude the possibility of estimating secondary binding, as in the case of chemical or spectographic determinations or consider secondary binding as an interference in the estimation of primary binding, as in the case of amperometric titrations.
Among organic mercurials of widely different structure, selective biological action, and different toxicities, it seems essential that the total protein binding be considered if there be hope of relating structure to biological action or hope of understanding the mode of their diverse actions. The polarographic method suggested by Hata (1) in 1951 offers the possibility of a differentiation of, and an estimation of, the total primary and secondary binding of organic mercurials on proteins. In this method, the depression of the polarographic wave height of the mercurial in the presence of increasing concentrations of protein is determined, and the amount of mercurial bound to the protein estimated from a standard concentration curve of the mercurial. The use-