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

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<ul><li><p>J BIOCHEM MOLECULAR TOXICOLOGYVolume 20, Number 1, 2006</p><p>Dissociation of Arsenite-Peptide Complexes:Triphasic Nature, Rate Constants, Half-lives,and Biological ImportanceKirk T. Kitchin and Kathleen WallaceEnvironmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Office of Research andDevelopment, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA; E-mail: kitchin.kirk@epa.gov</p><p>Received 3 November 2005; revised 8 December 2005; accepted 11 December 2005</p><p>ABSTRACT: We determined the number and the dis-sociation rate constants of different complexes formedfrom arsenite and two peptides containing either one(RVCAVGNDYASGYHYGV for peptide 20) or threecysteines (LECAWQGK CVEGTEHLYSMKCK for pep-tide 10) via radioactive 73As-labeled arsenite andvacuum filtration methodology. Nonlinear regressionanalysis of the dissociation of both arsenite-peptidecomplexes showed that triphasic fits gave excellent r2</p><p>values (0.9859 for peptide 20 and 0.9890 for peptide 10).The first phase of arsenite-peptide dissociation had thelargest span (decrease in binding), and the rate wastoo fast to be measured using vacuum filtration meth-ods. The dissociation rate constants of arsenite-peptidecomplexes for the second phase were 0.35 and 0.54min1 and for the third phase were 0.0071 and 0.0045min1 for peptides 20 and 10, respectively. For peptide20, the three spans of triphasic decay were 85%, 9%, and7% of the total binding of 16.1 nmol/mg protein. Forpeptide 10, which can bind in both an intermolecularand intramolecular manner, the three spans of triphasicdecay were 59%, 16%, and 25% of the total binding of43.7 nmol/mg protein. Binding of trivalent arsenicals topeptides and proteins can alter their structure and func-tion and contribute to adverse health outcomes such astoxicity and carcinogenicity. C 2006 Wiley Periodicals,Inc. *J Biochem Mol Toxicol 20:4856, 2006; Published on-line in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/jbt.20108</p><p>KEYWORDS: Arsenic; Arsenite; Binding; Dissociation;Dithiol; Sulfhydryl</p><p>INTRODUCTION</p><p>Human exposure to inorganic arsenic can lead tocarcinogenesis in urinary bladder, lung, skin, liver,</p><p>Correspondence to: Kirk T. Kitchin.c 2006 Wiley Periodicals, Inc. This article is a U.S. Government workand, as such, is in the public domain of the United States of America.</p><p>kidney, and to many other nonneoplastic health prob-lems (e.g., dermatological, cardiovascular, and neuro-logical effects) [1]. The modes of action of inorganicarsenic are not well understood at either a biologi-cal, cellular, or molecular level. When chemical mech-anisms of arsenics biological action are considered,three likely possible modes of action are (a) binding oftrivalent arsenicals to tissue sulfhydryls, (b) oxidativestress/reactive oxygen species/free radicals formedfrom arsenic exposure, and (c) nucleophilicity of triva-lent arsenicals which for, example, can lead to depletionof S-adenosylmethionine [2].</p><p>The binding of trivalent arsenicals to proteinsulfhydryl groups and the ensuing enzyme inhibitionand altered biological function can be seen as possiblechemical causes of many of the proposed modes of ac-tion of arsenic. Examples of other carcinogens whichare known to bind to proteins as part of their mode ofaction include 2,3,7,8-tetrachloro-p-dioxin, estrogens,and diethylstilbesterol [3]. Examples of enzyme inhibi-tion caused by trivalent arsenicals include pyruvate de-hydrogenase [4], glutathione reductase [5], and thiore-doxin reductase [6]. In some cases, enzyme inhibitionappears to be mediated via arsenical forms complexedto three sulfur atoms [7,8]. Arsenite and monomethy-larsonous acid (MMA(III)) occur in high