Cathodic stripping voltammetry of adenine nucleotides, polyriboadenylic acid and nicotinamide adenine dinucleotide coenzymes at the dropping mercury electrode

279

Bioelectrochemistry nnd Bioenergefics, 12 (1984) 219-293 A section of J. Electrounal. Chem., and constituting Vol. 173 (1984) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

682-CATHODIC STRIPPING VOLTAMMETRY OF ADENINE NUCLEOTIDES, POLYRIBOADENYLIC ACID AND NICOTINAMIDE ADENINE DINUCLEOTIDE COENZYMES AT THE DROPPING MERCURY ELECTRODE

H. KLUKANOVA

l, M.

STUDNICKOVA

l

* and J. KOVAR

l

Faculty of Science, J. E. Purkyne University, 61 I 37 Bmo, Kotkiiskb (Revised

manuscript

received

2 (Czechoslovakia)

May 15th 1984)

SUMMARY

The inverse voltammetry of twelve adenine nucleotides, poly r-A and seven nicotinamide adenine dinucleotide coenzymes was performed in 0.1 M sodium phosphate buffer (pH 7.0) at the d.m.e. using high scan rates (lo-100 V/s). The stripped phase formed at +0.15 V versus s.c.e. consists of insoluble mercurous salts (SLAMP, ADP, ATP, d-AMP) with 1: 1 nucleotide-Hg(1) stoichiometry or of mercuric complexes with 1: 2 (adenosine, ADPR) or 1: 1 (c-AMP) composition. Z-AMP probably forms a thick polymeric layer. Poly r-A forms a surface salt with 1: 1 compostiion in Hg(I) to the AMP monomer. Resolution of the adenine nucleotides in the mixture was not possible because of mixed phase formation. Nicotinamide adenine dinucleotide coenzymes form insoluble mercuric salts with 1: 2 composition. The influence of adenine ring substitution on the electroactivity in the inverse voltammetry was studied in the case of S-AMP and of NAD+.

LIST OF ABBREVIATIONS

C.&V.

h.p.1.c. c-AMP d-AMP Ade Ado ADPR NADH NAD+ NADP+ NADPH NMN poly r-A

l l

cathodic stripping voltammetry high-performance liquid chromatography 3’,5’-AMP deoxyadenosine monophosphate adenine adenosine adenosine diphosphoribose reduced form of nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate reduced form of nicotinamide adenine dinucleotide nicotinamide mononucleotide polyriboadenylic acid

Department of Biochemistry. * Department of Physical Chemistry.

0302-4598/84/$03&O

0 1984 Elsevier Sequoia

S.A.

phosphate

280 INTRODUCTION

The advantage of cathodic stripping voltammetry (c.s.v.) in the study and analysis of pyrimidine [1,2] and purine [3,4] bases has been pointed out by Palecek and co-workers. Studying the structural influence upon the electrochemical behaviour, they suggested that the mechanism involves the reaction of the studied substances with the surface of the mercury anode, resulting in the formation of organomercuric compounds. Florence [5] tested many organic substances, e.q. adenosine, ATP and NAD, searching for their C.S.V. response under invariable experimental conditions. The substances mentioned were found [5] to be inactive in the C.S.V. regime. The analysis of the influence of the phosphate groups on the electrochemical activity of adenine nucleotides and coenzymes and their responses in the inverse voltammetry on the d.m.e. in neutral phosphate medium is the main aim of this paper. The theory of inverse voltammetry by Brainina [6] is applied to the case of C.S.V. on the d.m.e. to give the mechanism of the phase formation and its stripping for the substances studied. The theory of electrosorption worked out by Conway and Angerstein-Kozlowska [7] has been used to obtain an estimate of the equilibrium electrosorption constants characterizing the nucleotide 1 : 1 salts with mercurous and mercuric surface cations. EXPERIMENTAL

