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The interfacial behaviour of guanosine mono-, di- and triphosphate in solutions of varying pH at charged interfaces
497
Bioelectrochemistry and Bioenergetics, 16 (1986) 497-W A section of J. Electroana/. Chem., and constituting Vol. 212 (1986) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
906 - THE INTERFACIAL BEHAVIOUR OF GUANOSINE MONO-, DIAND TRIPHOSPHATE IN SOLUTIONS OF VARYING pH AT CHARGED INTERFACES *
Y.M. TEMERK Chemistry (Revised
**, M.M. KAMAL,
Department, manuscript
M.E. AHMED
and Z.A. AHMED
Faculty of Science, Assiut University, Assiut (Egypt) received
April 2nd 1986)
SUMMARY A systematic study of the adsorption and interfacial behaviour of guanosine mono-, di- and triphosphate (.5’-GMP, 5’-GDP and 5’-GTP) at the h.m.d.e., has been carried out in different buffer solutions by phase-sensitive a.c. voltammetry. At low bulk concentrations, molecules of guanosine phosphate adsorbed at the maximum adsorption potential in a dilute adsorption layer are oriented planar to the electrode surface, where the interaction of w electrons with the interface favours adsorption. At bulk concentrations above a threshold value, the stacking interactions between vertically oriented molecules lead to association and formation of a compact layer. The adsorption can be described quantitatively for both types of adsorption layers by single- and doublestep Frumkin isotherms, respectively. The resulting adsorption parameters are evaluated and conclusions on the respective interfacial behaviour, orientations and interactions of these substances are discussed. The influence of the phosphate group on the surface reorientation of guanosine at the charged interface was also considered.
INTRODUCTION
The guanine nucleotides are substances of paramount biological and physiological significance in various aspects. In biochemical reactions in living systems, these guanine mononucleotides interact frequently as monomers or as units of the nucleic acids with charged biological interfaces. Therefore the use of electrochemical methods for the determination of the adsorption parameters has the advantage that the potential difference acting across the interface and thus the corresponding interfacial electric field becomes easily adjustable [l-4]. In this context, it has been shown that bases, nuckosides and nucleotides are adsorbed and undergo an
l Contribution presented at the VIIIth International Symposium its, Bologna, June 24th-29th 1985. l * To whom all correspondence should be addressed.
0302-4598/86/$03.50
0 1986 Elsevier Sequoia
S.A.
on Bioelectrochemistry
and Bioenerget-
498
association at the charged mercury-solution interface [5-201. Thus, the knowledge of the adsorption stages and interfacial orientation of the monomeric nucleoside or nucleotide units provides important information to help in disentangling the more complicated interfacial situation when these units are attached to the backbone of the strands of DNA. In continuation of our quantitative studies on the adsorption stages and association of guanosine [18] and methylated derivatives [19,20], it would be of interest to investigate the interfacial behaviour of guanosine mono-, di- and triphosphates with phase-sensitive a.~. voltammetry at the hanging mercury drop electrode (h.m.d.e.). The present paper is focused on the investigation of the interfacial behaviour and adsorption equilibria of guanosine phosphates (5’-GMP, 5’-DGP and 5’-GTP) in a wide range of bulk concentrations and at different pH values. The adsorption parameters were also computed at various pH values and the influence of the phosphate group on the interfacial behaviour and orientation of guanosine molecules is discussed. EXPERIMENTAL
Materials
and solutions
Guanosine mono-, di- and triphosphate were purchased from Serva, Heidelberg, F.R.G. Solutions of different concentrations of the investigated substances were prepared by dissolving a known amount of the chemically pure product in a definite volume of McIlvaine buffer [21]. The Mcllvaine buffer was brought to a constant ionic strength of 0.5 M by addition of KC1 and was adjusted to the desired pH. It also served as supporting electrolyte. All chemicals were reagent grade and KC1 was Merck “Suprapure”. Triply distilled water was used for preparing the solutions. The content of guanosine phosphate in the sample solution was determined with a Unicam SP 800 Spectrophotometer at 255-260 nm. pH was measured with digital Radiometer pH-meter, Model pH M 64. Apparatus
and methodr
A Princeton Applied Research (PAR) Model 174 polarographic analyzer interface and a PAR Model 5101 lock-in amplifier/phase detector were employed for a.~. voltammetric measurements. Phase-sensitive a.c. voltammograms were recorded with a phase angle adjusted to 90° corresponding to the out-of-phase component of the a.c. current (capacitive current component). The amplitude of the a.c. voltage was 10 mV,,, the scan rate of the d.c. ramp of the mean electrode potential was 2 mV s-l and the ax. frequency had a value of 330 Hz, unless stated otherwise. The working electrode was a Metrohm hanging mercury drop electrode (h.m.d.e.), type E 410, with a surface area of 1.75 x lop2 cm2. A saturated calomel electrode from Ingold, type 303-NS, served as reference electrode and the auxiliary electrode was a coiled platinum wire. The solution was deaerated by passing a slow stream of
499
nitrogen, and an inert atmosphere was maintained during the measurements passing nitrogen over the solution. All measurements were carried out at 5 o C.
by
RESULTS AND DISCUSSION
A survey of the adsorption behaviour of guanosine mono-, di- and triphosphate as a function of potential and pH is provided by the phase-sensitive a.c. voltammograms (Figs. l-4). The recorded capacitive a.c. component is proportional to the differential double-layer capacitance in potential regions where no faradaic process occurs [22]. In the pH range 3.4-8.0, the a.c. voltammograms of the respective mononucleotides indicate a progressive decrease in the capacitive current, with
(5':GMP),pH
3.4
1: 0.00 24.2 xVT6N 3:5.2 xlU% 4:6.1 x~O-~M 5:9.3 x lo-% 6:1.6x10-k
.' I'
Fig. 1. a.c. voltammetric curves of S-GMP at the h.m.d.e. 0.5 M McIlvaine buffer, 5”C, area of h.m.d.e. 1.75~10~’ cm’, scan rate 2 mV s-l, frequency 330 Hz, amplitude 10 rnVpp,phase angle 90°, adsorption time 180 s.
