Models for studying the binding capacity of albumin to zinc by stripping voltammetry

129

Adytlca

Chlmzca Acta, 259 (1992) 129-138 Elsevler Science Publishers B V , Amsterdam

Models for studying the binding capacity of albumin to zinc by stripping voltammetry Juan C Wdal *, Gemma Ceprla and Juan R Castdlo Department of Analytrcal Chemistry, Faculty of Sctences, Umversrty of Zaragoza, 50009 Zaragoza (Spam)

(Received 23rd May 1991, revised manuscnpt recewed 4th October, 1991)

Abstract

The complexatlon capacity of albumin to zmc was studied by different voltammetrlc methods Cycbc voltammetry was used with a hanging mercury drop electrode (HMDE) to diagnose quahtatlvely the electrode mechanisms occumng at a mercury electrode and to evaluate the charge-transfer rate constant of the electrochemical reactlon of zmc m the presence of different concentrations of albumin The condltlonal stability constant of the zinc-albumin complex was determined pseudo-polarographlcally by differential-pulse anodlc strlppmg voltammetry (DP-ASV) with a Nafion-coated mercury film electrode (Nf-MFE) This electrode permits the determmatmn of free (labde) zmc and rejection of the mterfenng adsorption of free albumin on the glassy carbon surface A mean conditional stability constant of log p’ = 6 10 k 0 16 was obtained by this method DP-ASV was used to calculate p’ by titrating an albumin solution wnth Zn(II) and by Ruzlc data treatment In this instance a mean of log /3’ = 5 49 + 0 28 was found with the HMDE and Nf-MFE The results are compared and discussed Keywords Anodlc strlppmg voltammetry, Cychc voltammetry, Dlfferentlal pulse voltammetry, Albumm, Complexation, Zinc

Zmc 1s a very important trace element m the body, and performs various important functions, It 1s the cofactor of several enzymes, as shown by the wide variety of zmc metalloenzymes that exist [ll Owmg to Its blologlcal importance, Its determmatlon 1s of interest m fluids such as blood and urme Nevertheless, the determmatlon of total zmc 1s not a significant value [2], and the health of individuals 1s related to the dlstrlbutlon of the metal m blood combined with different hgands, therefore, speclatlon studies of protein-bound zmc are of special unportance For instance, human blood serum contams ca 1 mg l- ’ of zmc, Foot and Delves [3] found that zmc m blood 1s 60% bound to albumin, 5% bound to ammo acids and low-molecular-weight hgands and 35% bound to high-molecular-weight proteins (mainly a-2macroglobuhn) 0003-2670/92/$05

An instrumental method for the speclflcatlon of the metal m a blologlcal fluid must not produce disturbances of the natural equlhbnum, and the highly sensitive anodlc strlppmg voltammetry (ASV) 1s an appropriate techmque for this purpose [4,51 However, the organic matter m blologlcal fluids 1s usually adsorbed on the surface of the electrodes and produces severe interferences m the voltammetrlc measurements In particular, proteins strongly mhlblt the electrochemical reactions of metal Ions at mercury electrodes [6] An interesting approach for avoiding mterferences from adsorption m ASV 1s to cover a mercury film electrode (MFE) with a dialysis membrane [71 or a permselectlve polymeric film [8,9] In particular, Naflon perfluormated films have been extensively used [ 10,111 A very thm film of Nafion (Nf) allows a rapid

00 0 1992 - Elsevler Science Pubhshers B V All nghts reserved

130

electrode response to the mass transport of the analyte concentrations, there being a compromise between the exclusion of organic matter and the unhmdered transport of the metal ions Its permselectlvlty and antlfoulmg properties permit, for example, the determmatlon of metals dn-ectly m body flmds by ASV by avoldmg these mterferences [lO,lll The aim of this work was to study the analytlcal utlhty of the highly sensltlve dlfferentlal-pulse anodlc stripping voltammetry (DP-ASV) with a Naflon-covered MFE (Nf-MFE) to determine zinc m the presence of bovme serum albumin The binding capacity of this protem to zinc (condotlonal stability constant) was evaluated with two approaches pseudo-polarography and titration of an albumin solution with Zn(I1) (RUZIC treatment) [121 The purpose was to contrlbute to studies of the speclatlon of this and other elements m solutions with a high protem content and of the apphcablhty of ASV with a chemically modified electrode (Naflon coating) to calculate the stability constants with the above two methods In the Ruzlc method, the results were compared with those obtained usmg a hanging mercury drop electrode (HMDE) and Nf-MFE