Materials The compounds studied were purchased from the companies quoted in parentheses: NAD+, NADH (Boehringer), NADP+ (Merck), NADPH (Calbiochem), ATP (Reanal), 5’-AMP (Sigma), ADP (Calbiochem), 2’-AMP (Serva), 3’-(2-)AMP (Calbiochem), c-AMP (Fluka), d-AMP (California Corp. for Biochem. Res.), NMN (Boehringer), ADPR (Sigma), poly r-A (Sigma). NAD+ derivatives substituted at the adenine ring, i.e. N(l)-carboxymethyl-NAD+ (IV), N(6)-carboxymethyl-NAD+ (V) and N(6)-(6-aminohexyl)carbamoylmethyl-NAD+ (VI), were synthesized as described in Ref. 8. Similar derivatives of AMP, i.e. N(l)-carboxymethyl-5’-AMP (I), N(6)-carboxymethyl-5’-AMP (II) and N(6)-(6-aminohexyl)-carbamoylmethyl-5’AMP (III), were synthesized analogously [S]. All the substances showed one peak in high-performance liquid chromatography (h.p.1.c.) except for the 2’-(3’-) AMP mixture and ADP (which contained a trace of .AMP). The components of the buffering solutions were of analytical-reagent grade (Lachema). C.S.v. measurements Cyclic voltammetry on the d.m.e. was performed using a PRG 4 instrument (Tacussel, France) connected to a Tektronix 5103 N storage oscilloscope. The C.S.V. curves were photographed. The analysed solutions were deaerated by a stream of nitrogen. A potentiostated cell was used with the s.c.e. as the reference and the

281

mercury bottom as the auxiliary electrode. The pre-polarization potential UP, equalled +0.15 V, the scan rate was lo-100 V/s. Three capillaries were used with flow rates of 1.3, 0.91 and 0.88 mg/s and with natural drop times of 5.2, 9.2 and 6.0 s in the open circuit with h = 60 cm into 0.1 M Na-phosphate buffer. The measured currents were converted into current density for comparison of the results. h.p. 1.c. anafysis

The liquid chromatograph consisted of an MC 100 pump (Mikrotechna, Czechoslovakia), a home-made stainless steel column (150 x 4 mm) packed with Separon Si C,, (5 pm) (Laboratory Instruments, Czechoslovakia) and a UV 254 detector (CSAV Workshops, Czechoslovakia). The mobile phase was 25 m M sodium phosphate buffer, pH 6.3, or the same buffer containing 10 % methanol (V/V). DATA

EVALUATION

Inverse voltammetry

of solid phases at the d.m.e.

This kind of voltammetry has been described (summarizing the previous work) by Brainina [6] for several types of electrode processes. The case of formation of an insoluble compound on the surface of an electroactive electrode can be applied to the systems studied in this paper. The formation of the surface layer proceeds in two steps, i.e. the ionization of the metal and the follow-up reaction of the surface cations with a suitable substance (A): Me+Me”++ne-

Me”+ + n A- + MeA,

The reacting substance ought to be a nucleophilic comprises the reduction: MeA,+ne-+Me+nA-

(1) reagent.

The dissolution

reaction

(2)

its product being free metal, and the diffusion of A- being the rate-determining step. The responses of the reaction of the electrode metal (Hg) with anions and bases can also be found in direct voltarnrnetry by the positive scan of potential (cf. Ref. 4). However, the sensitivity in this regime is much lower than that in the stripping mode with the electrode covered with the reaction product. The summarizing reaction (1) can be considered to be at equilibrium only at low scan rates in direct voltammetry. According to the thickness of the layer of MeA,, two theoretical cases can be met

[61:

(i) a thin layer whose dissolution is controlled by the diffusion of A- from the electrode; (ii) a thick layer of lower conductance; the reduced material must be transported inside the layer by a chemical reaction which is the step controlling the dissolution rate. During the pre-polarization time, a new phase is formed on the electrode surface,

282

its amount being proportional to the positive charge consumed. Owing to this proportionality, a limited part of the new phase is represented by the Hg(I) or Hg(II) complexes or precipitates with the studied nucleotides. The prevailing part of the phase consists of the salts with the components of the supporting electrolyte. Under these conditions (i.e. rather short delay times (< 2 s) and not too high concentrations), the peak current of the stripping peak is proportional to the concentration of the studied substance and the negative charge consumed for the stripping of the phase is divided into two parts, similar to the positive charge forming the phase. A minimal necessary concentration of the organic substance is needed in order to overcome the solubility product. Tis circumstance can enhance the detection limit in the analysis. The theory developed for a solid planar electrode [6] can be matched for the measurements on the d.m.e. by setting 0.85 m 2/3 ti’” for the electrode area (m is the flow rate, t, is the delay time) and 1.16 fi for the diffusion layer thickness. The charge consumed for the phase production is in relation to the growing drop anode. For the stripping made by a fast voltage pulse (cu 1 ms), the anode can be considered to be of constant area. The resulting equation for the peak current I, can be written in the following form for case (i): IP = 0.73n2.F2( RT)-‘cllm2’3t~/6D”2cu where n is the number of electrons exchanged; R, T, Sand D have their usual meanings; (Y is the charge transfer coefficient for the reduction process; c is the concentration of the studied substance in solution and u is the scan rate in V/s. The peak potential UP is shifted negatively by O.O58/arn V when the scan rate is increased by one order of magnitude. The peak width at the I,/2 height is approximately 0.108/w V. The slope of the log I, uersus log u dependence (X) has a value of X = 1 and the slope (p) of log 1, versus log t, is equal to 1.2 in this case ( IP is the peak current). The exact equation for IP has a much more complex form for case (ii) than that in the previous case [6]. It contains the activity of the stripped phase, the equilibrium and the forward rate constants of the transport reaction moving the reduced M centres away, and the reaction layer thickness as process parameters. If we assume that the transport reaction does not proceed during the deposition process, the peak height may be expressed as ZP = const.u’/2t~‘/6c The rate coefficient X is equal to 0.5 and the value of p is equal to 1.8 in this case. 17, is shifted negatively by half of the value described for the case (i) with increasing scan rate. Analysis of the surface complexes The reaction of the mercury anode with complexing substances to form Hg(II)A, was studied by means of d.c. polarography [9,10]. The anodic wave observed in the