4 (5':COP),pH
3.4
l:ooo 2:1.9x10-5M 3:2.8xWM 4:4.7 xlcr'lv 5: 7.1 x10-% 6:2.1 x10%
-1.2
-1.0
-0.8
3
-0.6
-0.4
-0.2
Fig. 2. (I.c. voltammetric curves of 5’-GDP at the h.m.d.e., pH 3.4. Other conditions as in Fig. 1.
500
(5’:
GDP),pH
1: 0.00 2:1.9xlcr% 3:4.7x10-4"/ 4:7.6x16% 5:1.2~lO-~M
-1.2
-1.0
5.1
6:1.4nld5,'f 7:1.8x10-% 8:2.3xKI-='/'I 9:3.o~Iuk 10:5.6~1O-~N
-0.8
-0.6
-0.4
-0.2
Fig. 3. CI.C.voltammetric curves of 5’-GDP at the HMDE, pH 5.1. Other conditions as in Fig. 1.
increasing concentration, at a potential U,,,,,, of the supporting electrolyte. The decrease corresponds to a progressive coverage of the electrode surface by the dilute adsorption layer. The r-electron interaction with the electrode will favour a flat adsorption of guanine moiety in the range of positive or zero charge. At pK, -CpHs -CpK, [23] (Table 1) and above the threshold value, a sudden sharp decrease in the a.~. capacitive current is observed, giving rise to a very sharply defined pit. The pit reflects the formation of a compact adsorption film due to pronounced lateral interaction of the adsorbed species in a certain potential range. At potentials more negative than -0.9 V, the U.C. voltammograms exhibit a desorption peak corresponding to the desorption of loosely bound species existing in the film at more negative potentials. The destabilization of the compact adsorption film in highly acidic (pH x pK,) or alkaline solutions (pH > pK,) indicates that the degree of association of the adsorbed species of guanosine phosphate is connected with the adsorption of species which have a neutral guanine moiety, according to their pK values (Table 1). Therefore the compact stage of adsorption is formed as a result of the interaction of the charged electrode with the phosphate group and the permanent dipole of neutral guanine residue between N(3) and N(7), depending on their charge and the sterical arrangement of the molecule.
(5':GTP),pH 8
:o.oo
1 2:1.9x10-% 3:3.7x10-k
-3
4: 5.7x10-% 5:6.6xlb'H 6:1.4x10-M
Fig. 4. a.=. voltammetric curves of 5’-GTP at the HMDE. Other conditions as in Fig. 1.
501 TABLE 1 Adsorption parameters of the dgute layer of GUO, 5’-GMP, 5’-GDP and 5’-GTP at various pH values 10’ x Threshold bulk concentration (mall-‘)
lo-’ B (cm3 mol-‘)
- AC, (kJ mol-‘)
a
1.08 3.94 1.98 1.33
34.8 31.6 35.6 35.2
0.90 0.50 1.15 1.13
5’-GMP (pK, = 2.4, pK, - 9.4) 3.4 - 0.55 0.2 5.1 - 0.55 0.4 7.1 - 0.55 4.8 - 0.57 1.5 8.0
2.98 3.08 0.698 0.605
38.8 38.9 35.4 35.1
0.62 0.11 0.79 0.88
5’-GDP ($4 = 2.9, pK, - 9.6) 3.4 - 0.50 2.4 5.1 - 0.50 2.6 1.1 - 0.50 10.4 8.0 - 0.50 8.6
1.81 1.70 0.151 0.0921
37.6 37.5 31.9 30.6
0.63 0.66 0.86 0.81
5’-GTP (PK, = 3.3, pK, = 9.3) 3.4 -0.4 5.1 - 0.4 7.1 - 0.52 2.8 - 0.52 8.0 4.8
0.0532 0.0716 0.633 0.253
31.9 32.3 35.3 33.2
0.56 0.61 0.63 0.54
UH
Potential of maximum adsorption (v)
GUO (pK, - I.6, pK, = 9.2) 3.2 -0.55 -0.52 5.2 -0.52 7.1 8.0 -0.52
2.1 0.52 0.41 0.55
The U.C. voltammetric behaviour of guanosine phosphate is similar to some extent to that of guanosine. However, the adsorption range of guanosine phosphate extends to more positive and negative potentials. This should be interpreted as a significant contribution to the adsorption by negatively charged phosphate groups. As model considerations reveal, the latter, under the electrostatic attraction of the electrode, can be turned into a position where the phosphate group comes into direct contact with the surface. The peak at -0.34 V (Fig. 2) corresponds to a specific reorientation to a position where the base comes into strong interaction with the surface, while the adsorption of the hydrophilic phosphate group attains a solution-sided position. The occurrence of this reorientation peak is to be expected, because the net charge of the guanosine phosphate is approximately balanced. This behaviour is in good agreement with results obtained for the adsorption of adenosine and cytidine phosphate [10,11,17]. Additional information on the course of the adsorption and interfacial association of guanosine phosphate at different pH values is offered by the time dependence of the U.C. capacitive response, shown in Fig. 5. At low bulk concentrations, the capacitive U.C. current, measured at a constant mean electrode potential (U,,,),
502
I z
3 -I 4
(5':GMPLpH l:O.OO 2:2.3xl(r% 3:71110+~
--------_______
7.1
4: 1.6x10-% 5:2.1x10-% 6:2.8~lO-~N
7:3.7riO-% 8:4 S~lO-~bf 9: 5.41 g-y 10:7.5xlo-=M ----------_-__--__,
-y
_____
2-:; ///
EEs
5678 9 10 0
20
I J====
,C(min)
I 60
40
Fig. 5. Time dependence conditions as in Fig. 1.
