Transcript

J BIOCHEM MOLECULAR TOXICOLOGYVolume 20, Number 1, 2006

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: [email protected]

Received 3 November 2005; revised 8 December 2005; accepted 11 December 2005

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

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 0.54min−1 and for the third phase were 0.0071 and 0.0045min−1 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 20:48–56, 2006; Published on-line in Wiley InterScience (www.interscience.wiley.com).DOI 10.1002/jbt.20108

KEYWORDS: Arsenic; Arsenite; Binding; Dissociation;Dithiol; Sulfhydryl

INTRODUCTION

Human exposure to inorganic arsenic can lead tocarcinogenesis in urinary bladder, lung, skin, liver,

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.

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 arsenic’s 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].

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 [10–12]. 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

48

Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 49

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.

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 arsenic’s possible modes of biological action.

MATERIALS AND METHODS

Efficiency of Arsenate Reductionto Arsenite/Stability of Arsenite

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.

Stability of Peptide Sulfhydryl GroupsDuring the Binding Experiments

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.

Dissociation Studies of Arsenite-PeptideComplexes

Peptides were synthesized, and mass spectroscopyand HPLC purity determinations (peptide 10 at 94%

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 37◦Cto remove the excess SO2 gas.

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 Tris·HCl. 73As-labeled arsenite and the targetpeptide were incubated together for 12 h (associationtime) at 2–8◦C 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 Tris·HCl 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.

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

50 KITCHIN AND WALLACE Volume 20, Number 1, 2006

nonspecific binding, filtered peptides were washedtwice with 2 mL of cold 150 mM NaCl, 100 mM Tris·HCl,pH 7.5, solution. The wash solution was kept on ice.

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 32–91 uMrange studied.

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

Y = Span1(exp − k−1t) + Span2(exp − k−2t)

+ Span3(exp − k−3t) + Plateau

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.

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 k−1, k−2, and k−3.The plateau term is nonspecific binding that does notdecay with time.

RESULTS

Arsenate Reduction and the Stabilityof Peptide Sulfhydryl Groups Duringthe Binding Experiments

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 0–35% loss of free sulfhydryls during the 27–36 h at 2–8◦C utilized for the association and dissocia-tion parts of these experiments.

Dissociation Studies

Figure 1 shows the triphasic time course of dis-sociation of 73As labeled arsenite dissociating frompeptide 20 which contains only one sulfhydryl group.

Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 51

TABLE 1. Regression Analysis of the Dissociation of Com-plexes of Arsenite and Peptide 20 with One Cysteine

95% ConfidenceBest Fit Standard Error Limits

Span 1 (nmol/mg) 13.6 0.498 12.5–14.6k−1 (min−1) 50.8 28.8 −8.07 to 110T1/2 (min) 0.0136Span 2 (nmol/mg) 1.39 0.471 0.429–2.35k−2 (min−1) 0.349 0.317 −0.299 to 0.996T1/2 (min) 1.98Span 3 (nmol/mg) 1.09 0.400 0.273–1.91k−3 (min−1) 0.0071 0.007 −0.0074 to 0.021T1/2 (min) 98.3Plateau (nmol/mg) 3.47 0.218 3.03–3.92r 2 0.9859

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.

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

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.

second (8.6%) and third (6.8%) phases of dissociation.However, the second and third rate constants are 0.35and 0.0071 min−1, 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.

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 0.0045 min−1, which correspondto half-lives of 1.29 and 155 min, respectively.

52 KITCHIN AND WALLACE Volume 20, Number 1, 2006

TABLE 2. Regression Analysis of the Dissociation of Com-plexes of Arsenite and Peptide 10 with Three Cysteines

95% ConfidenceBest Fit Standard Error Limits

Span 1 (nmol/mg) 25.9 1.01 23.9–27.9k−1 (min−1) 19.0 8.9 1.25–36.7T1/2 (min) 0.036Span 2 (nmol/mg) 6.80 0.865 5.07–8.53k−2 (min−1) 0.535 0.134 0.266–0.804T1/2 (min) 1.29Span 3 (nmol/mg) 11.0 0.541 9.92–12.1k−3 (min−1) 0.0045 0.001 0.0032–0.0057T1/2 (min) 155Plateau (nmol/mg) 1.99 0.517 0.955–3.02r 2 0.9890

For triphasic 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.