283

presence of the ligand A and the cathodic wave corresponding to the reduction of the Hg(II)A, complex present in solution were found to have identical half-wave potentials although the slopes of the two waves might be different. The shift of the half-wave potential of the anodic wave with the concentration of the ligand [9] obeys the equation

u,,2=

U’-+$.ln[A]“-’

where U’ is the concentration-independent part of the potential containing the standard potential U o (Hg(I1) 1Hg) and the logarithm of the ratio of the diffusion coefficients of A and HgA, multiplied by the stability constant of HgA,. The half-wave potential of the anodic wave is shifted negatively by 29 mV per one decadic order of magnitude of the increasing concentration in the case of Hg(II)A, complexes and does not depend on the ligand concentration in the case of Hg(II)A complexes. The concentration dependence of the half-wave potential of the anodic wave arising in the presence of anions forming mercurous precipitates, Hg(I)A, is expressed as follows: U,,z = U” -s

ln[ A]

where U” is again the constant part of the half-wave potential including the logarithm of the solubility product of Hg(I)A. The expressions for U,,2 p resented above also apply to the cathodic peak potential in the C.S.V.regime provided that the concentration of Hg(II)A, in reaction (2) obeys the Nernst equation. This means that the electroreduction of the surface complex is faster than the diffusion of the liberated A- anions and the X and p values fit those for case (i) described above. The found [9,10] stoichiometry of the mercurous and mercuric compounds formed at the electrode does not usually differ from that of the reaction of Hg(1) or Hg(I1) with ligands in solution. Surface complexes with 1: 1 composition can be characterized by their electrosorption constants using the theory of Conway and Angerstein-Kozlowska [7]. MtA-

“=^

MA+ne(3)

1 - 0, CA

@*(at 4)

The electrosorption process (3) leading to a two-dimensional monolayer of A chemisorbed on sites M of a metal electrode at the potential U, has, in the case of Langmuirian adsorption, the equilibrium isotherm n.FU, 64 = KAcA exp 7 i - eA where 0, is the fractional coverage of the electrodeposited A species, cA the concentration of reactant A, U, the electrode potential and K, the electrochemical

284

equilibrium adsorption constant scaled with respect to the reference potential for U,. The voltammetric current Z, divided by the scan rate was designated pseudo-capacitance and for KacA exp(n.%W,/RT) < 1 (i.e. 0, < 1) it is given by the equation

IA -= V

QAnF

n.FlJ,

w y

yKAcA

where QA is the charge for complete monolayer formation and the other symbols have their usual meanings. Using this theory, we suppose that the peak height (IA) of the C.S.V.peak is the measure of the extent of electrosorption of substance A at the positive pre-polarization potential Upp and the (U,, - U,) difference is U,, the measure of the Lewis basicity of the studied substance. The electrosorption behaviour of two substances, A and B, can be compared using equation (4). For the one-electron process with identical concentrations of both substances at room temperature, the following equation results

4o -0584

QEIKEI

+ log &a = log A

QAKA

The value of the denominator

in the first term is 0.029 for the two-electron

(5) process.