80
of the out-of-phase
, loo
, 160
component
of the a.c. current of 5’-GMP at U,,,.
Other
at first slowly decreases to the first equilibrium value, corresponding to the dilute adsorption layer. For concentrations higher than the threshold value (Table l), where a pit is observed on the a.~. voltammograms, a drastic decrease to a second equilibrium value is observed, corresponding to the formation of a compact layer. Once a compact adsorption film has been built up, there is no substantial influence from the solution and thus the extent of the capacitance change accompanying the film formation depends only on the first stage and is determined mainly by the steric arrangement of adjacent molecules. The effect of potential on the degree of association is reflected by Fig. 6, which shows the potential dependence of the adsorption for the dilute and compact adsorption stages of Y-GDP at pH 5.1. At the maximum adsorption potential, the resulting concentration dependence of the U.C. capacitive current decrease AI,,,,
0
2
4
6
Fig. 6. Dependence of the capacitive various adsorption potentials.
current decrease AZ,,,, on the bulk concentration
of 5’-GDP
at
503
(the decrease of the capacitive a.~. current with respect to the I,,,, value of the supporting electrolyte for a given bulk concentration) has the form of a two-step isotherm. On the other hand, at potentials more positive or more negative than the maximum adsorption potential, the threshold concentration value for compact film formation increases appreciably or the two-step isotherm is ill-defined. Therefore, the choice of a proper potential as an electrical variable is significant in studying the degree of association of guanosine phosphate and in the evaluation of adsorption parameters. In this context, on comparing the threshold concentration value at different pHs at a maximum adsorption potential (Table l), it becomes evident that the association of guanosine phosphate, which is manifested at pK, > pHs > pK,, occurs at a lower concentration. This indicates that the degree of association of the adsorbed species of guanosine phosphate depends predominantly on the stacking of the molecules, which have a neutral guanine residue. From the threshold concentration values at one and the same pH for guanosine and its phosphate derivatives (Table 1, Figs. 7 and S), one can conclude that the interfacial association of guanosine takes place at a lower threshold concentration than for guanosine phosphate. The tendency for the interfacial association in acidic medium diminishes in the order guanosine > 5’-GMP > 5’-GDP > 5’-GTP due to the compensating influence of the negatively charged phosphate groups at the positive end of the dipole at N(3). Obviously the diminution of the positive charge on N(3) decreases the stabilization by base stacking, which causes the interfacial association of adjacent adsorbed molecules and consequently the formation of the compact layer. This trend is in agreement with the increasing value of the protona-
c a,b
b
Fig. 7. Dependence of the capacitive current decrease A I.,,, on the concentration at pH 5.1 at the maximum adsorption potential. Other conditions as in Fig. 1.
of
phosphate
504
b
0
1
2
3
4
5
6
’
a
Fig. 8. Dependence of the capacitive current decrease AI,,, on the concentration of guanosine phosphate at pH 8.0 at the maximum adsorption potential. Other conditions as in Fig. 1.
tion constant of N(7) (pK,, Table 1). In neutral and alkaline solution, the tendency of the interfacial association has the order guanosine > 5’-GTP > 5’-GMP > 5’-GDP. This trend is supported by the lower ionization constant of N(1) of 5’-GTP compared with the corresponding values for 5’-GMP and 5’-GDP. It also reflects the highly donating power of the negatively charged phosphate groups of 5’-GTP (pK{ -K 1, pK; < 6) in the direction of the dipole, which leads to an increase of the electron density on the oxygen at C(6) and encourages the proton transfer process between N(1) and the oxygen at C(6). In order to calculate the adsorption parameters for the dilute and compact adsorption layers at various pH values, the equilibrium values of the U.C. capacitive current at a given bulk concentration were measured and the degree of coverage B was calculated using the relation: e=
2-t
= (AT;Ejm
(1)
where the Cs are the differential capacities in the supporting electrolyte (Cc), at a given bulk concentration of guanosine phosphate (C), and at bulk concentrations corresponding to full coverage (C,). A I,,,, is the decrease of the capacitive U.C. current (with respect to the I,,,, value of the blank supporting electrolyte) for a given bulk concentration, and (AI,,,,),,, is the maximum decrease corresponding to full coverage. It has been found that the experimental data fit well a Frumkin adsorption isotherm given by the equation: Bc= &exp(-2aB)
(2)
where 8 is the degree of coverage, a the interaction coefficient and c the bulk concentration of the substance investigated. In addition, the free enthalpy of adsorption -AGO can be calculated from the adsorption coefficient B. The calculated values of the adsorption parameters of guanosine phosphate at various pH
505 TABLE 2 Adsorption parameters of the compact layer of GUO, 5’-GMP, values
5’-GDP
and 5’-GTP
1O-4 B (cm3 mol - ’ )
- AG, (kJ mol-‘)
(1
GUO 3.2 5.2 7.1 8.0
5.16 1.99 2.10 1.90
34.0 31.2 31.2 29.8
2.1 2.0 2.1 2.2
5’-GMP 3.4 5.1 7.1 8.0
2.36 2.80 0.732 0.776
32.9 33.3 30.1 30.3
1.8 1.8 1.9 1.6
5’-GDP 3.4 5.1 7.1 8.0
0.92 0.75 0.71 0.127
30.7 30.2 30.1 26.0
1.8 1.8 2.0 2.0
5’-GTP 3.4 5.1 7.1 8.0
-
-
0.39 0.211
29.3 27.2
PH
at various pH