J C VIDAL ET AL

Reagents All chemicals were of analytical-reagent

grade (Merck) unless stated otherwise High-punty water was obtained from a Mdhpore M&-Q water purlflcatlon system All sample vials were rmsed m 0 5 M mtrlc acid and high-punty water before use Bovme serum albumin (contammg 12% of water and less than 0 005% of fatty acids) was obtained from Sigma A stock solution of 1000 mg 1-l was prepared by dilution with 0 01 M phosphate buffer (pH 7 4)-O 1 M KC1 Thus solution was stored at 4°C m a refrigerator and dlluted to room temperature when used A 5 wt % solution of Nafion (Aldrich, prepared from Nafion 117 perfluormated membrane) was dduted m ethanol-water (0 9 + 0 1, v/v) to obtain a 0 40 wt % coating solution Supporting electrolyte buffers of 0 1 M phosphate (Suprapur, Merck) (pH 7 4) and 0 1 M acetic acid-acetate (pH 4 50) were prepared by suitable weighing and dilution to 250 ml These solutions were purified by passing them through a Chelex-100 column (sodmm form) and stored at 4°C

Nafwn coatzng of the GCE EXPERIMENTAL

Apparatus

Electrochemical experunents and ASV measurements were performed with an Inelecsa 1212 potentlostat Interfaced with an Acer 500 + computer (8088 microprocessor, IBM-compatible) and a Star LC-10 printer for recording the voltammograms The instrument parameters of the voltarnmetric techmques, stlrrmg and data treatment were controlled by compded BASIC programs All electrodes were from Methrom A saturated calomel electrode (SCE) and platinum wu-e were used as reference and counter electrodes, respectively A Kemula HMDE and a glassy carbon electrode (GCE, 3 * 0 05 mm dlameter) were used as working electrodes A Crlson Model 525 dlgltal conductlmeter with conductlvlty cells of K = 1 cm- ’ was used to test the ohmic resistance of solutions

Prior to coating, the GCE was pohshed for 60 s with 0 3-pm a-alumina particles, rinsed with ethanol and water, somcated m water for 5 mm and dried with a filter-paper The electrode was coated as described previously [13] by applying with a mlcrohtre pipette 10 ~1 of a 0 40% (w/v) Naflon solution m ethanol-water (0 9 + 0 1) to cover the active carbon disk The solvent was then allowed to evaporate for 20 mm and the film formed was heated with a hair-dryer at 60°C for 2 mm Mercury was deposited on the electrode substrate through the Naflon film by electrolysis at -850 mV vs SCE from a preplatmg solution containing 40 pg ml-’ of Hg(I1) m 0 1 M acetate buffer (pH 4 50) for 5 mm These freshly prepared Naflon and mercury films were washed thoroughly with water and precondltloned by two depoatlon-stnppmg cycles m a sample solution before use

BINDING

CAPACITY

OF ALBUMIN

131

TO ZINC

The coated electrode was useful for at least 4 h d care was taken not to expose the Hg-Nf film to air between changes of solutions, m order to prevent oxldatlon of the mercury Therefore the tip of the electrode was always immersed m de-

oxygenated water when the workmg solution was changed Between measurements, the mercury was cleaned from remaining amalgamated zmc with a potential step imposed on the workmg electrode (- 50 mV vs SCE for 1 mm and - 100 mV for 5 s)