RESULTS AND DISCUSSION

Properties of the mercury anode in the presence of the substances under study in the sodium phosphate buffer medium The positively polarized mercury electrode reacts with many kinds of anions (see [lo] for references). This reaction, which shows capacitive responses, proceeds in one or more steps at more negative potentials than the potential of the faradaic dissolution of mercury. The electrochemical study of the anode in phosphate buffers [11,12] confirmed that the electrode was covered with the insoluble mercurous phosphate Hg,HPO,. The presence of hydroxide anions causes surface oxide formation and enhancement of the dissolution of mercury by forming the more soluble mercuric hydroxide [13], as was shown for alkaline solutions of fluoride [14] and sulfide [15]. The state of the mercury anode is pH-dependent, its dissolution being fast both in an acidic [ll] and in an alkaline medium [13]. At a I!&, equal to +0.15 V in the 0.1 M Na-phosphate buffer (pH 7) (Fig. l), mixed layers of Hg,HPO, and HgO are apparently formed, which are reduced on scanning the potential negatively. At U,, = +0.2 V, the reduction current of the surface at 0 V is about three times higher than that at U,, = +0.15 V. In the presence of inorganic diphosphate or glucose-6-phosphate, the reduction current at 0 V (with Upp = + 0.15 V) is enhanced. The presence of 5’-AMP and other adenine derivatives causes the lowering of this reduction current and the formation of a peak on the descending part of the current-voltage curve (Figs. 1 and 2, Table 1). This behaviour can be explained by the complex formation between the surface cations and the electron-donating centres of the nucleotide. The dependence of the peak

285

height of 5’-AMP with U,, = +0.15 V on pH (Fig. 2) goes through a maximum at pH 7.8. At higher pH values, the enhanced dissolution of mercury (higher reduction current at 0 V) causes a decrease in the peak. Because of the change in the properties of the electrode surface, the more negative potential (U,, = + 0.1 V) must be chosen at pH > 7.8. The stripping peak of 5’-AMP is then shifted negatively and increases again with increasing pH (Fig. 2a). The influence of the oxide coverage of the

l-

Fig. 1. Voltammetric curves of 1 mM 5’-AMP in 0.1 M sodium phosphate buffer, pH 7 (curve 1) and of the buffer alone (curve 2) at the d.m.e. Flow rate 0.88 mg/s, drop time 3 s, delay time 2 s, pre-polarization potential I!& equal to +0.15 V versus s.c.e., scan rate 100 V/s.

Fig. 2. Influence of buffering conditions on the stripped phase formation at the d.m.e. (a) Dependence of the peak height Zp (open symbols) and of the peak potential U, (0) of the C.S.V. peak of 5’-AMP on pH. 0.5 mM AMP in 0.1 M Na-phosphate buffer, d.m.e. with flow rate 0.91 mg/s, drop time 3 s, delay time 2 s, pre-polarization at +0.15 V uerw.r s.c.e. (curves 1 and 3) or at +O.lO V (curve 2, A), scan rate 20 V/s. The pH region of the start of oxidation of the surface is indicated by broken lines. (b) Dependence of the peak height Ip (0) and of the peak potential Up (0) of the stripping peak of NADH on the concentration (c) of the phosphate buffer, pH 7.0. 1 mM NADH, other conditions as (a).

1

and constants

characterizing

the behaviour

of the studied

compounds

in the C.S.V. regime

0” 0” -

-0.19 -0.18 -0.42 -0.39 -0.20

NADPH NADP IV V VI

’ Decreasing concentration dependence. ’ Values obtained with 5’-AMP chosen as substance ’ ADPR chosen as substance A.

65 65 0 35-40 35 35 80 50-55 50-55

-0.17 -0.16 - 0.20 0.00 - 0.20 -0.16 -0.32 -0.14 - 0.16 - 0.25 - 0.25

ATP d-AMP c-AMP ‘2’-AMP ADPR Ado I III poly r-A NADH NAD

1 3 3 2 3 3 3 3 3

70 60

5’-AMP ADP

1 1

(mv)

Alogc

“up

-0.10 -0.15

Substance

Group No.

A.