With respect to peptide 20 with one cysteine, non-linear regressions were attempted using models with1, 2, 3, and 4 spans. The fit with four spans gave highlysimilar values for r2 and absolute sum of squares com-pared to the three span fit, so there was no advantagein going to the more complex model. The fit with threespans had a r2 of 0.9859 and absolute sum of squares of9.201. The fit with either two or one spans gave some-what lower r2 values (0.9839 and 0.9532) and higherabsolute sum of squares (10.49 and 30.48), respectively,so these fits did not explain the data as well as a three-span model does.

With respect to peptide 10 with three cysteines,nonlinear regressions were also done using modelswith 1, 2, 3, and 4 spans. The fit with three spans had a r2

of 0.9890 and absolute sum of squares of 66.06. Similarlyto peptide 20 data, the peptide 10 fit with 4 spans gavequite similar values for r2 and absolute sum of squares,so there was no advantage in going to the more com-plex model. If there is a fourth span of arsenite-peptidedissociation in these experiments, the fourth span is sosmall as to not be easily found in up to 1440 min (24 h) ofobservation. The fit with either two or one spans gavesomewhat lower r2 (0.9754 and 0.7920) and higher ab-solute sum of squares (148 and 1252), respectively, sothese fits did not explain the data as well as a three-spanmodel does.

Overall, the second and third phases of arsenite-peptide complex decay are discernable for peptide 20and obvious for peptide 10. Therefore, three spans fitsare a better interpretation than two span fits for the dis-sociation of arsenite-peptide complex(es). The choice ofusing three spans in our regression analysis presentedin Table 1 has the advantage of also matching the the-oretical principle that three is the maximum amount ofcomplexes that can form from arsenite- and cysteine-containing peptides [16,17].

DISCUSSION

Interactions between Arsenite and EitherCys or GSH

A combined scheme that shows both the reduction(pentavalent → trivalent oxidation state for arsenic)[18] and the complexing properties of thiol (RSH)for inorganic and methylated arsenicals has been pre-sented by Cullen’s group [17]:

(CH3)xAsO(OH)3−x + (5–x)RSH

→ (CH3)xAs(SR)3−x + RSSR

Monodentate GSH or cysteine complexes have beenobserved with dimethylarsinic acid (DMA(V)) [17,20].Bidentate GSH or cysteine complexes have been ob-served with methylarsonic acid (MMA(V)) [16,17].Tridentate complexes have been seen with arseniteand glutathione [12,16,17,19]. The common trivalentforms of arsenic, arsenite, MMA(III), and dimethy-larsinous acid (DMA(III)) all bind to metallothioneinwith stoichiometry consistent with the known coordi-nation chemistry of arsenic (monodentate for DMA(III),bidentate for MMA(III) and tridentate for arsenite) [21].

We favor the binding scheme presented inFigure 3 because (a) it fits with the known coordinationchemistry of arsenic, (b) intermolecular complexesof this type of structure are known for glutathioneand cysteine [12,17,19,22–24], and (c) intramolecularbidentate complexes of trivalent arsenicals and smallchelator molecules such as dimercaptol (British Anti-Lewisite), mesodimercaptosuccinic acid (DMSA), and2,3-dimercapto-1-propanesulfonic acid, sodium salt(DMPS) are known [12,22,25]. Interestingly, as arsenicadds methyl groups, the arsenic atom becomes morenegatively charged (increased electron density) and thisweakens the bonds that arsenic can form with eitheroxygen or sulfur atoms [26].

Trivalent arsenic complexes containing three(As(GS)3) or two (CH3As(GS)2) glutathione moietieshave been observed in the urine of arsenite treated(0.5–5 mg/kg sc) mice deficient in the enzyme gamma-glutamyl transpeptidase which cleaves most GSH con-jugates [24]. This enzyme is abundantly expressed inthe microvilli of the proximal tubule of the kidney.No complexes of one GSH and dimethylated formsof arsenic were observed in spite of large amounts ofDMA(V) being present in the mouse urine. This is con-sistent with our observation that monodentate com-plexes of Cys-containing peptides and arsenite dissoci-ate very rapidly (Figures 1 and 2, k−1 values presented inTables 1 and 2). Not surprisingly, the authors observedthat the As(GS)3 species was unstable during ion ex-change separation conditions (i.e., dissociation of the

Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 53

FIGURE 3. Schematic representation of arsenite-peptide complexes associating and dissociating (A) in an intermolecular manner with peptide20 with one cysteine as an example and (B) in an intramolecular manner with peptide 10 with three cysteines as an example. Peptide 10 can bindarsenite in both the intermolecular and intramolecular manner. The Kd values of 190, 124 and 1.32 uMolar are based on the saturation-bindingstudies of peptide 20, 10 and 19 by Kitchin and Wallace [13]. Estimates of the arsenite-peptide complexes dissociation rates k−1, k−2 and k−3

come from this study. The three respective association rates, k1, k2 and k3 are calculated by the using the equation Kd = k−1/k1 = koff/kon while thehalf-lives are estimated by using the equation T1/2 = 0.693/dissociation rate constant.

As(GS)3 complex occurred) [24]. Overall there is a sub-stantial amount of evidence that shows tridentate com-plexes of GSH with arsenite and bidentate complexesbetween MMA(III) and GSH, thus arguing that theschematic representation of arsenite-peptide bindingpresented in Figure 3 is reasonable.

Arsenite Binding to Monothiol, Dithiol, andTrithiol Peptides—Affinities, Half-lives, andConsequences

Trivalent arsenic compounds bind more stronglyto dithiol sites than to monothiol sites [10–13]. Arsenitedithiol sites have a slower dissociation rate constant andlonger half-lives than monothiol sites (data of Tables 1and 2). Although the affinity of arsenite for trithiol- anddithiol-binding sites is about the same [13], the dissoci-ation rate constant of the third phase of dissociation issmaller (and thus the half-live is longer) for trithiol-binding sites than for either monothiol- or dithiol-binding sites (Tables 1 and 2). These data suggest that

dithiol- and trithiol-binding sites will be the most likelycausal triggers of biological effects because of theirstronger affinity and because the bi- and tri-dentatecomplexes last so much longer than the rapidly disso-ciating and reforming binding of arsenite to monothiolsites. Even though monothiol-binding sites do not holdon to arsenite for long, they are still abundantly presentin life-forms such as mammals. At any one given time,∼99% of arsenite is bound to tissue sulfhydryls, mostlyto monothiol sites [13].

The literature on arsenic has recognized the biolog-ical importance of higher affinity dithiol-binding sitesfor many years, but it has largely ignored the possibleimportance of trithiol containing binding sites. For tri-and bi-dentate arsenicals (i.e., arsenite and MMA(III)),binding can crosslink two or three different peptides orproteins forming new homo or hetero dimer or trimerforms of the original peptides and proteins. Arsenite iscapable of binding with high affinity to two cysteinemoieties that are a surprisingly large distance (14 inter-vening amino acids) apart [27].

54 KITCHIN AND WALLACE Volume 20, Number 1, 2006

In this study, we found a low degree of arsenitebinding to peptide 20 (at 91.3 uMolar) in a bidentate(9%) and tridentate (7% of the total binding) manner(Table 1). The concentration of protein sulfhydrylin vivo is approximately 12 mMolar [13]. This is over130-fold more concentrated than our in vitro dissocia-tion of binding studies so more arsenite is expected tocrosslink different proteins in a bidentate and tridentatemanner in vivo. Some of the biological effects observedafter inorganic arsenic exposure may be due to binding-induced crosslinking of peptides and proteins in ad-dition to the well-appreciated conformational changes.Thus, arsenite and MMA(III) should be considered pro-tein crosslinkers as well as protein-binding agents. In-tramolecularly, bridging between two cysteines by ar-senite will form a ring structure and effectively changethe conformation of a peptide or protein [12,28]. Therehas been a general view that the ring structures formedby bidentate arsenicals are important in mediatingthe peptide and protein effects of arsenicals. How-ever, it seems that the slower dissociation rate (andhence longer half-life) of the second and third sulfuratoms complexing to the arsenic is more importantthan the geometry of arsenic-containing complex. Inother words, the number of arsenic-sulfur coordina-tions is more important than if the coordinations oc-cur in an intermolecular or intramolecular manner. Thepresence of two sulfhydryls close enough together tomake a high affinity arsenite binding site does greatlyincrease the amount of arsenite bound to two or threesulfur atoms (e.g., the much larger values for span 2and span 3 for peptide 10 compared to peptide 20). Insomewhat similar fashion, when the number of methylsubstituents on the arsenic atom is increased, the DNA-damaging potency of trivalent arsenicals is greatlyincreased [29].