v 02

1

I

I

-650

-960

I -1070 Potentul

RESULTS AND DISCUSSION

Cychc voltammemc study of Zn-albumm complex with the HMDE First a cyclic voltammetrlc study of the mfluence of albumm on the zmc charge transfer was carried out with the HMDE The cyclic voltammograms for Zn(I0 and zmc-albumin (Zn-ALB) complex solutions were recorded from -950 to - 1350 mV with different scan rates In all mstances the concentration of zmc was 5 mg 1-l and the concentrations of albumm varied from 0 to 1115 mg 1-l (supportmg electrolyte 0 1 M phosphate buffer, pH 7 02) Solutions of zmc with albumin were allowed to equilibrate for 24 h before measurements, and samples were deareated for 5 mm with oxygen-free mtrogen prior to recording the cyclic voltammograms As can be seen m Fig 1, albumm decreases the rate of the zmc charge transfer (decrease m charge-transfer constant, k,) and the cathodic peak of the cychc voltammogram (the supporting electrolyte was 0 05 M phosphate buffer) In this potential range (from about -900 to - 1350 mV> no direct reduction of the Zn-ALB complex occurs It would be necessary to use more negative potentials, but these are difficult to apply owmg to a conslderable increase of background and H+ reduction currents The two cathodic and anodlc processes display Randles-Sevclk behavlour [zP = K(scan rate)‘/* with R > 0 99 m all mstances], mdlcatmg that the electrochemical reactions of zmc are controlled solely by dtiuslon and no adsorption voltammet-

I

I

-11.50 mV vs

-1290

I -1400

SCE

Fig 1 Illustratwecyclicvoltammograms for (1) albumm (400 mg ml-‘), (2) zmc(II) (5 pg ml-‘) and (3) zmc-albumin (5 pg ml-’ and 240 mg ml-‘, respectwely) solutions Scan rate, 20 mV s-l

rlc peaks [of Zn(II> or Zn-ALB complex] were observed with the HMDE at scan rates below 60 mV s-l However, the Intensity of the cathodic peakfor a Zn(II)-AL,B solution 1s less than that for the same process m a Z&I) solution without ALB, owing to the adsorption of free AL,B on the mercury (which hinders the charge transfer for zinc) and also to a decrease m free zmc (lablle) because of Its complexatlon This complexatlon effectively decreases the labile (free) zinc and is the basis for studymg complexatlon of albumm with this metal and calculation of the condltlonal stablhty constant with strlppmg voltammetry by the RUZK mathematical method The anodlc peak corresponds to the strtppmg of amalgamated (preconcentrated) zmc m the mercury drop Hence, although the amount of metal deposited m the electrode depends on the charge-transfer rate, usmg potentials up to - 1350 mV the mtenslty of this peak wdl mamly depend on the preconcentratlon time, 1e , the potential scan rate, and varies slightly with albumin To estabhsh the reverslblhty of the charge transfer for zmc m the presence of albumm, values of k, (charge-transfer constant) were evaluated by the Nicholson method [14] Briefly, a scan rate 1s chosen to obtain m the cychc voltammogram a peak separation (A E,,, = E,, - EP,,) approximately between 120/n and 60/n mV, cor-

132

J C VIDAL

responding to a quasi-reversible charge transfer This value (obtamed with several scan rates) corresponds m a tabulated form to a dlmenslonless parameter y (as Indicated m Fig 3 m 11411,y = where a = nFv /RT, v IS the scan k,/(?raDY’2, rate (V s-l), D IS the mean diffusion coefficient [D = (D&&D&,o.#2], (Y1s the charge-transfer coefflclent and the other terms have their usual meanings To characterize the u-reversible response by (Y, this value can be obtained by the shift of E,, E p,C,2(potentials of the cathodic peak maximum and half-height peak, respectmely) because the shape of the cyclic voltammogram IS independent of the scan rate [15], where E,, - Ep,c,2 = - 1857 RT/anF Nevertheless, through the proper selection of condmons as Indicated, AE,, becomes almost independent of (Yand a maxTmum vananon of about 5% IS expected for AE,, obtained with y by not usmg cr m the calculations Hence values of AE,, close to 105/n mV are nearly Independent of CYm the range 0 3 < (Y < 0 7, and then the useful range for determmatlon of k, IS 0 5 < y < 5 By this calculation, hmns for y > 7 and y < 0 01 correspond to the reversible and totally lrreverslble charge-transfer cases, respectlvely (n A E,, < 62 and nAE,, > 300 mV, respectively) Table 1 lists the calculated k, (charge-transfer rate constant, cm sP1) for Zn(I1) with increasing concentrations of ALB As indicated, ALB de-

TABLE 1 Charge-transfer rate constants (k,) for solutrons of ik(lI) (5 mg I-‘) wrth increasing concentratrons of albumin, obtained wrth CV and HMDE a Albumm (mg ml-‘)