_ _

-

_ _

W&W-l

(1.8 b, 2.1 c _

0.0 c

(1.3 b) 2.0 b

(-0.6’)

I

QAKA

PeK,

0.0 b 1.3 b

log

P-WW.1

Hg(II)L, [Hg(II)L,l

V-WY4

_

Hg(I)L Hg(I)L Hg(II)L [Hg(II)L, Hg(II)L, Hg(II)L,

WNW1

Hg(I)L

Complex composition

1.1 0.8 1.2 0.8 _

0.7 1.0 0.7 1.7 0.8 0.8 1.3 1.0 2.0 0.8 0.8

1.7 1.0 0.9 0.5 0.9 0.9 0.7 1.1 1.0 1.1 0.8 1.4 1.2 1.0 1.1

0.9 _

P

1.2

X

_ _

_ _ _

_ 123 109 87 127

74

(mV)

ALb Alogu

i

R R

_

i

-

i

i

i

ii

i

i

R

i

SPe

10e4 to 10W3 M substances in 0.1 h4 sodium phosphate buffer, pH 7.0; Upp equal to +O.lS V versus s.c.e. (the pre-polarization potential); (b is the peak potential for 1 mM substance, and the scan rate 20 V/s; the group number denotes the type of peak shape (cf. Fig. 3); AUp /A log c was measured with a scan rate of 20 V/s; log(Q,K,/Q,K,) v al ues were obtained using equation (5); X = d log I,/d log u( f 0.1) for 1 m M substance, delay time 2 s, drop time 3 s; p = d log 2,/d log td (kO.1). drop time 3 s, scan rate 20 V/s; AU,/A log u for 1 mM substance, drop time 3 s, delay time 2 s. Layer thickness: type (ii) thick, X = 0.5, p = 1.8; type (i) thin, X = 1, p = 1.2; layers showing the repulsive influence are denoted R; the suggested complex compositions for layers with X and p values deviating by more than + 0.1 from type (i) are given in parentheses.

Parameters

TABLE

287

mercury anode is probably also reflected in the peak height dependence of NADH on the buffer concentration (Fig. 2b). The mechanism of the interaction of NADH with the anode is essentially similar to that of 5’-AMP (see below). At low phosphate buffer concentrations (0.01-0.05 M), the peak is shifted negatively, its height being less than that at medium buffer concentrations. With a 0.2 A4 buffer concentration, the interaction of inorganic phosphate with the anode competes with that of NADH. Adenine nucleotides and poly r-A at the mercury anode The influence of the location of the phosphate group and of the substitution of the adenine ring is expressed in the U, and Z, values, in the X and p parameters, and in the log QBKB/QAKA values (Table 1). The values of the parameters characterizing the thickness of the adsorbed layer (X and p) indicate that in the series of compounds tested both thin and thick layers can be formed. The former case is typical of 5’-AMP and d-AMP; the latter type can be found in the case of 2’-AMP. The layers of the other adenine derivatives cannot be classified unequivocally as typical cases. Repulsive interactions can be expected between the molecule of oligo-anions in the adsorbed layer, which might enhance the dissolution rate (higher X values) and slow down the deposition of the layer (smaller p values). Layers showing these repulsive interactions were observed in the case of ATP. The estimate of the QK ratios relative to that for 5’-AMP for some of compounds studied are also given in Table 1. The description of the electrosorption system (3) resembles that of the homogeneous complex formation process. However, the adsorption equilibrium at the charged electrode surface involves: (1) higher concentrations, than those in solution, not only of the adsorbable Lewis bases (as was demonstrated by Bond and Hefter [la] for d.c. polarography of complexes) but also of the Lewis acids, i.e. surface cations; (2) an additional charge forced through the interface into the electrosorbed layer as a difference to the balanced interaction of charges during the formation of complexes in solution (obeying the law of conservation). Consequently, the electrosorption behaviour could not be simply and generally related to mercury compound formation in the solution. On the other hand, the electrosorption of anions forming sparingly soluble mercuric and mercurous salts (e.g. Hg,HPO, with the solubility product pS = 12.4 [17]) can be supposed to proceed through formation of these salts, as will be shown below. The correlation between the pS values of mercuric compounds of purine and pyrimidine bases and their activity in the C.S.V. regime was confirmed at the h.m.d.e. in alkaline medium

m* The substances studied can be divided into three groups according to the shapes and potentials of their stripping peaks (Fig. 3). The first group consists of 5’-AMP, ADP and ATP. The stripping peaks of these substances are situated on the depressed current of the Hg,HPO,-HgO mixed layer. ADP shows only a small indentation on the reduction current of this layer so that only the concentration dependence of this response can be evaluated. 2’-AMP with its pronounced stripping

