Dissociation of arsenite‐peptide complexes: Triphasic nature, rate constants, half‐lives, and biological importance

Abstract

We determined the number and the dissociation rate constants of different complexes formed from arsenite and two peptides containing either one (RVCAVGNDYASGYHYGV for peptide 20) or three cysteines (LECAWQGK CVEGTEHLYSMKCK for peptide 10) via radioactive 73As-labeled arsenite and vacuum filtration methodology. Nonlinear regression analysis of the dissociation of both arsenite-peptide complexes showed that triphasic fits gave excellent r2 values (0.9859 for peptide 20 and 0.9890 for peptide 10). The first phase of arsenite-peptide dissociation had the largest span (decrease in binding), and the rate was too fast to be measured using vacuum filtration methods. The dissociation rate constants of arsenite-peptide complexes for the second phase were 0.35 and 0.54 min(-1) and for the third phase were 0.0071 and 0.0045 min(-1) for peptides 20 and 10, respectively. For peptide 20, the three spans of triphasic decay were 85%, 9%, and 7% of the total binding of 16.1 nmol/mg protein. For peptide 10, which can bind in both an intermolecular and intramolecular manner, the three spans of triphasic decay were 59%, 16%, and 25% of the total binding of 43.7 nmol/mg protein. Binding of trivalent arsenicals to peptides and proteins can alter their structure and function and contribute to adverse health outcomes such as toxicity and carcinogenicity.

Arsenite Interacts Selectively with Zinc Finger Proteins Containing C3H1 or C4 Motifs

“…Our observation of arsenite binding selectivity for peptides and cellular target proteins with three or more cysteine residues is consistent with chemical studies demonstrating substantial differences in the K d values for radioactive arsenite binding to model peptides based on the number of free cysteines (32). Another important consideration is demonstrated by the elegant work of Kitchin and Wallace (43). Using rapid vacuum filtration assays and radioactive arsenite, dramatic differences in the chemical kinetics of arsenite-peptide interactions were revealed.…”

“…Our studies demonstrate arsenite binding selectivity in both peptide and protein with a link between interaction and functional consequences for protein activity. Although arsenite may be capable of binding to a C2H2 zinc finger (32,33,43), the final product may not persist for a sufficient length of time to be detected by MALDI-TOF-MS. This difference in chemical kinetics could have major impact in cells, where a more stable interaction of arsenite with a C3 or C4 zinc finger protein may be necessary to sustain biological impact.…”

Arsenic's Interactions with Macromolecules and its Relationship to Carcinogenesis

“…About 91% of the total arsenite binding is to the more numerous monothiol sites. The association rate constant of ligand receptor complex (k on ), dissociation rate constant of ligand receptor complex (k off ) and the half life (T 1/2 ) for arsenite binding to monothiol sites were too fast to be measured [29]. The overall picture that emerges is one of arsenite having a very short occupancy time on particular monothiol binding sites [29].…”

“…For dithiol sites the K d can be 1-20 mM, much lower than the value for monothiol sites. Estimates of the k off rates are between 0.3-0.5 min À1 which leads to a measurable half life (t 1/2 ) of about 1.3-2.0 min for arsenite binding to dithiol sites [29]. Such dithiol sites on important proteins and peptides can easily be occupied by trivalent arsenicals long enough and the resulting complex stable enough to cause some of the adverse health effects of arsenic.…”

“…Sites that are effectively trithiol sites can occur when arsenite binds to a dithiol protein site containing two cysteines from a protein and then the tridentate arsenite coordinates to either a glutathione or to the free amino acid cysteine. This would result in a tridentate coordination that would be expected to have a long T 1/2 of about 1-3 h [29].…”