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ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES
Citation:
KITCHIN, K. T. AND K. WALLACE. ARSENITE BINDING TO SYNTHETIC PEPTIDES: THE EFFECT OF INCREASING LENGTH BETWEEN TWO CYSTEINES. JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 20(1):35-38, (2006).
Impact/Purpose:
to determine the binding affinity of arsenite to 8 synthetic peptides
Description:
Binding of trivalent arsenicals to peptides and proteins can alter peptide/protein structure and enzyme function and thereby contribute to arsenic toxicity and carcinogenicity. We utilized radioactive 73As- labeled arsenite and vacuum filtration methodology to determine the binding affinity of arsenite to 8 synthetic peptides ranging from 13 to 24 amino acids long and containing 1 or 2 cysteines separated by 0 to 17 intervening amino acids. Six of the eight peptides were highly similar in amino acid sequence and were based on cysteine containing regions of the hormone binding site of the human estrogen receptor-alpha (for example the sequence of peptide 28 is LFGAWCGKGVEGTEHLYSMKCKNV). The peptides with 0 to 14 intervening amino acids between two cysteines bound arsenite with Kd values of 2.7 to 20.1 uM and with Bmax values from 36 to 103 nmol/mg protein (0.083 to 0.19 nmol/nmol of protein). Thus, conformationally, these peptides effectively contain a dithiol high affinity binding site for arsenite. Peptide 17 with two C separated by 19 amino acids bound arsenite with a Kd of 123 uM and a Bmax of 41.8 nmol/mg. The monothiol peptide 19 bound arsenite with a Kd of 124 uM and a Bmax of 26 nmol/mg protein. Thus, peptides containing a single sulfhydryl or two sulflhydryls spaced 17 amino acids apart acted as a monothiol and had higher Kd values in the 100-200 uM range, demonstrating lower affinity for binding arsenite. The binding capacity of the studied peptides was roughly proportional to the number of free cysteines; amino acid sequence and peptide conformation had a limited impact on the maximal binding capacity for arsenite. All experimental binding curves fit well to a one site binding model.