values are given in Tables 1 and 2. Comparing the values of the adsorption parameters of guanosine phosphate at different pH values shows a significant dependence on the pH values either for the dilute or the compact layer. In acidic buffer solution, the magnitude of the adsorption coefficient B is higher than that of the neutral and alkaline solutions for guanosine mono- and diphosphate. The decrease of hydrophobicity of 5’-GMP and Y-GDP in neutral and alkaline media is due to the highly compensating effect of the completely ionized phosphate groups (pK; < 1, pK; < 6) on the dipole moment of the guanine moiety. However, the increase in B value of 5’-GTP in neutral and alkaline media may be ascribed to the appearance of the counteracting effect as a result of the proton transfer process, which may decrease to some extent the excess negative charge on the dipole of the guanine residue. The rather large standard free energy of adsorption - AGOin acidic medium shows that the moderately charged interface and large adsorption forces prevail. In this context the interaction coefficient a is rather small and indicates moderate lateral attractive interaction. For the compact adsorption stage, the magnitude of the adsorption coefficient B is significantly lower than that in the dilute stage. This significantly lower adsorptivity of the compact film correlates with its sensitivity to larger alterations of the
506
adsorption potential and its tendency to collapse suddenly at a certain value of the interfacial electric field at the positive and negative side of the region of U,,,. Nevertheless, the magnitude of Acindicates that the same unit of the molecule, i.e. guanine moiety, still remains predominantly responsible for the adsorption in the compact film stage at the potential of maximum adsorption. The value of a generally increases in the compact stage due to the enhanced possibilities for intermolecular attractive interactions, resulting from the perpendicular orientation and greater population of the adsorbed molecules in the compact stage. The value of the maximum surface concentration r,,, was calculated using the relation [24] r = l.l28c(
D#”
where c is the bulk concentration of guanosine or guanosine phosphate in mol cmW3, D the diffusion coefficient, t the time and I’ the surface concentration of the adsorbed compound. The diffusion coefficients have been evaluated using the Stokes-Einstein equation [25]. The value of the maximum surface concentration I’,,, for the compact layer was obtained from equation (3) by taking the time t, as the extrapolated time at which the linearized first portion of the time dependence of the a.~. current reaches the final horizontal part, i.e. the full coverage for the compact layer (See Fig. 5, curve 9). From the I?, for the compact layer and the ratio of the two steps of the isotherm obtained from U.C. voltammetric measurements, the surface concentration for the dilute layer has been evaluated as given previously [14,15]. The results reveal that the surface concentration .r,,, for guanosine is higher than for guanosine mono- and diphosphate (Table 3). Consequently, the average surface area per molecule A, increases in the same way. This behaviour is reflected by the smaller value of B for guanosine phosphate as compared with guanosine. Also, the interaction coefficient a is larger for guanosine than for guanosine phosphate. In general, and as will be seen from the afore-mentioned results, it is
TABLE 3 Maximum surface concentration phosphate at pH 5.1 Substance
Dilute stage Guanosine 5’-GMP 5’-GDP
r,
and surface area per molecule
A,,, for guanosine
10’0 r -2 (molcm#VI )
A,
2.21 2.03 1.71
0.71 0.83 0.91
5.23 4.99 4.63
0.31 0.34 0.36
(nm2)
compactstage Guanosine 5’-GMP 5’-GDP
and guanosine
507
remarkable that small steric differences in the adsorbed molecules yield a distinct change in the adsorption behaviour. This underlines the particular power of the U.C. voltammetric approach with phase-sensitive detection for the study of intermolecular forces which govern the formation of a compact film. REFERENCES 1 P. Valenta and H.W. Niimberg, Biophys. Stmct. Mech., 1 (1974) 17. 2 E. PaJecek, Collect. Czech. Chem. Commun., 39 (1974) 3449. 3 H.W. Ntimberg and P. Valenta, Proc. 29th Symp. Colston Res. Sot., Bristol, 1977, D.H. Everett and B. Vincent (Editors), Scientechnica, Bristol, 1978, p. 201. 4 J.M. Sequaris, P. Valenta, H.W. Ntmberg and B. Malfoy in ref. 2, p. 230. 5 V. Vetterl, Collect. Czech. Chem. Commun., 31 (1966) 2105. 6 V. Vetterl, J. Electroanal. Chem., 19 (1968) 169. 7 T.W. Webb, B. Jan& and P.J. Elving, J. Am. Chem. Sot., 95 (1973) 991. 8 J.W. Webb, B. Janik and P.J. Elving, J. Am. Chem. Sot., 95 (1973) 8495. 9 U. Retter, H. Jehring and V. Vetterl, J. Electroanal. Chem., 57 (1974) 391. 10 D. Krznaric, P. Valenta and H.W. Ntimberg, J. Electroanal. Chem., 65 (1975) 863. 11 P. Valenta, H.W. Niiberg and D. Ktznaric, Bioelectrochem. Bioenerg., 3 (1976) 418. 12 V. Vetted, Bioelectrochem. Bioenerg., 3 (1976) 338. 13 M.A. Jensen, T.E. Cummings and P.J. Elving, Bioelectrochem. Bioenerg., 4 (1977) 447. 14 Y.M. Temerk and P. Valenta, J. Electroanal. Chem., 93 (1978) 635. 15 Y.M. Temerk, P. Valenta and H.W. Nbmberg, J. Electroanal. Chem., 100 (1979) 77. 16 Y.M. Temerk, Can. J. Chem., 57 (1979) 1136. 17 Y.M. Temerk, P. VaJenta and H.W. Niimberg, Bioelectrochem. Bioenerg., 7 (1980) 705. 18 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 8 (1981) 671. 19 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 11 (1983) 457. 20 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 12 (1984) 205. 21 P.J. Elving, J.M. Markowitz and I. Rosenthal, Anal. Chem., 28 (1956) 1179. 22 H. Jehring, Electrosorptionsanalyse mit der Wechselstrompolarographie, Akademie Verlag, Berlin, 1974. 23 G.D. Fasman (Editor), Handbook of Biochemistry and Molecular Biology, Vol. 1, (3rd ed.), p. 169. 24 S.L. Phillips, J. Electroanal. Chem., 12 (1966) 294. 25 A. Einstein, Ann. Phys., 17 (1905) 549; 19 (1906) 371; Z. Elektrochem., 14 (1908) 235.
