You are here:
ARSENITE BINDING TO SUBSETS OF THE HUMAN ESTROGEN RECEPTOR-ALPHA
Citation:
Kitchin, K T. AND K. A. Wallace. ARSENITE BINDING TO SUBSETS OF THE HUMAN ESTROGEN RECEPTOR-ALPHA. Presented at Society of Toxicology, New Orleans, LA, March 6-10, 2005.
Description:
Enzyme inhibition by arsenicals has been described many times, but the underlying binding of trivalent arsenicals to peptides and proteins has received little attention. The purpose of this study was to determine Kd and Bmax values for arsenite binding to nine synthetic peptides (up to 25 amino acids long) which contained between 0 and 4 sulfhydryls. We selected the human estrogen receptor-alpah protein for study because arsenite is a potential nonsteroidal environmental estrogen and several interactions between arsenic exposure, estrogenicity and carcinogenicity are known. We utilized radioactive 73As labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to synthetic peptides based on a zinc finger region containing up to 4 free sulfhydryls and the estrogen binding region containing up to 3 free sulfhydryls. In our studies, amino acids other than cysteine (including methionine and histidine) did not bind arsenite. Peptides modeled on the estrogen receptor with two or more nearby free sulfhydryls (2 or 5 intervening amino acids) had Kd values in the 1-4 ?Molar range. Peptides containing a single sulfhydryl or two sulfhydryls spaced 17 amino acids apart had higher Kd values in the 100-200 ?Molar range, demonstrating lower affinity. With the exception of peptide 24 which had an unusually high Bmax value of 234 nmol/mg, the binding capacity of the studied peptides was proportional to the number of free cysteines. Based on our experimental Kd values and published literature values for tissue sulfhydryls, 99% or more of arsenite should be bound to tissue sulfhydryls and not free in vivo. The binding of trivalent arsenicals to protein sulfhydryl groups and the ensuing enzyme inhibition and altered biological function can initiate at least five proposed modes of arsenic's carcinogenic action - induced chromosomal abnormalities, altered DNA repair, altered DNA methylation patterns, altered growth factors and enhanced cell proliferation.