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Toxicol Appl Pharmacol. 2005 Aug 1;206(1):66-72.

Arsenite binding to synthetic peptides based on the Zn finger region and the estrogen binding region of the human estrogen receptor-alpha.

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  • 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA. kitchin.kirk@epa.gov


We selected the estrogen receptor protein for study because of prior results indicating that arsenite is a "potential nonsteroidal environmental estrogen". We utilized radioactive (73)As-labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to synthetic peptides. A zinc finger region containing four free sulfhydryls and the hormone binding region containing three free sulfhydryls based on the human estrogen receptor-alpha were studied. Peptide 15 (RYCAVCNDYASGYHYGVWSCEGCKA) bound arsenite with a K(d) of 2.2 microM and B(max) (maximal binding capacity) of 89 nmol/mg protein. Peptide 10 (LECAWQGKCVEGTEHLYSMKCKNV) had a K(d) of 1.3 microM and B(max) of 59 nmol/mg protein. In contrast, the monothiol peptide 19 (LEGAWQGKGVEGTEHLYSMKCKNV) bound arsenite with a higher K(d) of 124 microM and a B(max) of 26 nmol/mg protein. In our studies, amino acids other than cysteine (including methionine and histidine) did not bind arsenite at all. Peptides modeled on the estrogen receptor with two or more nearby free sulfhydryls (two or five intervening amino acids) had low K(d) values in the 1-4 microM range. Peptides containing single sulfhydryls or two sulfhydryls spaced 17 amino acids apart had higher K(d) values in the 100-200 microM range, demonstrating lower affinity. With the exception of peptide 24 which had an unusually high B(max) value of 234 nmol/mg, the binding capacity of the studied peptides was proportional to the number of free cysteines. Binding of trivalent arsenicals to peptides and proteins can contribute to arsenic toxicity and carcinogenicity via altered peptide/protein structure and enzyme function.

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