tissue levels inmammals and possess at least two positions availableto bind to sulfhydryls. Therefore, these two arsenicalsmay contribute to adverse health effects via bindingand the ensuing peptide- and protein-induced confor-mational effects. The ring hypothesis of arsenical tox-icity centers on the formation of complexes of trivalentarsenicals and two sulfhydryls of one molecule whichresults in a cyclic system [9]. Dithiols, such as lipoicacid, have long been known to have a higher affinitythan monothiols for trivalent arsenicals [1012]. But thestability of the complexes formed by arsenite and thecysteine moieties of peptides and proteins is not known.Furthermore, it has also never been clear if arsenic could</p><p>48</p></li><li><p>Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 49</p><p>easily form complexes with three sulfhydryls of onemolecule, what the half-life of such a complex mightbe and if arsenic-trithiol complexes could be importantmediators in the adverse health outcomes caused byarsenic exposures.</p><p>The purpose of this study was to determine thedissociation rates of radioactive arsenite-peptide com-plexes with peptides that contained one or moresulfhydryl groups. The two amino acid sequences wereRVCAVGNDYASGYHYGV for peptide 20 with one cys-teine and LECAWQGK CVEGTEHLYSMKCK for pep-tide 10 with three cysteines. The two studied peptideswere based on zinc finger region and the hormone bind-ing region of the human estrogen receptor alpha [13].Experimentally, we allowed radioactive arsenite andthese two cysteine-containing peptides to come intobinding equilibrium, and then added excess cold ar-senite and determined the span size, the dissociationrate, and the half-life of the radioactive arsenite-peptidecomplex(es) via receptor dissociation techniques. Byspan size, we mean the quantitative amount of anarsenite-peptide complex (in nmol/mg protein), sim-ilar to the concept of compartment size in pharmacoki-netics. The results are interpreted with respect to thekinetics and half-lives of arsenicals binding to inter-molecular and intramolecular mono-, di-, and tri- thiol-binding sites, and the insight that this can give us interms of arsenics possible modes of biological action.</p><p>MATERIALS AND METHODS</p><p>Efficiency of Arsenate Reductionto Arsenite/Stability of Arsenite</p><p>To ascertain how efficient the reduction of arseniteby SO2 was, we determined the ratio of arsenite to arse-nate by separating the arsenate from the arsenite usinga strong anion exchange cartridge [14]. The presenceof oxygen during the reduction of arsenate to arsenitemay prevent complete reduction of arsenate. In addi-tion, once the SO2 is removed, the arsenite may oxidizeback to arsenate via oxygen exposure.</p><p>Stability of Peptide Sulfhydryl GroupsDuring the Binding Experiments</p><p>The o-phthalaldehyde fluorescence technique wasused for determining the free sulfhydryl group concen-tration [15] both before and after the 12-h incubationsfor the association and after the time required for thedissociation of the arsenite-peptide complexes.</p><p>Dissociation Studies of Arsenite-PeptideComplexes</p><p>Peptides were synthesized, and mass spectroscopyand HPLC purity determinations (peptide 10 at 94%</p><p>and peptide 20 at 100% purity) were performedby a commercial laboratory (Alpha Diagnostics, SanAntonio, TX). The NH2 terminal ends were labeledwith flouresceinisothiocyanate (FITC). 73As (arsenate)was obtained from the Brookhaven National Labora-tories and reduced to arsenite by bubbling with SO2gas (Matheson Gas Products, Inc.) into the arsenatesolution, waiting at least 1 h and then warming to 37Cto remove the excess SO2 gas.</p><p>Binding incubations included the test peptide orprotein diluted in cold water and 73As arsenite di-luted in cold 150 mM NaCl, pH 7.5 buffer containing100 mM TrisHCl. 