288

peak is the only representative of the second group. Not so sharp, but well-defined stripping peaks are observed in the presence of nucleotides belonging to the third group. The U, values of the first group in Fig. 3 (5’-AMP, ADP, ATP) follow the order of the pK values [18] for the phosphate group (6.2 for AMP, 6.4 for ADP, 6.5 for ATP) so that interaction of the phosphate group with the surface cations can be supposed to prevail. The stripped layers of AMP and ADP have X and p parameters approaching case (i) whereas the layer of ATP shows the repulsive effects (Table 1). The peak potential shift for 5’-AMP (about 70 mV per decade) with the scan rate confirms formation of the mercurous complex of 1 : 1 composition (n = 1, (Y= 0.8, evaluated from the U, uersus u shift). The dependence of Zp on concentration (Fig. 4) has a similar shape for group 1 (the low Zp values are nearly equal for AMP, ADP and ATP). The saturation limit of Z, is attained at comparatively low concentrations. It can be supposed that the charge required for monolayer formation is almost equal for these substances so that the increments of QK products are due to the increase in K. The increase in the stability of complexation of Hg(1) by ADP, as compared to AMP, by about 1.3 orders of magnitude (Table 1) can be compared with the increase in stability [18] of soft complexes of Mg(I1) in solution (A log K = 1.4 for ADP-Mg(I1) as compared to AMP-Mg(I1)). Similar values of the stability increments support the suggestion that the phosphate group of the nucleotide is bound preferentially to the mercurous surface cation in analogy to the Mg(I1) complexes in the solution. The ill-defined shape of the ADP response (Fig. 3) cannot be explained with certainty 14 a --__

1

-0.2

0 -0.2 U(V)’

-\, 1

\\ a sI I Q

1 \ \ \ \

0

-\ \ Jr I:

0.15\ -.0.4

-0.2

0

U(V)

3

b -0.4 -0.2 _x._ +---YE+ ‘\

0

Ir

\

ct a

8 I

\

\ \

1

,-/ ‘aal

0' $2:

Q

a I3

Fig. 3. Typical shapes of the C.S.V. peaks of the substances studied. (a) Adenine nucleotides. 1 mM nucleotides in 0.1 M Na-phosphate buffer (pH 7.0), pre-polarization potential + 0.15 V, scan rate 20 V/s, delay time 2 s, drop time 3 s (flow rate 0.88 mg/s). (1) Full line: 5’-AMP; broken line: ADP; (2) 2’-AMP; (3) c-AMP. (b) Nicotinamide coenzymes. Conditions identical to those in (a). Full line: NADH (flow rate of the capillary equal to 1.3 mg/s); broken line: NADPH (flow rate of 0.91 mg/s).

289

because of the presence of AMP in the sample. The response of a mixture in the C.S.V. cannot be generally and simply related to the response of its components, as will be shown below. The value of A log QK for ATP is only a rough estimate due to the presence of repulsive interactions in the adsorbed layer. Adenine ring substitution on the N(1) site does not disturb the surface complex formation, as can be expected from the primary role of the phosphate group in binding of 5’-AMP (Table 1). It is interesting that substitution of the amino group in derivative II causes the disappearance of the studied response, which is recovered by replacing the amino group by a longer chain containing a basic group (III). One additional anionic charge in N(6)-carboxymethyl-5’-AMP might strengthen the repulsive interaction (as apparent from the X and p values of N(6)carbamoylmethyl-5’-AMP, i.e. III in Table 1) so that the adsorbed layer does not arise. It seems that the adenine ring is not as important for complex formation of the studied compounds belonging to the first group as it is for the other two groups (see below). Nevertheless, its presence in the molecule is a prerequisite for the occurrence of the stripping peak since the phosphorylated sugar alone does not give this signal. The binding of Hg(1) to the adenine ring in the adenosine complex in solution is characterized by a log K value of about 2.8 [19]. This additional stabilization of the nucleotide salt might ensure the advantageous competition of nucleotides with the phosphate buffer for the electrode surface. 2’-AMP (the only member of the second group with U, at 0.0 V-cf. Fig. 3) exhibits behaviour which differs from that of the other studied nucleotides in several

Fig. 4. Concentration dependence of the C.S.V.peak current density of adenine nucleotides. Scan rate 20 V/s, delay time 2 s, drop time 3 s. (1) ATP, (2) 5’-AMP, (3) C-AMP, (4) d-AMP, (5) ADPR, (6) 2’-AMP.