ALB Zn molar ratio

90 130 27.5 405 815 1115

0 018 0 025 0 054 0 079 0 159 0 218

k, knl s-r) 678x10-’ 106x10-’ 7 10x 10-4 5 44x10-4 3 78~10-~ 2 17x 10-4 3 10x 10-6

a Scan rates vaned from 0 1 to 600 mV s-l Drffusron coeffrcients D,u,, = 0 666~ 1O-5 cm* s-l, D = 1060x 1O-5 cm* s-r Charge-transfer coefficrent ((Y)~?%

ET AL

-0 4 r

-02

-

i s ? 5 0

oo-

02

04L

-

’ -850

I -960

I -1070

I

I

-1180

-1290

Potentml mV vs

I -1400

SCE

Frg 2 Influence of scan rate on cychc voltammograms of a solutton contaming zmc and albumin (5 pg ml-’ and 150 mg l-l, respecttvely) Scan rate (1) 10, (2) 60, (3) 90, (4) 120 mV s-1

creases the reverslblhty of the zmc charge transfer by decreasing k,, because of the adsorption of free albumin on the mercury electrode and mhlbmon of the charge transfer For instance, Zn(I1) has a quasi-reversible behavlour at about 60 mV of albumin of, e g , S -l, but with a concentration 130 mg I-’ this behavlour is observed from as low as 10 mV s- ’ upwards Cychc voltammograms of Zn-ALB solunons at scan rates higher than 60 mV s-l also show a cathodic prepeak at potentials of about - 1060 mV, more anodlc than that corresponding to a Faraday process This peak has a characterlstlc adsorption shape (symrnetrlcal) and ns maximum current 1s directly proportional to scan rate (v) (I~ = Ku) This behavlour indicates adsorption of ALB which produced this current at these scan rates above 60 mV s-l This 1s illustrated m Ag 2 at several scan rates (supportmg electrolyte 0 05 M phosphate buffer) This adsorption peak did not Increase with higher ALB concentrations, mdlcatmg saturation of the electrode surface by a monolayer of the protein Pseudo-polarographlc determnatton of the ZnALB condrtlonal stablkty constant wzth a Nafoncoated MFE A variety of voltammetrlc techmques have been developed for the study of metal complexatlon since the classical polarographlc technique of

BINDING

CAPACITY

OF ALBUMIN

133

TO ZINC

Heyrovsky and Lmgane based on the shift of the half-wave potential (E,,,) with hgand concentration [16] Nevertheless some of these techniques lack the sensltlvlty needed for dilute solutions (ca pg ml-’ levels or lower) To overcome this problem, the use of highly sensltlve ASV has been used to evaluate stability constants (usually m natural waters), e g , m the so-called pseudo-polarography [17,181 Pseudopolarograms plot the anodlc ASV strlppmg peak current as a function of the apphed pre-electrolySIS(deposltlon) potential Hence the potential at half maximum current of the obtained wave (El*/2 of the pseudo-polarogram) is analogous and can be related to the polarographlc half-wave potential (El,,), and it 1s assumed to reflect the overall actlvatron energy of the electrode reaction and 1s used for evaluating the complexatlon capacity In the theory developed for pseudo-polarography on a thm glassy carbon MFE [171, the followmg equation IS applicable AE;,, = E&(free) = (RT/nF)ln

- E,*/,(complex) p’ + (RT/nl;)ln

uL

hence plots of AE,*/, vs -1n a,_ provide metal complexatlon data through an analysis similar to those used m classical polarography, but with metal concentrations up to the ng ml-’ range To obtain the pseudo-polarogram, ASV peak heights can be used as they are directly proportional to total charge passed durmg the plating period The error due to the kmetlc current contnbutlon to the peak height (due to dlssoclatlon of the complex m the diffusion layer) m obtaining E& 1s negligible with the high rotation speed (and hence a thm dlffuslon layer) and the metal concentration used m strlppmg voltammetry A short plating time IS also advisable to avoid metal depletion As the electrochemical reaction can be non-reverslble owing to the presence of albumin, E;“,2 was estimated directly from the wave for a current value equal to half the hmltmg current, and by extrapolating the residual current from the foot of the wave upwards The relative error m can be about 3-5% with this proceW&rev dure [17]