Bioelectrochemistry and Bioenergetics, 16 (1986) 497-W A section of J. Electroana/. Chem., and constituting Vol. 212 (1986) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
906 - THE INTERFACIAL BEHAVIOUR OF GUANOSINE MONO-, DIAND TRIPHOSPHATE IN SOLUTIONS OF VARYING pH AT CHARGED INTERFACES *
Y.M. TEMERK Chemistry (Revised
**, M.M. KAMAL,
Department, manuscript
M.E. AHMED
and Z.A. AHMED
Faculty of Science, Assiut University, Assiut (Egypt) received
April 2nd 1986)
SUMMARY A systematic study of the adsorption and interfacial behaviour of guanosine mono-, di- and triphosphate (.5’-GMP, 5’-GDP and 5’-GTP) at the h.m.d.e., has been carried out in different buffer solutions by phase-sensitive a.c. voltammetry. At low bulk concentrations, molecules of guanosine phosphate adsorbed at the maximum adsorption potential in a dilute adsorption layer are oriented planar to the electrode surface, where the interaction of w electrons with the interface favours adsorption. At bulk concentrations above a threshold value, the stacking interactions between vertically oriented molecules lead to association and formation of a compact layer. The adsorption can be described quantitatively for both types of adsorption layers by single- and doublestep Frumkin isotherms, respectively. The resulting adsorption parameters are evaluated and conclusions on the respective interfacial behaviour, orientations and interactions of these substances are discussed. The influence of the phosphate group on the surface reorientation of guanosine at the charged interface was also considered.
INTRODUCTION
The guanine nucleotides are substances of paramount biological and physiological significance in various aspects. In biochemical reactions in living systems, these guanine mononucleotides interact frequently as monomers or as units of the nucleic acids with charged biological interfaces. Therefore the use of electrochemical methods for the determination of the adsorption parameters has the advantage that the potential difference acting across the interface and thus the corresponding interfacial electric field becomes easily adjustable [l-4]. In this context, it has been shown that bases, nuckosides and nucleotides are adsorbed and undergo an
l Contribution presented at the VIIIth International Symposium its, Bologna, June 24th-29th 1985. l * To whom all correspondence should be addressed.
0302-4598/86/$03.50
0 1986 Elsevier Sequoia
S.A.
on Bioelectrochemistry
and Bioenerget-
498
association at the charged mercury-solution interface [5-201. Thus, the knowledge of the adsorption stages and interfacial orientation of the monomeric nucleoside or nucleotide units provides important information to help in disentangling the more complicated interfacial situation when these units are attached to the backbone of the strands of DNA. In continuation of our quantitative studies on the adsorption stages and association of guanosine [18] and methylated derivatives [19,20], it would be of interest to investigate the interfacial behaviour of guanosine mono-, di- and triphosphates with phase-sensitive a.~. voltammetry at the hanging mercury drop electrode (h.m.d.e.). The present paper is focused on the investigation of the interfacial behaviour and adsorption equilibria of guanosine phosphates (5’-GMP, 5’-DGP and 5’-GTP) in a wide range of bulk concentrations and at different pH values. The adsorption parameters were also computed at various pH values and the influence of the phosphate group on the interfacial behaviour and orientation of guanosine molecules is discussed. EXPERIMENTAL
Materials
and solutions
Guanosine mono-, di- and triphosphate were purchased from Serva, Heidelberg, F.R.G. Solutions of different concentrations of the investigated substances were prepared by dissolving a known amount of the chemically pure product in a definite volume of McIlvaine buffer [21]. The Mcllvaine buffer was brought to a constant ionic strength of 0.5 M by addition of KC1 and was adjusted to the desired pH. It also served as supporting electrolyte. All chemicals were reagent grade and KC1 was Merck “Suprapure”. Triply distilled water was used for preparing the solutions. The content of guanosine phosphate in the sample solution was determined with a Unicam SP 800 Spectrophotometer at 255-260 nm. pH was measured with digital Radiometer pH-meter, Model pH M 64. Apparatus
and methodr
A Princeton Applied Research (PAR) Model 174 polarographic analyzer interface and a PAR Model 5101 lock-in amplifier/phase detector were employed for a.~. voltammetric measurements. Phase-sensitive a.c. voltammograms were recorded with a phase angle adjusted to 90° corresponding to the out-of-phase component of the a.c. current (capacitive current component). The amplitude of the a.c. voltage was 10 mV,,, the scan rate of the d.c. ramp of the mean electrode potential was 2 mV s-l and the ax. frequency had a value of 330 Hz, unless stated otherwise. The working electrode was a Metrohm hanging mercury drop electrode (h.m.d.e.), type E 410, with a surface area of 1.75 x lop2 cm2. A saturated calomel electrode from Ingold, type 303-NS, served as reference electrode and the auxiliary electrode was a coiled platinum wire. The solution was deaerated by passing a slow stream of
499
nitrogen, and an inert atmosphere was maintained during the measurements passing nitrogen over the solution. All measurements were carried out at 5 o C.
by
RESULTS AND DISCUSSION
A survey of the adsorption behaviour of guanosine mono-, di- and triphosphate as a function of potential and pH is provided by the phase-sensitive a.c. voltammograms (Figs. l-4). The recorded capacitive a.c. component is proportional to the differential double-layer capacitance in potential regions where no faradaic process occurs [22]. In the pH range 3.4-8.0, the a.c. voltammograms of the respective mononucleotides indicate a progressive decrease in the capacitive current, with
(5':GMP),pH
3.4
1: 0.00 24.2 xVT6N 3:5.2 xlU% 4:6.1 x~O-~M 5:9.3 x lo-% 6:1.6x10-k
.' I'
Fig. 1. a.c. voltammetric curves of S-GMP at the h.m.d.e. 0.5 M McIlvaine buffer, 5”C, area of h.m.d.e. 1.75~10~’ cm’, scan rate 2 mV s-l, frequency 330 Hz, amplitude 10 rnVpp,phase angle 90°, adsorption time 180 s.
