Studies on the surface orientation of uracil, uridine, thymine and thymidine at the mercury electrode surface by electrocapillary measurements

137

Bioelecirochemistry and Bioenergetics, 25 (1991) 137-144 A section of J. Electroanal. Chem., and constituting Vol. 320 (1991) Elsevier Sequoia S.A., Lausanne

Short communication

Studies on the surface orientation of uracil, uridine, thymine and thymidine at the mercury electrode surface by electrocapillary measurements M.M. Kamal, Z.A. Ahmed, M.E. Ahmed, M.S. Ibrahim and Y.M. Temerk Chemistry

Department,

(Received

20 April 1990; in revised form 15 August

Faculty of Science, Assiut

University, Assiui (Egypt) 1990)

INTRODUCTION

In living cells, nucleic acids come into contact with various types of boundaries. The interfacial situation in the living cell, however, is very complex and not easily adjustable. Thus, versatile models of the biological interface and more simple molecules than the macromolecules of DNA and RNA are needed to contribute to a deeper understanding of the general biophysicochemical behaviour of these substances at charged interfaces. Therefore the present work is focussed on the surface activity of some nucleic acid components. Surface activity and interfacial phenomena of some nucleosides and nucleotides at the mercury/solution interface have been studied by various authors in the past [l-12]. The potentialities of polarography and voltammetry to study the interfacial behaviour of these biological compounds at charged interfaces have been established recently [13-191. The adsorption and association of uracil, thymine and their nucleosides and nucleotides at the mercury surface has been investigated by differential capacitance and ac voltammetric measurements [8,9,18,19]. The adsorption of these compounds was shown and factors which govern the ability of such compounds to promote the surface reorientation process were elucidated. At pH 9.0 and 25 o C, thymidine and thymidine mononucleotides differ significantly from the nucleosides and mononucleotides of uracil, since they do not exhibit the surface reorientation effect at the mercury/solution interface [9]. Continuing our quantitative studies on the interfacial behaviour of nucleic acid components [ll-191, we were further interested in investigating the surface activity of uracil, uridine, thymine and thymidine by electrocapillary measurements in order to study the surface orientation process of the investigated compounds at pH 7.0, which corresponds to the pH of the living cell. 0302-4598/91/$03.50

0 1991 - Elsevier Sequoia

S.A.

138 EXPERIMENTAL

Chemicals and solutions Uracil, uridine, thymine and thymidine were obtained from Sigma. Solutions containing different concentrations of the compounds investigated were prepared by dissolving a known amount of the chemically pure product in a definite volume of a B&ton-Robinson buffer of pH 7.0. Triply distilled water was used for preparing the solutions. The concentrations of the investigated compounds in the sample solutions were determined with a Unicam SP 8000 Spectrophotometer. The pH was measured with a digital Radiometer pH meter Model pH M64. Apparatus and methods Electrocapillary measurements were made using a PAR Model 174 A polarographic analyzer equipped with a PAR Model 172 A drop timer and electrode assembly. A thermostatted Metrohm cell equipped with a three-electrode system was used. The working electrode was a dropping mercury electrode (DME), the saturated calomel electrode (SCE) was used as the reference electrode and a coiled platinum wire as the auxiliary electrode. The measurements were carried out with a non-sihconized capillary with a natural drop time t = 4.8 s at 0.0 V with open-circuit potential in 1 M KC1 and at a mercury height h = 65 cm. To avoid interference by catalytic hydrogen evolution, all measurements were taken at 5’C, where the catalytic hydrogen responce is shifted to sufficiently negative electrode potentials. RESULTS AND DISCUSSION

The electrocapillary curves at various concentrations of uracil, uridine, thymine and thymidine at pH 7.0 we obtained by accurately measuring the drop time as a function of the electrode potential (Figs. 1 and 2). The recorded curves reflect the adsorption effects of the investigated biological compounds across the whole potential range, from the more positive potential up to the more negative potential region. There is a systematic depression of the electrocapillary curves in the whole potential range with increasing concentrations of the compounds investigated, corresponding to the progressive coverage of the electrode surface by the adsorbed molecules. A sharp depression of the electrocapillary curves is observed in a potential range around the potential of maximum adsorption (E,,). Further informative conclusions on the surface orientation of the investigated compounds at the mercury electrode surface can be drawn from the dependence of the surface tension u on the bulk concentrations at constant adsorption potential. The relative surface tension values were calculated from the results of the drop time curves by using a version of the equation derived by Kimmerle [20]:

&=

[+-]“[&+-I”’

exp[-K3(y-xef)l

139

6.05 -

5.60 -

- 0.2

-04

- 0.6

-0.8

-1.0

Fig. I. Electrocapillary curves at various 4.4x10-~; (3) 9.9~10~~; (4) 3.5~10-~;

E/V

uridine concentrations at pH 7.0 and T= 5OC. (1) 0.0; (2) (5) 8.4~10-~; (6) 1.3~10-~; and (7) 3.8X1O-3 mol 1-l of

5.5

1053 E ; 8 6 5.1

4.9

- 0.2

-

0.4

-06

-0.8

-1.0

E/V

Fig. 2. Electrocapillary curves at various thymine concentrations at pH 7.0 and T = 5O C. (1) 0.0; (2) 2.9x10-~; (3) 1.1x10-~; (4) 5.4~10~~; (5) 1.3~10~~; (6) 1.7~10~~ (7) 2.9x10m3, and (8) 5.2x10-’ mol 1-l of thymine.

140 d

b

c

I

I

I

a

L

3.5 4.0

3.5 4.0

3.0 , 3.5

2.5 3.0 , 3.0 -lcgKlmM)

2.0 , 1.5 c,d 2.5 , 2.0 b 25

c for the investigated compounds Fig. 3. Surface tension (0) vs. -log potential: (a) uracil; (b) uridine; (c) thymine; and (d) thymidine.

at the maximum

adsorption

where a,,, is the reference value of the interfacial tension for the specific electrolyte solution at the corresponding drop time tref, volume flow rate ( y,,_) and density d,,r. u is the relative surface tension of the investigated compounds at drop time t and volume flow rate y. D and d are the densities of mercury and the solution. K, K, and K, have been defined previously [21] and are taken to be 0.973, 1 and 0.00, respectively. A plot of the calculated value of the surface tension as a function of -log c (concentration) at constant adsorption potential is shown in Fig. 3. The precise isotherm is obtained at the potential of maximum adsorption around I?,,,. The surface excess values at E,,, are obtained according to the Gibbs adsorption equation (da/d&o

= r

(2)

where CI is the interfacial tension, p is the chemical potential p = pLo+ R lin a, r is the surface excess, and a is the activity of the compounds investigated. The slope (d a/d log c) of the vs. u (-log c) * plots at one concentration multiplied by (N/2.303RT) gives the number of adsorbed molecules per unit area at a given concentration, N being Avogadro’s number, R the universal gas constant (8.314 X 10’ erg mol-’ degree-‘) and T the absolute temperature. The calculated values of the surface tension cr or surface excess I? are plotted as a function of the bulk concentration (Figs. 3 and 4). The observed isotherm curve at maximum adsorption potential has the form of a double step Frumkin adsorption isotherm for uracil, uridine and thymine, corresponding to two adsorption stages on

l The low bulk concentration were taken diagnostic tests outlined elsewhere [22].

as equal

to the activity

throughout

this study,

based

on the

141

Concentration

Fig. 4. Surface excess (r) dependence of the bulk concentration maximum adsorption potential: (a) uracil; (b) uridine; (c) thymine;

! mM

of the different compounds and (d) thymidine.

at the

the electrode surface. The first stage reflects the dilute adsorption layer for relative low bulk concentrations of uracil, uridine and thymine. At higher bulk concentration, above a threshold concentration value, one obtains the second stage of adsorption of uracil, uridine and thymine which is due to reorientation of the adsorbed molecules on the electrode surface. This indicates that at the maximum adsorption potential uracil, uridine and thymine are oriented in a dilute adsorption layer parallel to the electrode surface, where interaction of the n-electron system with the surface favours the adsorption. With an increase of the bulk concentration above the threshold value, the stacking interactions between vertically oriented molecules lead to association and formation of a compact layer around E,,. The ratio at full coverage for the first and second steps of the isotherm indicates that the number of adsorbed molecules is increased by a factor of ca. 2 for the compact layer. This is in good agreement with the conclusions of Parry and Parsons [23] and Damaskin et al. [24] that the number of water molecules displaced from the surface by adsorption of hydrophobic species is doubled for a change in adsorption orientation from the parallel to the vertical position. The dependence of the surface excess P on the bulk concentration of thymidine is more interesting (Fig. 4). The resulting concentration dependence of the surface excess for thymidine has the form of a one-step isotherm in the range of potential of E mm’ The isotherm corresponds to one adsorption stage of thymidine on the

142 TABLE

1

The adsorption parameters for initially flat and perpendicularly thymine and thymidine at the mercury/solution interface Compound