Solutions of Zn(I1) (2 ng ml-‘) wth mcreasmg concentrations of albumm Qgand) were studied by the pseudo-polarographlc technique with a Nafion-covered MFE (to overcome the adsorption of the albumm) and by DP-ASV In all mstances the lonrc strength was 0 1 M (supportmg electrolyte phosphate buffer, pH 7 40) and complex solutions were allowed to equilibrate for 24 h The MFE 1s mtrmslcally more sensltlve than the HMDE and is used m stripping voltammetry wth low metal concentration levels (range ca l-40 ng ml-‘) It was therefore chosen for this study owing to low concentration of metals used m pseudo-polarography, but this electrode 1smore prone than the HMDE to interferences from adsorption of the albumin, and so rejection of albumin matter was effected by the Naflon layer coated on the glassy carbon surface Coating of workmg electrodes with polymeric permselectlve membranes 1s a common procedure introduced to circumvent the adsorption of surface-active compounds on a planar glassy carbon surface m ASV [S-113 The attractive features of Naflon (chemical inertness, non-electroactive, hydrophlhc and msoluble m water) have been particularly useful for voltammetrlc sensing

mu11 Covering of the electrode was done quickly and simply as described earlier by applymg a droplet of a Nf solution to the glassy carbon surface with a mlcrohtre pipette The thickness of the Nf film IS estimated to be about 170 nm, assuming the density of this film to be ca 158 g -3 and uniform coverage of the electrode area r&3] It 1s so small that mass transport of Z&I) through the film 1s hardly impeded This thickness can be controlled by varying the concentration of the Nf solution and/or the volume of this solution applied The values reported earlier [13] are the optimized valves, and there was a compromise between protection against adsorption of ALB and free dlffuslon of Zn(II) towards the electrode surface Through this Nafion layer, mercury was deposited on the glassy carbon by electrolysis m a separate solution The film of mercury adhered firmly to the carbon substrate, and can be used

J C VIDAL ET AL

134

throughout the lrfetlme of the chemically modlfled electrode (at least 4 h) takmg care with anodlc losses Nevertheless, repeated preparations can modify the sensltlvlty of the electrode (by about 5-lo%), and so the standard addition method IS advisable m all determmatlons of free zmc for obtaining reproducible data concentrat1ons

Figure 3 shows typical voltammograms of zmc and Zn-ALB complex solutions with bare and Naflon-covered MFEs The instrument parameters were AE, (change in pulse potential) = 100 mV, t, (time before pulse applied) = 1000 ms, t, (pulse width) = 20 ms, AE (step height) = 5 mV, scan rate (AE/t, + t,) = 490 mV s-l, t, (electrolysis time) = 2 mm, and t, (rest time before stnppmg) = 5 s, the supportmg electrolyte was 0 01 M Trls-acetate buffer (pH 7 02) Owmg to its low formation kmetlcs, the Zn-ALB complex was allowed to equlhbrate ovemlght (24 h) before the voltammetrlc measurements From Fig 3, the followmg observations can be made Increased sensltlvlty for zmc 1s obtamed with the Naflon electrode with respect to the bare electrode (voltammograms 1 and 3), as a result of its ion-exchange propertles The ASV signal for zinc m the complex solution 1s less than that for Zn alone (voltammograms 1 and 2, revi-

<

=10-

z e

2 0

D

2 4

05-

ooI -1300

I

I

-1225

-1150 Potentd,

mV vs

I -1075

I -1000

SCE

Fig 3 Comparison of DP-AS voltammograms obtamed with bare and Naflon-covered GCEs for zmc and zmc-albumm solutions (1) zmc, 2 X lo-’ M and Nf-GCE, (2) same as (1) plus 10 ppm of albumm, (3) zmc, 2X lo-’ M and bare GCE, (4) same as (3) plus 10 ppm of albumm

TABLE 2 Shifts of E;* m the pseudo-polarograms with albumm concentration a

for zmc (2 ng ml- ‘)

E& (mV)

[Albumm] (1O-3 M)

Lo~albumm]