290

aspects. The concentration dependence (Fig. 4) in addition to the deep depression of the mercury anode response supports the assumption that the phosphate group together with the adjacent ring (3) nitrogen is involved in the complex formation in this case. The composition of the complex cannot be determined unambiguously because the parameters X and p correspond to the layer of the (ii) type. It is likely that the polymeric layer of Hg(I1) (2’-AMP), is formed. The high current observed in the presence of 2’-AMP can be explained assuming an increased interaction of this compound with the electrode surface due to its enhanced hydrophobicity. This explanation is supported by the observed behaviour of this compound in reversedphase h.p.1.c. (the retention of 2’-AMP is substantially greater than that of 5’-AMP). The formation of the more pronounced stripping peaks of the third group of the compounds studied is also connected with the complexation to divalent centres (cf. Fig. 3 and Table 1). The only exception is d-AMP, which forms the Hg(1) (d-AMP) salt. Although the pK value of the phosphate group of d-AMP is the same as that of 5’-AMP [17], the C.S.V. peak of d-AMP can be detected at more negative potential values than that of 5’-AMP. The peak current of d-AMP exceeds that for identical concentrations of 5’-AMP. The increase in current (A log I = 0.5) might be ascribed to the increase in hydrophobicity due to the loss of one hydroxyl group. Accordingly, the interaction of d-AMP with hydrophobic surfaces essentially surpasses that of 5’-AMP as indicated by a far higher retention of d-AMP in reversed-phase h.p.1.c. in comparison with that of 5’-AMP. The most important compound of the third group is adenosine, which apparently forms the Hg(I1) (Ado), complex as can be seen from the peak potential shift with concentration (Table 1). Two adenine rings of adenosine might be involved in the surface complex with Hg(I1) with their amino groups, as supposed in the case of adenine itself [4]. The binding of Hg(I1) to adenosine (to the N(1) and C(6)-NH, sites) in the solution was proved by Simpson [19] to be more stable than that of Hg(1). This additional stabilization contributes apparently to the preference of Hg(I1) centres in cooperation with the enhanced insolubility of Hg(I1) (Ade), (pS of about 13.2, Ref. 2) as compared with that of Hg,HPO, (pS of about 12.4, Ref. 17). ADPR behaves similarly to adenosine probably because of the screening of the pyrophosphate group with two ribose residues, the composition of the complex being Hg(II)(ADPR), (Table 1). c-AMP (the last low-molecular member of the third group) also forms a mercuric complex but of 1 : 1 composition, i.e. Hg(II)(c-AMP), as follows from the zero, U, shift with concentration (Table 1). This anomalous behaviour might be incidental to the specific character of the phosphate group of c-AMP. The concentration dependence of the C.S.V.peak height of c-AMP is shifted to greater values, indicating the highest surface salt solubility of the compounds studied (cf. Fig. 4). The specific electrochemical behaviour of the individual substances is not maintained in their mixture. Mixed phase formation diminishes the resolution ability of the C.S.V. method. For example, the mixture of 5’-AMP, ADP, d-AMP and c-AMP (each compound 0.4 mM) in 0.1 M phosphate buffer, pH 7.0, gives only one peak at U,, = +0.15 V with Up = -0.07 V (20 V/s). The mixture of 3’-AMP and 2’-AMP

291

gives the stripping peak at - 0.2 V with X = 1 and p = 0.9. The shift of 70 mV per one decadic order of magnitude of concentration (lop4 to lop3 M) indicates Hg(I)L composition of the surface complexes. The pronounced stripping peak of 2’-AMP is not shown by the mixture. This non-additivity of the signals of the individual components of the mixture is the main reason for the limited importance of the observed phenomena for analytical purposes. The found type of interaction of the adenine nucleotides (through the phosphate residues and the adenine ring) with a positively charged mercury electrode has also been described in the case of their adsorption on a positively charged silver electrode. Ervin et al. [20] measured the Raman spectra of adenosine, AMP, ATP and poly r-A adsorbed on a Ag electrode from their 2 mM solutions in 0.1 M KC1 with 1 mM Na,HPO,, pH 8.2. The adsorption was induced by the polarization of the electrode at - 0.1 V. The surface Raman frequencies of the studied substances resembled mostly the frequencies found in solutions. The new band at 245 cm-’ found with AMP and ATP was assigned to the Ag-phosphate bond. Adenosine produced substantially weaker Raman scattering than AMP. The adsorption of the nucleotides on the d.m.e. at the potential of zero charge as studied by a.~. polarography and differential capacitance measurements reflects another type of interaction in the adsorbed layer. The enhanced surface concentration and small charge result in strong mutual interactions of the adsorbed molecules whereas the interaction between the electrode and the adsorbed layer is very small. Detailed studies have been published giving the Frumkin adsorption parameters obtained by phase sensitive a.~. voltammetry [21] or single sweep voltammetry [22] for a series of protonated adenine nucleotides (AMP, 3’-AMP, c-AMP, ADP and ATP). The adenine moiety was found [21,22] to be responsible for the interaction in the adsorbed layer also in the case of deoxyadenosine nucleotides in alkaline medium [23]. The adsorption of deoxyadenosine, d-AMP, d-ADP and d-ATP was studied [24] by means of specular reflectivity measurements at a gold electrode polarized around - 0.1 V uersus Ag 1AgCl (potential of zero charge) in a medium of 0.1 M NaClO,. Deoxyadenosine showed the most pronounced adsorption effect, as compared to those of d-AMP, d-ADP and d-ATP. Hydrophilic phosphate groups were supposed to lower the adsorption ability. The composition of mercury complexes in solution was studied only with adenosine. Apart from the cited study by Simpson [19] dealing with CH,-Hg(I)(Ado) and Hg(II)(Ado) complexes, only one other paper has been found in the literature. Yamane and Davidson reported [25] that Hg(II)(Ado), is formed in an excess of adenosine while in an excess of Hg(I1) only Hg(II)(Ado) is formed. The latter complex composition was regarded as unexpected not only by these authors [25] but also by the reviewer Philips [26]. The presence of the synthetic polynucleotide, poly r-A, at the mercury anode in the C.S.V. regime is manifested by the appearance of one peak at about - 0.2 V. The shape of the signal belongs to the third group (cf. Fig. 3). The high value of p (= 2) might be due to the co-operativity in the adsorption of the polyanion segments at the positively charged surface. The interaction with the anode probably results in the