4 (5':COP),pH
3.4
l:ooo 2:1.9x10-5M 3:2.8xWM 4:4.7 xlcr'lv 5: 7.1 x10-% 6:2.1 x10%
-1.2
-1.0
-0.8
3
-0.6
-0.4
-0.2
Fig. 2. (I.c. voltammetric curves of 5’-GDP at the h.m.d.e., pH 3.4. Other conditions as in Fig. 1.
500
(5’:
GDP),pH
1: 0.00 2:1.9xlcr% 3:4.7x10-4"/ 4:7.6x16% 5:1.2~lO-~M
-1.2
-1.0
5.1
6:1.4nld5,'f 7:1.8x10-% 8:2.3xKI-='/'I 9:3.o~Iuk 10:5.6~1O-~N
-0.8
-0.6
-0.4
-0.2
Fig. 3. CI.C.voltammetric curves of 5’-GDP at the HMDE, pH 5.1. Other conditions as in Fig. 1.
increasing concentration, at a potential U,,,,,, of the supporting electrolyte. The decrease corresponds to a progressive coverage of the electrode surface by the dilute adsorption layer. The r-electron interaction with the electrode will favour a flat adsorption of guanine moiety in the range of positive or zero charge. At pK, -CpHs -CpK, [23] (Table 1) and above the threshold value, a sudden sharp decrease in the a.~. capacitive current is observed, giving rise to a very sharply defined pit. The pit reflects the formation of a compact adsorption film due to pronounced lateral interaction of the adsorbed species in a certain potential range. At potentials more negative than -0.9 V, the U.C. voltammograms exhibit a desorption peak corresponding to the desorption of loosely bound species existing in the film at more negative potentials. The destabilization of the compact adsorption film in highly acidic (pH x pK,) or alkaline solutions (pH > pK,) indicates that the degree of association of the adsorbed species of guanosine phosphate is connected with the adsorption of species which have a neutral guanine moiety, according to their pK values (Table 1). Therefore the compact stage of adsorption is formed as a result of the interaction of the charged electrode with the phosphate group and the permanent dipole of neutral guanine residue between N(3) and N(7), depending on their charge and the sterical arrangement of the molecule.
(5':GTP),pH 8
:o.oo
1 2:1.9x10-% 3:3.7x10-k
-3
4: 5.7x10-% 5:6.6xlb'H 6:1.4x10-M
Fig. 4. a.=. voltammetric curves of 5’-GTP at the HMDE. Other conditions as in Fig. 1.
501 TABLE 1 Adsorption parameters of the dgute layer of GUO, 5’-GMP, 5’-GDP and 5’-GTP at various pH values 10’ x Threshold bulk concentration (mall-‘)
lo-’ B (cm3 mol-‘)
- AC, (kJ mol-‘)
a
1.08 3.94 1.98 1.33
34.8 31.6 35.6 35.2
0.90 0.50 1.15 1.13
5’-GMP (pK, = 2.4, pK, - 9.4) 3.4 - 0.55 0.2 5.1 - 0.55 0.4 7.1 - 0.55 4.8 - 0.57 1.5 8.0
2.98 3.08 0.698 0.605
38.8 38.9 35.4 35.1
0.62 0.11 0.79 0.88
5’-GDP ($4 = 2.9, pK, - 9.6) 3.4 - 0.50 2.4 5.1 - 0.50 2.6 1.1 - 0.50 10.4 8.0 - 0.50 8.6
1.81 1.70 0.151 0.0921
37.6 37.5 31.9 30.6
0.63 0.66 0.86 0.81
5’-GTP (PK, = 3.3, pK, = 9.3) 3.4 -0.4 5.1 - 0.4 7.1 - 0.52 2.8 - 0.52 8.0 4.8
0.0532 0.0716 0.633 0.253
31.9 32.3 35.3 33.2
0.56 0.61 0.63 0.54
UH
Potential of maximum adsorption (v)
GUO (pK, - I.6, pK, = 9.2) 3.2 -0.55 -0.52 5.2 -0.52 7.1 8.0 -0.52
2.1 0.52 0.41 0.55
The U.C. voltammetric behaviour of guanosine phosphate is similar to some extent to that of guanosine. However, the adsorption range of guanosine phosphate extends to more positive and negative potentials. This should be interpreted as a significant contribution to the adsorption by negatively charged phosphate groups. As model considerations reveal, the latter, under the electrostatic attraction of the electrode, can be turned into a position where the phosphate group comes into direct contact with the surface. The peak at -0.34 V (Fig. 2) corresponds to a specific reorientation to a position where the base comes into strong interaction with the surface, while the adsorption of the hydrophilic phosphate group attains a solution-sided position. The occurrence of this reorientation peak is to be expected, because the net charge of the guanosine phosphate is approximately balanced. This behaviour is in good agreement with results obtained for the adsorption of adenosine and cytidine phosphate [10,11,17]. Additional information on the course of the adsorption and interfacial association of guanosine phosphate at different pH values is offered by the time dependence of the U.C. capacitive response, shown in Fig. 5. At low bulk concentrations, the capacitive U.C. current, measured at a constant mean electrode potential (U,,,),
502
I z
3 -I 4
(5':GMPLpH l:O.OO 2:2.3xl(r% 3:71110+~
--------_______
7.1
4: 1.6x10-% 5:2.1x10-% 6:2.8~lO-~N
7:3.7riO-% 8:4 S~lO-~bf 9: 5.41 g-y 10:7.5xlo-=M ----------_-__--__,
-y
_____
2-:; ///
EEs
5678 9 10 0
20
I J====
,C(min)
I 60
40
Fig. 5. Time dependence conditions as in Fig. 1.
80
of the out-of-phase
, loo
, 160
component
of the a.c. current of 5’-GMP at U,,,.