-1159 - 1181 - 1191 - 1211

0005 0 010 0 050

-530 -500 -430

a Optimized instrument parameters of DP-ASV AE, = 60 mV, t, = 1000 ms, fp = 40 ms, AE = 5 mV, scan rate = 4 70 mV s-l

spectmely) owmg to a decrease m lablle zmc concentration A shght shift m peak potential for zmc voltammograms compared with bare and Naflon-covered electrodes 1s obtained (voltammograms 1 and 3), as a result of the finite rewstance of the Naflon layer on the electrode A severe decrease m the zmc peak m the presence of albumin 1s obtained with the bare MFE (voltammogram 4) owmg to the adsorbed surfactant (protein) on the glassy carbon surface DP-ASV was chosen as the best voltammetrlc technique for the pseudo-polarographlc study owmg to its higher sensltlvlty and good reproduabdlty for the strrppmg analysis of zmc m comparison with linear-sweep ASV (LS-ASV) and squarewave ASV (SQW-ASV> SQW-ASV 1s also more prone to lrreverslblhty m solutions with surfaceactwe agents such as proteins, owing to its high frequency Table 2 lists the shift of E& for zmc as a function of the albumm concentration m the pseudo-polarograms obtamed Presumably the Nf layer should permit the determination of Zn(I1) wlthout interference from ALB, and should not modify the thermodynamlc/kmetlc voltammetrlc evaluation of the bmdmg of the complex In Fig 4, typlcal pseudo-polarograms are shown for Zn(II> solutions alone and with 3350 mg 1-l of ALB From the experimental results, the leastsquares fit gave a condltlonal stablhty constant of log p’ = 6 10 f 0 16 for the Zn-ALB complex at pH740 There 1s a dlverslty of values m the literature when comparmg the bmdmg constants for the Zn-ALB complex, depending on the technique,

BINDING

-1350

CAPACITY

OF ALBUMIN

-1250 Deposhn

135

TO ZINC

-1150 potentml

mV vs

-1050 SCE

Fig 4 Pseudo-polarograms for (1) Zn(II) (2 ng ml-‘) and (2) Zn(II)-ALB (2 ng ml-’ and 3350 mg ml-‘, respectively) solutions obtamed wth DP-ASV and an Nf-MFE In all instances Te= 4 mm

type of albumin and chemical conditions Guthans and Morgan [19] reported an apparent dlssoaatlon constant (K,) for Zn2+, N12+ and Cd2+ with human serum albumin (HSA) m the range of 10 FM (log B’ = 5 0, I e , HSA bmds 2-3 mol of these ions per mol of protein), the relative affmlties bemg Zn2+> N12+> Cd2+, by equlhbrmm dialysis and mununoadsorbent chromatographlc techniques Smgh et al [201 reported an apparent average assoaatlon constant (K,,) for Zn with BSA at pH 6 6 of log K,, = 3 45, by eqmhbrmm dialysis (Scatchard plots) Wu et al 1211obtained a dlssoclatlon constant for the Zn-ALB complex (from cord serum) of (l-3) X low5 M by Sephadex G-50 gel-filtration chromatography However, Glroux [22] gave log K, = 7 0 for the Zn-HSA complex by gel chromatography at pH 7 4 Fmally, Magneson [231 obtained a bmdmg constant of log p = 7 5 at pH 7 45 with Zn-horse albumin complex by titrating a protein solution with the metal and by atomic absorption spectrometry Measurement of the bmdmg capacrty of albumin for zmc by the RUZK treatment with HMD and Najion-covered MF electrodes

DP-ASV was also used with an HMDE and NEMFE to calculate the conditIona stab&y con-

stant of the Zn-ALB complex by titrating an ALB solution with Z&I), followmg the theoretlcal Ruzlc data treatment [12] For this purpose, Increasing amounts of zmc(I1) were added to a solution containing a fared concentration of ALB, and the free (labile) zinc concentrations were experimentally calculated by mterpolatlon on a cahbratlon graph of Zn(I1) (without protem) dlssolved m the same buffer (supportmg electrolyte 0 05 M phosphate buffer, pH 7 02) and with the HMDE The standard addition method was also used for calculatmg the Zn(II) concentration Using the RUZICdata treatment m the titration of zmc with albumin (which 1s based on a 1 1 complex), the followmg relationship applies [Zn],/[Zn]t

- [Znlf = l/j?’