73As-labeled arsenite and the targetpeptide were incubated together for 12 h (associationtime) at 28C on ice prior to vacuum filtration. Muchshorter incubation times of 15 min or 1 h did not al-low maximal amounts of slower decay arsenite-peptidecomplexes to form. Solutions were deoxygenated bybubbling nitrogen gas through them. Separations ofbound and free 73As arsenite employed 0.45 uM ni-trocellulose filters (Brandel, Inc.) soaked in a pH 7.5solution containing 150 mM NaCl, 0.3% polyethylen-imide, and 100 mM TrisHCl and a model M-24Cmembrane harvestor (Brandel Inc., Gaithersburg, MD).The conditions used in the dissociation experimentfor peptide 20 were 91.3 uMolar peptide concentra-tion, 30 uMolar nonradioactive arsenite (Fischer Sci-entific) concentration, and a specific activity of 1 uCiper 30 uMolar cold arsenite. Sampling was done at 18different time points between 0 and 1440 min (24 h) togive 40 samples and 42 uCi of radioactive arsenic to-tal per experiment. The experimental conditions usedfor peptide 10 were 32.1 uMolar peptide concentra-tion, 10 uMolar nonradioactive arsenite concentration,and a specific activity of 1 uCi per 5 uMolar cold ar-senite. Sampling was done at 27 different time pointsbetween 0 and 1440 min to give 70 samples and 146 uCiof radioactive arsenic total used per experiment. Du-plicate or triplicate samples were used for dissociationmeasurements. All dissociation experiments were re-peated at least twice on different days. Representativedissociation span and rate values are presented in thispaper.</p><p>After the 12-h incubation, a 104 excess of nonra-dioactive carrier arsenite was added to the incubationmedia, rapidly mixed at various times ranging fromseveral seconds to as much as 24 h later and subjectedto vacuum filtration to separate bound from free 73As-labeled arsenite. The zero time point of dissociation didnot have 104 excess of cold carrier arsenite added toit. Only when the 73As-labeled arsenite has dissociatedfrom the last binding site of peptide(s), will the bindingassay used in this study find that the complex has disso-ciated. Thus, the combined rates measured from morethan one dissociation are determined by the slower ofthe two or three sequential dissociations. To reduce</p></li><li><p>50 KITCHIN AND WALLACE Volume 20, Number 1, 2006</p><p>nonspecific binding, filtered peptides were washedtwice with 2 mL of cold 150 mM NaCl, 100 mM TrisHCl,pH 7.5, solution. The wash solution was kept on ice.</p><p>Gamma counting was done in a Packard MinaxiAuto gamma 5000 for 30 min. Protein was strippedfrom the nitrocellulose filters in a pH 10 buffer of62.5 mM Na2CO2 containing 5% sodium lauryl sulfate.The concentration of protein was determined spec-trophotometrically using the Pierce bicinchoninic acidprotein determination kit (using weighed bovine serumalbumin as the standard) and a Wallac Victor 1420multilabel counter. Neither measurement day effects ornonspecific binding produced difficulties in perform-ing these experiments. Nonspecific binding was lowin our studies probably because the synthetic peptideswere high in both purity and sufficient in concentrationto allow substantial arsenite binding in the 3291 uMrange studied.</p><p>Nonlinear regression for one, two, three, and fourphase dissociation rates was performed by the softwareprogram Prism 4. For triphasic decay of the arsenite-peptide complexes, the equation being fit was</p><p>Y = Span1(exp k1t) + Span2(exp k2t)+ Span3(exp k3t) + Plateau</p><p>FIGURE 1. Dissociation of 73As-labeled arsenite-peptide 20 complexes. The two inserts show the second and third phase of the triphasic modelof dissociation of arsenite-peptide 20 complexes. The values shown are means standard error bars for two or three samples per time point. Fortriphasic decay the equation of best fit was Y = 13.6(exp 50.8t) + 1.39(exp 0.349t) + 1.09(exp 0.0071t) + 3.47.