292

insoluble salt formation with 1 : 1 stoichiometry of Hg(I): monomer AMP (Table 1). The estimate of the electrosorption constant of poly r-A is similar to that of ADP and ATP. The stability constant [27] of the Mg(II)-poly r-A complex in the solution is equal to that of ATP (log K = 4.2, [18]). This coincidence suggests the similarity of the binding mode of poly r-A in both heterogeneous Hg(1) and homogeneous Mg(I1) complexes, i.e. the metal ion binding to phosphate residues with a slight contribution of the adenine rings. A similar binding mode of poly r-A to the positively charged silver electrode was suggested [20] from the surface Raman spectrum. Surface complexes

of nicotinamide

adenine dinucleotide coenzymes

All the studied coenzymes form surface compounds with mercuric cations, as can be seen from the shifts of their peak potentials with concentration (Table 1) and the shapes of the peaks (Fig. 3) which resemble that of 2’-AMP. The electrosorption constant of NADH is higher than that of ADPR although NMN+ alone does not show electroactivity in the C.S.V. regime. The pyrophosphate group does not apparently play a decisive role in the complexation (similarly as in ADPR binding) so that the metal binding proceeds predominantly through the adenine and nicotinamide rings. The peak potentials for the oxidized forms carrying one additional positive charge do not differ substantially from those of the reduced forms. The X and p parameters for the NADH layer approach case (i); the layers of NADP coenzymes show repulsion effects. The concentration dependence of the peak height for the coenzymes NAD+ and NADH is linear in the range of lop4 to 10e3 M. For NADP+ and NADPH, the peak height has a nearly constant value in the range of lop4 to 1O-3 M with a slight decrease at concentrations above lop3 M. The peak height is about three times lower than that of the NADH complex (Table 1). This behaviour may be due to greater mutual electrostatic repulsion of NADPH molecules in the surface layer. Adenine ring substitution leads to a negative U, shift in the presence of IV, V and VI (Table 1). The presence of the nicotinamide ring again shows the co-operativity in the complexation of the surface cations (cf. AMP substitution in I and III). At variance with II, the substitution of the amino group of adenine in V does not suppress its electroactivity in the C.S.V. regime. The adsorption of NADf on the mercury electrode has been studied by Elving et al. using the d.c. polarographic capacitive step and the competitive adsorption of Et4Nf [28]. The major role of adenine in the adsorption at -0.6 V resulted from comparison of the behaviour of NAD+ and NMN+, which was also reflected in our results. The orientation and conformation of NADH adsorbed on solid (carbon) electrodes in aqueous and non-aqueous media have been investigated and discussed by Elving et al. in many papers (see review article [29]). A folded conformation was suggested for the NADH molecule near the electrode surface with possible perpendicular and planar orientations of the adsorbed molecules. A folded conformation perpendicular to the electrode surface is also indicated by the observed increase in the electrosorption constants of NADH as compared to ADPR (cf. Table 1).

293

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