CL + [Zn],/CL

where [Zn], 1s the total concentration of zmc m solution, [Zn], 1s the concentration of the free (labile) zmc, p’ 1s the condltlonal stability constant of the Zn-ALB complex and CL IS the complexatlon capacity of albumin With large multi-functional molecules such as proteins, CL represents the concentration of mdlvldual functional groups acting independently as hgands In all instances, solutions of Zn with ALB were allowed to equrhbrate for 24 h before the voltammetrlc determmatlon of [Zn], The mmlmum electrolysis potentials for Zn (E, = - 1225 mV with HMDE and E, = - 1200 mV with NfMFE) were used so as not to increase the lablhty of the Zn-ALB complex by kmetlcs effects m the subsequent determmatlon of [Zn], For companson, titrations of albumin with zmc were made with two working electrodes, HMDE and Nf-MFE Mercury electrodes are generally more disturbed than polymer-coated electrodes by adsorption of proteins and surfactants and so the results are expected to be different [13] The optlmlzed instrumental parameters for free zmc determmatlon by DP-ASV were AE, = 40 mV, t, = 500 ms, t, = 30 ms, AE = 5 mV and scan rate = 9 40 mV se1 with the HMDE and AE, = 60 mV, t, = 1000 ms, t, = 40 ms, AE = 5 mV and scan rate = 4 70 mV s-l with the Nf-MFE The experimental results for an HMDE are plotted m Fig 5 (ratio of free to bound vs free zinc), obtained by titrating a 0 120 g 1-l ALB

JC

136 25 r

lot----05

15

25

35

m, . M Fig 5 Plot of the ratio of free zmc to bound vs free zmc (Ruzlk plot) obtamed from the experimental results m expenments wth 0 120 g 1-l of an ALB solution by DP-ASV and an HMDE (regression coefficient R = 0 9987)

solution with concentrations of Zn(lI> from 15 x lop6 to 5 5 x 10m6 M From the results of three independent tltratlons, the calculated values were CL = 3 51 X 10S6 mol 1-l (1 e , 2 92 X 10m3 mol ofZnperlOOgofALB)andlog jY=538fO23 The number of bmdmg sites (n) was also evaluated assuming for ALB an average relative molecular mass of 67 000 [n = CL (moles of Zn per 100 g of ALB) x 67 OOO/lOO) In this case n=196 The Ruzlc plot of a titration of 0 060 g 1-l ALB with Z&I) (from 2 x lop7 to 1 x 10e6 M) and with the Nf-MFE gave the followmg results (mean of three titrations) CL = 3 80 X 10m7 mol I-’ (le, 633 x 10m4 mol of Zn per 100 g of protern), IZ= 0 42 and log /3’ = 5 61 f 0 26 The correspondmg Ruzlc plot 1s shown m Fig 6 The small difference m #3’ between the HMDE (log /Y = 5 38 + 0 23) and Nf-MFE (log p’ = 5 61 f 0 26) obtained by the Ruzlc procedure could be attributed to a shght change m kmetlcs, I e , the increased degree of dlssoclatlon (lablhty) of the Zn-ALB complex at the HMDE than with Nf-MFE, as a result of the smaller thickness of the diffusion layer (6) around the MFE compared with 6 for the HMDE [5,13] Nevertheless, It can be seen that the mean of p’ 1s smular for the HMDE and Nf-MFE electrodes, and conslstent with that obtamed m pseudo-polarography

VIDALETAL

As indicated, the Naflon layer would hmlt the diffusion rate for Z&I), which wfi be decreased An apparent diffusion coefflclent U&J can be defined for Zn(I1) m the polymer layer (composed of contrlbutlons from actual diffusion of the electroactlve metal and electron transfer [24]), and this DaP,, 1s usually smaller than D m a solution layer However, the Nafron coatmg is considerably thmner than the dlffuslon layer, and usually the recast Naflon IS not shown to hinder appreciably the diffusion of metals to the electrode surface [ll] Then permeation of a solution of electroactlve species mto a polymer-modified electrode IS not a lunltmg factor, and a Cottrelban convectme-dlffunon behavlour 1s still obtamed as a conclusion from Levlch plots The dlffuslonal lumtatlons wlthm the coatmg will also affect equally the solutions of Zn(I1) alone and Zn(I1) with albumin m the voltammetric measurement of free zinc The results of titrations are not expected to change by makmg these assumptions, and p’ ~111agree with the two electrodes From the results, it can be seen that log j3’ 1s similar with the HMDE and Nf-MFE m spite of the differences m CL and n This could be due to the different range of total zmc concentrations used with the two electrodes because of Its sensltlvlty The relative ratio of [Zn], to [Znlboundvaries with the two ranges of [Zn], and hence the slope of the hnear graph of [Zn],/[Zn],,,,, vs [Znlf so

r

t

751 45

0

’