</p><p>where Y is the total amount of arsenite-peptide bindingin nmol/mg protein, t is for time, spans 1, 2, and 3 arethe amounts of arsenite-peptide complex that are dis-sociating and the three exponential decay terms consistof the three dissociation rate constants k1, k2, and k3.The plateau term is nonspecific binding that does notdecay with time.</p><p>RESULTS</p><p>Arsenate Reduction and the Stabilityof Peptide Sulfhydryl Groups Duringthe Binding Experiments</p><p>In the experiments with peptides 20 and 10, 91.5%and 79.4% of the arsenate were reduced to arsen-ite, respectively [14]. Sulfhydryl stability experimentsshowed 035% loss of free sulfhydryls during the 2736 h at 28C utilized for the association and dissocia-tion parts of these experiments.</p><p>Dissociation Studies</p><p>Figure 1 shows the triphasic time course of dis-sociation of 73As labeled arsenite dissociating frompeptide 20 which contains only one sulfhydryl group.</p></li><li><p>Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 51</p><p>TABLE 1. Regression Analysis of the Dissociation of Com-plexes of Arsenite and Peptide 20 with One Cysteine</p><p>95% ConfidenceBest Fit Standard Error Limits</p><p>Span 1 (nmol/mg) 13.6 0.498 12.514.6k1 (min1) 50.8 28.8 8.07 to 110T1/2 (min) 0.0136Span 2 (nmol/mg) 1.39 0.471 0.4292.35k2 (min1) 0.349 0.317 0.299 to 0.996T1/2 (min) 1.98Span 3 (nmol/mg) 1.09 0.400 0.2731.91k3 (min1) 0.0071 0.007 0.0074 to 0.021T1/2 (min) 98.3Plateau (nmol/mg) 3.47 0.218 3.033.92r 2 0.9859</p><p>For triphasic decay the equation of best fit was Y = 13.6 (exp 50.8t) + 1.39(exp 0.349t) + 1.09 (exp 0.0071t) + 3.47.</p><p>Nonlinear regression analysis was used to estimate therate constants and half-lives for arsenite dissociationfrom peptide 20, and the values are presented in Table 1.Because the first dissociation process is simply too fastto measure, neither the dissociation rate or the half-life estimates for the first phase are really meaningful.Only small percentages of the total binding arsenite-peptide 20 binding (16.08 nmol/mg total) were in the</p><p>FIGURE 2. Dissociation of 73As-labeled arsenite-peptide 10 complexes. The two inserts show the second and third phase of the triphasic modelof dissociation of arsenite-peptide 10 complexes. The values shown are means standard error bars for two or three samples per time point. Fortriphasic decay the equation of best fit was Y = 25.9(exp 19.0t) + 6.8(exp 0.535t) + 11.0(exp 0.0045t) + 1.99.</p><p>second (8.6%) and third (6.8%) phases of dissociation.However, the second and third rate constants are 0.35and 0.0071 min1, which correspond to half-lives of 1.98and 98 min, respectively. The amounts of the arsenite-peptide 20 complexes which dissociated during theexperiment (i.e., the spans) were 13.6, 1.39, and 1.09nmol/mg. A plateau of 3.47 nmol/mg of binding didnot dissociate and is at least partly accounted for asradioactive arsenite binding to the nitrocellulose filteritself.</p><p>Figure 2 and Table 2 present the results of the disso-ciation experiment with arsenite and peptide 10 whichcontains three cysteines. In sharp contrast to peptide20 (with only one cysteine), the results with peptide 10showed substantially larger spans for the second andthird phases of dissociation. The three phases of disso-ciation accounted for 59%, 16%, and 25% of the totaldissociation of arsenite-peptide 10 complexes, respec-tively (Table 2). Again the dissociation rate constant forthe first phase of dissociation was too fast to measure byour technique, but the span amounted to 25.9 nmol/mgprotein of binding. The nonlinear regressions best fitvalues for the second and third dissociation rate con-stants were 0.54 and...</p></li></ul>

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