Other
at first slowly decreases to the first equilibrium value, corresponding to the dilute adsorption layer. For concentrations higher than the threshold value (Table l), where a pit is observed on the a.~. voltammograms, a drastic decrease to a second equilibrium value is observed, corresponding to the formation of a compact layer. Once a compact adsorption film has been built up, there is no substantial influence from the solution and thus the extent of the capacitance change accompanying the film formation depends only on the first stage and is determined mainly by the steric arrangement of adjacent molecules. The effect of potential on the degree of association is reflected by Fig. 6, which shows the potential dependence of the adsorption for the dilute and compact adsorption stages of Y-GDP at pH 5.1. At the maximum adsorption potential, the resulting concentration dependence of the U.C. capacitive current decrease AI,,,,
0
2
4
6
Fig. 6. Dependence of the capacitive various adsorption potentials.
current decrease AZ,,,, on the bulk concentration
of 5’-GDP
at
503
(the decrease of the capacitive a.~. current with respect to the I,,,, value of the supporting electrolyte for a given bulk concentration) has the form of a two-step isotherm. On the other hand, at potentials more positive or more negative than the maximum adsorption potential, the threshold concentration value for compact film formation increases appreciably or the two-step isotherm is ill-defined. Therefore, the choice of a proper potential as an electrical variable is significant in studying the degree of association of guanosine phosphate and in the evaluation of adsorption parameters. In this context, on comparing the threshold concentration value at different pHs at a maximum adsorption potential (Table l), it becomes evident that the association of guanosine phosphate, which is manifested at pK, > pHs > pK,, occurs at a lower concentration. This indicates that the degree of association of the adsorbed species of guanosine phosphate depends predominantly on the stacking of the molecules, which have a neutral guanine residue. From the threshold concentration values at one and the same pH for guanosine and its phosphate derivatives (Table 1, Figs. 7 and S), one can conclude that the interfacial association of guanosine takes place at a lower threshold concentration than for guanosine phosphate. The tendency for the interfacial association in acidic medium diminishes in the order guanosine > 5’-GMP > 5’-GDP > 5’-GTP due to the compensating influence of the negatively charged phosphate groups at the positive end of the dipole at N(3). Obviously the diminution of the positive charge on N(3) decreases the stabilization by base stacking, which causes the interfacial association of adjacent adsorbed molecules and consequently the formation of the compact layer. This trend is in agreement with the increasing value of the protona-
c a,b
b
Fig. 7. Dependence of the capacitive current decrease A I.,,, on the concentration at pH 5.1 at the maximum adsorption potential. Other conditions as in Fig. 1.
of
phosphate
504
b
0
1
2
3
4
5
6
’
a
Fig. 8. Dependence of the capacitive current decrease AI,,, on the concentration of guanosine phosphate at pH 8.0 at the maximum adsorption potential. Other conditions as in Fig. 1.
tion constant of N(7) (pK,, Table 1). In neutral and alkaline solution, the tendency of the interfacial association has the order guanosine > 5’-GTP > 5’-GMP > 5’-GDP. This trend is supported by the lower ionization constant of N(1) of 5’-GTP compared with the corresponding values for 5’-GMP and 5’-GDP. It also reflects the highly donating power of the negatively charged phosphate groups of 5’-GTP (pK{ -K 1, pK; < 6) in the direction of the dipole, which leads to an increase of the electron density on the oxygen at C(6) and encourages the proton transfer process between N(1) and the oxygen at C(6). In order to calculate the adsorption parameters for the dilute and compact adsorption layers at various pH values, the equilibrium values of the U.C. capacitive current at a given bulk concentration were measured and the degree of coverage B was calculated using the relation: e=
2-t
= (AT;Ejm
(1)
where the Cs are the differential capacities in the supporting electrolyte (Cc), at a given bulk concentration of guanosine phosphate (C), and at bulk concentrations corresponding to full coverage (C,). A I,,,, is the decrease of the capacitive U.C. current (with respect to the I,,,, value of the blank supporting electrolyte) for a given bulk concentration, and (AI,,,,),,, is the maximum decrease corresponding to full coverage. It has been found that the experimental data fit well a Frumkin adsorption isotherm given by the equation: Bc= &exp(-2aB)
(2)
where 8 is the degree of coverage, a the interaction coefficient and c the bulk concentration of the substance investigated. In addition, the free enthalpy of adsorption -AGO can be calculated from the adsorption coefficient B. The calculated values of the adsorption parameters of guanosine phosphate at various pH
505 TABLE 2 Adsorption parameters of the compact layer of GUO, 5’-GMP, values
5’-GDP
and 5’-GTP
1O-4 B (cm3 mol - ’ )
- AG, (kJ mol-‘)
(1
GUO 3.2 5.2 7.1 8.0
5.16 1.99 2.10 1.90
34.0 31.2 31.2 29.8
2.1 2.0 2.1 2.2
5’-GMP 3.4 5.1 7.1 8.0
2.36 2.80 0.732 0.776
32.9 33.3 30.1 30.3
1.8 1.8 1.9 1.6
5’-GDP 3.4 5.1 7.1 8.0
0.92 0.75 0.71 0.127
30.7 30.2 30.1 26.0
1.8 1.8 2.0 2.0
5’-GTP 3.4 5.1 7.1 8.0
-
-
0.39 0.211
29.3 27.2
PH
at various pH