Dissociation of Arsenite-PeptideComplex(es)

The data of Figures 1 and 2 are dominated by thelarge amount of arsenite-peptide complex dissociatingat a rate too fast for the technique of vacuum filtrationto adequately determine. We can be confident about thepool or span size for the rapidly dissociating complex,but not the rate constant for the most rapid phase ofcomplex decay. As it requires several seconds to addcold carrier arsenite, mix the resulting solution, andthen load and wash the material twice on the nitro-cellulose filter, the rapid phase of dissociation appearsto be ∼100% finished within about 15 s after adding theexcess cold carrier arsenite. Thus the first rate of disso-ciation rate might be as large as 433 min−1or as slow as4.33 min−1, corresponding to 0.01 and 1.0 s as the half-lives, respectively. The first phase of dissociation kinet-

ics of Figure 1 is probably very similar to the dissocia-tion kinetics of other monodentate complexes of arsen-ite and either cysteine, glutathione, or isolated proteinsulfhydryls.

A determination of the second and third spansizes and the dissociation rate constants for peptide20 is somewhat difficult, because the total number ofcounts available to work with is quite limited. We used42 uCuries and additional nonradioactive carrier ar-senite in the experiment displayed in Figure 1. In ourexperiments with peptide 10, intramolecular bindingof one arsenite to two or three sulfhydryls of the samepeptide can account for the much larger spans of thesecond and third phases of dissociation that was ob-served. For peptide 10, both intermolecular dissocia-tion of arsenite-(peptide 10)2 and arsenite-(peptide 10)3and the intramolecular bi- and tri-dentate complexes ofarsenite and peptide 10 are being experimentally ob-served at the same time.

Dividing the second and third dissociation rate con-stants for peptide 20 gives a value of about 49 (rates of0.349/0.0071). Doing similarly for peptide 10 gives usa value of about 119 (rates of 0.535/0.0045). For both ofthese two cases, it seems that the second and third sulfuratoms that the arsenite complexes to greatly slow downthe rate of dissociation of the resulting arsenite-peptidecomplex(es). If this pattern also held true for the differ-ences in dissociation rate for complexes of one or twosulfur atoms and arsenite, then it would predict dissoci-ation rates for an arsenite-monothiol complex of about17.1 and 63. 6 min−1 (and half-lives of about 0.041 and0.011 min) for peptides 20 and 10, respectively. Thus,with possible half-lives in the order of magnitude ofabout 1.0 s for an monodentate arsenite peptide com-plex, it is not surprising that the vacuum filtration tech-nique was not successful in directly determining themost rapid dissociation rate of arsenite- and cysteine-containing peptide complexes.

We have performed additional dissociation experi-ments in an attempt to learn more about the first rapidlydissociating phase of the arsenite-peptide complex. Weused a 1-h association of arsenite and peptide 10 inwhich the 104 carrier arsenite was added either duringthe sample load step or during a first, second, andthird wash steps. We observed between 54% and 89%of the arsenite-peptide 10 complexes being dissociatedby the added carrier. A short half-life of <0.1 s for themonodentate arsenite-peptide complex would have re-sulted in a similar amount of reduced binding (probably89% or higher) in all four cases, and this result was notexperimentally observed. In another experiment, 76%and 60% of the first phase of complex dissociation oc-curred when 104 carrier arsenite was added during thesample load step to complexes of 73As-labeled arsen-ite and peptide 20 and 10, respectively. Thus, for the

Volume 20, Number 1, 2006 TRIPHASIC DISSOCIATION OF ARSENITE COMPLEXES 55

rapid first phase of arsenite-peptide complex dissocia-tion, we believe that a possible half-life in the order ofmagnitude of about 1.0 s is more likely than half life of<0.1 s. If the half-life of the first phase of dissociationwas as large as 10 s, then the technique of vacuum filtra-tion should have been able to measure this rate withoutdifficulty.