55

’

’

65

8

EnI, M

’

75

t

’

65

’

’

(x ,","7,

Fig 6 RUZICplot for a tltratlon of a 0 060 g 1-l albumin solution wth zmc by DP-ASV wth a Naflon-covered MFE (regression coefficient R = 0 9902)

BINDING CAPACIIY OF ALBUMIN TO ZINC

(see Figs 5 and 6) However, although l/CL (slope of the fitted curve) 1s dtierent, the mtercept l//3’ CL gives slmdar p’ values From the experimental results, a different complexatlon (metal-bmdmg) capacity with [Znl, and [ALBI concentrations range could be suggested (see Ag 2 in [12]) Some workers indicate that bmdmg sites of BSA typically vary from strong to weak as the metal concentration increases m tttrations of a metal (copper) with this protein, by using the Schuman procedure [25] Then, when [Cu], < [ALB], the tltratlon can only represent the stronger bmdmg sites, not the average for all hgand sites on the macromolecule By the Ruzlc procedure, however, p’ 1s determined by using the whole tltratlon curve, leading to more accurate results than m the &human method BSA has also been reported to have one very strong bmdmg site and at least eleven other weak bmdmg sites, some of which act more like lon-exchange sites than bmdmg sites [25]

Conclusions

DP-ASV IS a highly sensitive voltammetrlc techmque that has been shown to be sultable for speclatlon studres and the determination of zmc m the presence of albumm by usmg an HMDE and a Nf-MFE The advantages of the electrode chemically modlfled with Naflon are its resistance to adsorptlon of albumin and the good mechamcal stablhty of the preformed mercury film The electrode IS prepared simply and qmckly and the voltammetrlc determmatlon could be applied m a simple way to the speclatlon of other metals m solution without interference from organic matter and/or surfactants The use of an HMDE and Naflon-covered MFE shows consistent results m the experlmental evaluation of the condltlonal stability constant (p’) of the Zn-ALB complex followmg the Ruzlc procedure, and assummg it IS a 1 1 complex In both mstances, log p’ 1s close to 5 45 at pH 7 02 m a phosphate buffer solution This value 1s shghtly higher with the Nf-MFE than the HMDE, probably as a result of a slight change m kmetlcs, 1 e , the effectwe measurement time IS slightly

137

different as a result of a different dlffuslon layer thickness m the two electrodes and therefore a different residence time of the complex molecule m this reaction layer around the electrode IS obtained The Naflon layer appears not to change this assumption (complex and free ALB cannot dlffuse through It) and then only kmetlc parameters of the metal complex dlssoclatlon and diffusion layer, both m solution, govern this ASV process Nevertheless, because of the stlmng of the solution, the residence time of the complex m this layer 1s very short and hence the fraction of labile metal 1s small As the diffusion coefflclents of ALB and the Zn-ALB complex are small (less than 10e7 cm* s-l), it IS also presumable that they would not contrlbute appreciably to the peak currents A slmllarly calculated /3’ was obtained with the very different method of pseudo-polarography (log p’ = 6 10 f 0 161, camed out wth DPASV and an Nf-MFE In this instance, the shift 1s of El*/2 m the so-called pseudo-polarogram only a measurement that reflects the overall actlvatlon energy of the electrode reaction, and depends on (can be related to) the hgand (albumm) concentration and the rate of the reduction with different E, of labile (free) zmc m the deposition step m ASV The preferred method to obtain a condItiona stab&y constant, from comparison of the three methods studled (based on rehabUy and speed), is the tltratlon of the protein solution with metal by the Ruzlc method, and by usmg an Nf-MFE to overcome adsorptlon problems The methods could be applicable to obtammg stability constants for other metals (such as Cu, Cd and Pb) with proteins such as casem and albumm It 1s necessary to ensure m these cases that the direct reduction of the complex does not contribute to the strlppmg currents at the potentials used (e g , this can be confirmed by the shape of the pseudo-polarogram, with a steady-state current instead of exponential growth with mcreasmg electrodeposltlon potentials), which would produce erroneous results It must be remembered than speclatlon studies by strlppmg voltammetry (and other analytical techmques) must always be

.JC VIDAL

138

operationally with care

defined and the results interpreted

This work was financed by project PB 88/0385 of the DGICYT (Spanish Educatmn and Science Department)

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