values are given in Tables 1 and 2. Comparing the values of the adsorption parameters of guanosine phosphate at different pH values shows a significant dependence on the pH values either for the dilute or the compact layer. In acidic buffer solution, the magnitude of the adsorption coefficient B is higher than that of the neutral and alkaline solutions for guanosine mono- and diphosphate. The decrease of hydrophobicity of 5’-GMP and Y-GDP in neutral and alkaline media is due to the highly compensating effect of the completely ionized phosphate groups (pK; < 1, pK; < 6) on the dipole moment of the guanine moiety. However, the increase in B value of 5’-GTP in neutral and alkaline media may be ascribed to the appearance of the counteracting effect as a result of the proton transfer process, which may decrease to some extent the excess negative charge on the dipole of the guanine residue. The rather large standard free energy of adsorption - AGOin acidic medium shows that the moderately charged interface and large adsorption forces prevail. In this context the interaction coefficient a is rather small and indicates moderate lateral attractive interaction. For the compact adsorption stage, the magnitude of the adsorption coefficient B is significantly lower than that in the dilute stage. This significantly lower adsorptivity of the compact film correlates with its sensitivity to larger alterations of the
506
adsorption potential and its tendency to collapse suddenly at a certain value of the interfacial electric field at the positive and negative side of the region of U,,,. Nevertheless, the magnitude of Acindicates that the same unit of the molecule, i.e. guanine moiety, still remains predominantly responsible for the adsorption in the compact film stage at the potential of maximum adsorption. The value of a generally increases in the compact stage due to the enhanced possibilities for intermolecular attractive interactions, resulting from the perpendicular orientation and greater population of the adsorbed molecules in the compact stage. The value of the maximum surface concentration r,,, was calculated using the relation [24] r = l.l28c(
D#”
where c is the bulk concentration of guanosine or guanosine phosphate in mol cmW3, D the diffusion coefficient, t the time and I’ the surface concentration of the adsorbed compound. The diffusion coefficients have been evaluated using the Stokes-Einstein equation [25]. The value of the maximum surface concentration I’,,, for the compact layer was obtained from equation (3) by taking the time t, as the extrapolated time at which the linearized first portion of the time dependence of the a.~. current reaches the final horizontal part, i.e. the full coverage for the compact layer (See Fig. 5, curve 9). From the I?, for the compact layer and the ratio of the two steps of the isotherm obtained from U.C. voltammetric measurements, the surface concentration for the dilute layer has been evaluated as given previously [14,15]. The results reveal that the surface concentration .r,,, for guanosine is higher than for guanosine mono- and diphosphate (Table 3). Consequently, the average surface area per molecule A, increases in the same way. This behaviour is reflected by the smaller value of B for guanosine phosphate as compared with guanosine. Also, the interaction coefficient a is larger for guanosine than for guanosine phosphate. In general, and as will be seen from the afore-mentioned results, it is
TABLE 3 Maximum surface concentration phosphate at pH 5.1 Substance
Dilute stage Guanosine 5’-GMP 5’-GDP
r,
and surface area per molecule
A,,, for guanosine
10’0 r -2 (molcm#VI )
A,
2.21 2.03 1.71
0.71 0.83 0.91
5.23 4.99 4.63
0.31 0.34 0.36
(nm2)
compactstage Guanosine 5’-GMP 5’-GDP
and guanosine
507
remarkable that small steric differences in the adsorbed molecules yield a distinct change in the adsorption behaviour. This underlines the particular power of the U.C. voltammetric approach with phase-sensitive detection for the study of intermolecular forces which govern the formation of a compact film. REFERENCES 1 P. Valenta and H.W. Niimberg, Biophys. Stmct. Mech., 1 (1974) 17. 2 E. PaJecek, Collect. Czech. Chem. Commun., 39 (1974) 3449. 3 H.W. Ntimberg and P. Valenta, Proc. 29th Symp. Colston Res. Sot., Bristol, 1977, D.H. Everett and B. Vincent (Editors), Scientechnica, Bristol, 1978, p. 201. 4 J.M. Sequaris, P. Valenta, H.W. Ntmberg and B. Malfoy in ref. 2, p. 230. 5 V. Vetterl, Collect. Czech. Chem. Commun., 31 (1966) 2105. 6 V. Vetterl, J. Electroanal. Chem., 19 (1968) 169. 7 T.W. Webb, B. Jan& and P.J. Elving, J. Am. Chem. Sot., 95 (1973) 991. 8 J.W. Webb, B. Janik and P.J. Elving, J. Am. Chem. Sot., 95 (1973) 8495. 9 U. Retter, H. Jehring and V. Vetterl, J. Electroanal. Chem., 57 (1974) 391. 10 D. Krznaric, P. Valenta and H.W. Ntimberg, J. Electroanal. Chem., 65 (1975) 863. 11 P. Valenta, H.W. Niiberg and D. Ktznaric, Bioelectrochem. Bioenerg., 3 (1976) 418. 12 V. Vetted, Bioelectrochem. Bioenerg., 3 (1976) 338. 13 M.A. Jensen, T.E. Cummings and P.J. Elving, Bioelectrochem. Bioenerg., 4 (1977) 447. 14 Y.M. Temerk and P. Valenta, J. Electroanal. Chem., 93 (1978) 635. 15 Y.M. Temerk, P. Valenta and H.W. Nbmberg, J. Electroanal. Chem., 100 (1979) 77. 16 Y.M. Temerk, Can. J. Chem., 57 (1979) 1136. 17 Y.M. Temerk, P. VaJenta and H.W. Niimberg, Bioelectrochem. Bioenerg., 7 (1980) 705. 18 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 8 (1981) 671. 19 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 11 (1983) 457. 20 Y.M. Temerk and M.M. Kamal, Bioelectrochem. Bioenerg., 12 (1984) 205. 21 P.J. Elving, J.M. Markowitz and I. Rosenthal, Anal. Chem., 28 (1956) 1179. 22 H. Jehring, Electrosorptionsanalyse mit der Wechselstrompolarographie, Akademie Verlag, Berlin, 1974. 23 G.D. Fasman (Editor), Handbook of Biochemistry and Molecular Biology, Vol. 1, (3rd ed.), p. 169. 24 S.L. Phillips, J. Electroanal. Chem., 12 (1966) 294. 25 A. Einstein, Ann. Phys., 17 (1905) 549; 19 (1906) 371; Z. Elektrochem., 14 (1908) 235.