CONCLUSIONS

The data of this study have (a) demonstrated thelong half lives of tridentate arsenite-peptide complexes;(b) demonstrated the large dissociation rate constantfor a monodentate complex of a trivalent arsenic anda single sulfhydryl group; (c) shown that the kineticsof dissociation of intermolecular and intramolecularcomplexes of arsenite and one, two, or three cysteinesof peptides is somewhat similar; (d) demonstrated thegreat difference (almost four orders of magnitude) be-tween the rates of dissociation of a single arsenic-sulfurcoordination versus an arsenic atom coordinated tothree sulfur atoms; (e) shown that arsenite can crosslinkpeptides and proteins and thus form long-lived com-plexes of two and three cysteine containing peptides(Figure 3A); (f) presented evidence that shows that thenumber of coordinations to sulfur is the matter of great-est importance in determining the stability of the over-all arsenite-peptide complex, not if the coordinationsare in a ring geometry or not; and (g) shown why arsen-ite (with up to tridentate binding), MMA(III) (with up tobidentate binding), and DMA(III) (with only monoden-tate binding) are expected to be very different in theirpeptide and protein-binding affinities, dissociation rateconstants, and the arsenic-peptide complex half-lives.

A schematic version of arsenite-peptide binding in-cluding values for the dissociation rates, associationrates, and half-lives of the various arsenite-peptidecomplexes is presented in Figure 3. Overall, this andprior studies [13,27] have given us a more comprehen-sive view (both in respect to kinetics of dissociation andassociation of complexes and the Kd and Bmax values)of the intermolecular and intramolecular way in whicharsenite binds to cysteine-containing peptides and thus,proteins.

The kinetics (both dissociation and association),affinity and capacity of the binding of trivalent arseni-cals to peptides and proteins, the ensuing conforma-tional changes, peptide and protein crosslinking, andenzyme inhibition can contribute to cell damage anddeath. This in turn can contribute to better understand-ing of the pharmacokinetics, pharmacodynamics, andthe mode of action of arsenic in causing adverse bio-logical effects such as toxicity and carcinogenicity. Bet-ter understanding trivalent arsenical-macromolecular

interactions can positively impact on environmentalrisk assessment of arsenicals.

ACKNOWLEDGMENTS

We thank Drs. Mike Hughes and Carl Blackmanfor reviewing this manuscript as part of EPA clearanceprocedures. This manuscript has been reviewed in ac-cordance with the policy of the National Health andEnvironmental Effects Research Laboratory, U.S. Envi-ronmental Protection Agency, and approved for pub-lication. Approval does not signify that the contentsnecessarily reflect the views and policies of the Agency,nor does mention of trade names or commercial prod-ucts constitute endorsement or recommendation foruse.

REFERENCES

1. National Research Council. Health effects of arsenic. In:Arsenic in drinking water. Washington, DC: NationalAcademy Press, 1999. pp 83–149.

2. Kitchin KT, Wallace K, Andrews P. Some chemical proper-ties underlying arsenic’s biological activity. In: ChappellWR, Abernathy CO, Calderon RL, Thomas DJ, editors.Arsenic exposure and health effects—V. Amsterdam:Elsevier, 2003. pp 345–354.

3. National Toxicology Program. Report on carcinogens,11th edition. U.S Department of Health and Human Ser-vices, Public Health Service.

4. Petrick JS, Jagadish B, Mash EA, Aposhian HV.Monomethylarsonous acid (MMA(III)) and arsenite:LD(50) in hamsters and in vitro inhibition of pyruvatedehydrogenase. Chem Res Toxicol 2001;6:651–656.

5. Styblo M, Serves SV, Cullen WR, Thomas DJ. Com-parative inhibition of yeast glutathione reductase byarsenicals and arsenothiols. Chem Res Toxicol 1997;10:27–33.

6. Lin S, Del Razo LM, Styblo M, Wang C, Cullen WR,Thomas DJ. Arsenicals inhibit thioredoxin reductasein cultured rat hepatocytes. Chem Res Toxicol 2001;3:305–311.

7. Wu C. Glutamine synthetase VI. Mechanism ofthe dithiol-dependent inhibition by arsenite. BiochemBiophys Acta 1965;96:134–147.