- Es

103qh

/mV

/M

a

oriented

molecules

P/ 1 mol-’

- AGo/ kJ mol-’

10-‘4r,/ molecule

of uracil,

cm - *

uridine,

%I/ nm2

Initially fZat orientation Uracil 500 Uridine 500 Thymine 600 Thymidine 500

5.10 1.58 3.00 -

0.39 0.80 0.34 0.25

5.9x102 3.7 x 103 3.6 x lo3 2.4~10~

23.8 28.5 28.4 22.1

1.39 1.56 1.44 0.87

0.71 0.64 0.69 1.15

Perpendicular Uracil Uridine Thymine

_ -

2.00 1.32 1.60

20.0 102.0 46.9

16.3 20.1 18.3

1.92 2.38 2.04

0.52 0.42 0.49

orientation 500 500 600

electrode surface. This reflects the dilute adsorption region where the molecules of thymidine are adsorbed flat on the electrode surface. As shown in Table 1, for the dilute adsorption stage (flat orientation) the area covered by thymidine molecules (S,,,) is very large in comparison with the other compounds. This suggests that probably an appreciable fraction of the electrode surface is covered by the deoxyribose moiety in addition to the thymine base. However, in the case of uridine the ribose moiety is tilted away from the electrode surface [9]. At more elevated bulk concentrations of thymidine the reorientation of the adsorbed molecules on the electrode surface is hindered by the steric effect of the deoxyribose moiety, whereas thymidine is not effective in promoting base-base stacking interactions and no condensed film formation is obtained. The results indicate that the presence of the ribose moiety in the nucleoside facilitates the surface reorientation of the adsorbed molecules and formation of a stacked layer as in the case of uridine, whereas a less rigid structure is provided if the 0H(2’) is replaced by a hydrogen atom with respect to the furane ring, like in the thymidine molecule [25]. A necessary prerequisite for the application of electrocapillary measurements to the determination of adsorption parameters of the compounds investigated is that the adsorption equilibrium is attained at the dropping mercury electrode. In this context, as the molar masses of the investigated compounds are relatively low it can be expected that this will be the case for a drop time of several seconds, as was shown previously [13]. The dependence of 8 (I/T,,) on the bulk concentration of the investigated compounds is shown in Fig. 5. The variation of 19 with the concentration of each compound is given by the Frurnkin adsorption isotherm: f3(1 - 0)-r

exp( -2afI)

= j3c

where a is the interaction parameter, p the adsorption coefficient, concentration and 8 is the relative coverage degree. The experimental

(3) c the bulk data were

143

0,4 0.2, , 0.1

0,8 0.4

,

1.,2 0.6

$6 , 0.8

2.0 0.3

, 3.0 , 0.4 0.5

Concentration

/mM

1.0 , 0.2

d a b

Fig. 5. Dependence of 6’ (degree of coverage) for a dilute layer on the bulk concentration compound investigated at the maximum adsorption potential: (a) uracil; (b) uridine; (c) thymine; thymidine.

of the and (d)

subjected, to a least-squares refinement and the values of the regreesion coefficient of the fit were found to lie in the range 0.991 to 0.998 for all the investigated compounds. This indicates that the experimental data fit a Frumkin adsorption isotherm well. The interaction coefficient a was determined from the slope of a logarithmic plot of the Frumkin the value at half coverage. The Gibbs energy of adsorption ( - AC” ) was then calculated from the adsorption coefficient /3 using the equation j3 = &exp(

-E&‘/RT)

(4)

Typical values of the adsorption parameters are calculated and given together with the average values of the surface coverage per molecule (S,) in Table 1. The results indicate that the magnitude of the adsorption coefficient p for a compact layer is significantly lower than for the dilute stage. Nevertheless, the interaction coefficient a increases in the compact stage due to enhanced possibilities for intermolecular attractive interactions, resulting from the perpendicular orientation and the greater population of adsorbed molecules in the compact stage. Moreover, the values of average area covered per adsorbed molecules for a compact adsorption film are very close to those obtained for natural and synthetic polynucleotides [26]. This indicates that the structure of the condensed film is similar in all cases and corresponds to a stacked orientation of the base residues due to base-base interactions. It can be concluded that the calculated areas covered by one molecule for the various compounds investigated are in a good agreement with the reported corresponding values calculated from ac voltammetric [l&19] and the differential capacitance measurements [8,9]. This supports the application of the electrocapillary method for quantitative treatment of the adsorption and reorientation behaviour of the studied compounds at the mercury electrode surface.

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