8. Styblo M, Thomas DJ. In vitro inhibition of glutathionereductase by arsenotriglutathione. Biochem Pharmacol1995;7:971–977.

9. Whittaker VP. An experimental investigation of the ‘ringhypothesis’ of arsenical toxicity. Biochem J 1947;41:56–62.

10. Johnstone RM. Sulfhydryl agents: Arsenicals. In:Hochster RM, Quastel JH, editors. Metabolic inhibitors:A comprehensive treatise. New York: Academic Press;1963. Vol 2, pp 99–118.

11. Aposhian HV. Biochemical toxicology of arsenic. In:Hodgson E, Bend JR, Philpot RM, editors. Reviewsin biochemical toxicology. Amsterdam: Elsevier; 1989.Vol 10, pp 265–299.

12. Delnomdedieu M, Basti MM, Otvos JD, Thomas DJ.Transfer of arsenite from glutathione to dithiols: A modelof interaction. Chem Res Toxicol 1993;6:598–602.

56 KITCHIN AND WALLACE Volume 20, Number 1, 2006

13. Kitchin KT, Wallace KW. Arsenite binding to syntheticpeptides based on the zinc finger and the estrogen bind-ing region of the human estrogen receptor-alpha. ToxicolAppl Pharmacol 2005;206:666–672.

14. Yalcin S, Le XC. Speciation of arsenic using solid phaseextraction cartridges. J Environ Monit 2001;3:81–85.

15. Hissin PJ, Hilf R. A fluorometric method for determina-tion of oxidized and reduced glutathione in tissues. AnalBiochem 1976;74:214–226.

16. Winski SL, Carter DE. Interactions of rat red blood cellsulfhydryls with arsenate and arsenite. J Toxicol EnvironHealth 1995;46:379–397.

17. Cullen WR, McBride BC, Reglinski J. The reactionof methylarsenicals with thiols: Some biologicalimplications. J Inorg Biochem 1984;21:179–194.

18. Carter DE. Oxidation-reduction reactions of metal ions.Environ Health Perspect 1995;103(Suppl 1):17–19.

19. Delnomdedieu M, Basti MM, Otvos JD, Thomas DJ.Reduction and binding of arsenate and dimethylarsinateby glutathione: A magnetic resonance study. Chem BiolInteract 1994;90:139–155.

20. Scott N, Hatlelid KM, MacKenzie NE, Carter DE. Re-actions of arsenic(III) and arsenic(V) species with glu-tathione. Chem Res Toxicol 1993;6:102–106.

21. Jiang G, Gong Z, Li XF, Cullen WR, Le XC. Interaction oftrivalent arsenicals and metallothionein. Chem Res Tox-icol 2003;16:873–880.

22. Aposhian HV. DMSA and DMPS—Water soluble anti-dotes for heavy metal poisoning. Annu Rev PharmacolToxicol 1983;23:193–215.

23. Rey NA, Howarth OW, Pereira-Maia EC. Equilibriumcharacterization of the As(III)-cysteine and the As(III)-glutathione systems in aqueous solution. J Inorg Biochem2004;98:1151–1159.

24. Kala SV, Kala G, Prater CL, Sartorelli AC, LiebermanMW. Formation and urinary excretion of arsenic triglu-tathione and methylarsenic diglutathione. Chem ResToxicol 2004;17:243–249.

25. Aaseth J. Recent advance in the therapy of metal poi-sonings with chelating agents. Human Toxicol 1983;2:257–272.

26. Carter DE, Aposhian HV, Gandolfi AJ. The metabolismof inorganic arsenic oxides, gallium arsenide andarsine: A toxicochemical review. Toxicol Appl Pharmacol2003;193:309–334.

27. Kitchin KT, Wallace K. Arsenite binding to syntheticpeptides: The effect of increasing length between twocysteines. J Biochem Molecular Toxicology 2006;20:35–38

28. Dagett V, Fersht A. The present view of the mecha-nism of protein folding. Nat Rev Mol Cell Biol 2003;6:497–502.

29. Andrewes P, Kitchin KT, Wallace KW. Dimethyarsineand trimethyarsine are potent genotoxins in vitro. ChemRes Toxicol 2003;16:994–1003.


Recommended