Interfacial behavior of thymine, thymidine and thymidine mononucleotides at the mercury-solution interface

J. Electroanal. Chem., 85 (1977) 377--388 © Elsevier Sequoia S.A., Lausanne -- Printed in The Netherlands

377

INTERFACIAL BEtIAVIOR OF THYMINE, THYMIDINE AND THYMIDINE MONONUCLEOTIDES AT THE MERCURY-SOLUTION INTERFACE

H. KINOSHITA, S.D. CHRISTIAN and G. DRYHURST * Depart~nent of Chemistry, University of Oklahoma, Norman, Okla. 73019 (U.S.A.)

(Received 3rd November 1976; in revised form 26th January 1977)

ABSTRACT The adsorption of thymine, thymidine and thymidine mononucleotides has been studied by a differential capacitance method at pH 9 at the dropping mercury electrode. The area occupied by one molecule of thymine adsorbed at the mercury-electrolyte solution interface is 55 -+ 2 A 2 which corresponds closely to the area expected for a thymine molecule adsorbed flat on the electrode surface. The area occupied by thymidine, TMP, TDP and TTP ranges from 100--> 200 A 2. This suggests that an appreciable fraction of the electrode surface is covered with deoxyribose or deoxyribosephosphate. In this respect the interfacial behavior of thymidine and thymidine mononucleotides differs significantly from the deoxynucleoside and monodeoxynucleotides of adenine where the deoxyribose or deoxyribosephosphate groups are largely tilted away from the electrode surface. INTRODUCTION A r e c e n t r e p o r t f r o m this l a b o r a t o r y d e s c r i b e d t h e interfacial b e h a v i o r o f adenine, d e o x y a d e n o s i n e a n d d e o x y a d e n o s i n e m o n o n u c l e o t i d e s at t h e m e r c u r y a q u e o u s s o l u t i o n i n t e r f a c e [1 ]. This was t h e first in a series o f investigations o f t h e interfacial b e h a v i o r o f t h e basic p u r i n e a n d p y r i m i d i n e c o m p o n e n t s o f nucleic acids. In nucleic acids a d e n i n e is h y d r o g e n b o n d e d to its c o m p l e m e n t a r y p y r i m i d i n e base, t h y m i n e . This r e p o r t is c o n c e r n e d w i t h t h e interfacial b e h a v i o r o f t h y m i n e , t h y m i d i n e and t h y m i d i n e m o n o n u c l e o t i d e s at t h e m e r c u r y - a q u e o u s s o l u t i o n i n t e r f a c e u n d e r e x a c t l y t h e s a m e c o n d i t i o n s as t h o s e e m p l o y e d f o r t h e s t u d y o f a d e n i n e species, i.e., p H 9 a t 25°C. All a d e n i n e species a p p e a r to be a d s o r b e d in a fiat o r i e n t a t i o n o n t h e elect r o d e s u r f a c e w i t h a n y s u b s t i t u e n t d e o x y r i b o s e or d e o x y r i b o s e p h o s p h a t e tilted a w a y f r o m t h e surface. T h u s , t h e area o c c u p i e d b y o n e a d s o r b e d a d e n i n e m o l e cule at t h e e l e c t r o d e surface is close t o 55 A 2, d e o x y a d e n o s i n e o c c u p i e s t h e s a m e area while dAMP, d A D P and d A T P o c c u p y o n l y 7 0 - - 8 0 A 2. A t high c o n c e n t r a tions, a d e n i n e and d e o x y a d e n o s i n e ( b u t n o t t h e d e o x y m o n o n u c l e o t i d e s at p H 9) e x h i b i t a surface r e o r i e n t a t i o n e f f e c t w h e r e t h e y a p p e a r to r e a r r a n g e f r o m a flat o r i e n t a t i o n to a p e r p e n d i c u l a r o r i e n t a t i o n [2 ]. Because a d e n i n e is a l w a y s h y d r o g e n b o n d e d t o t h y m i n e in d o u b l e - s t r a n d e d D N A this investigation was initiated so t h a t t h e interfacial b e h a v i o r o f t h e s e * To whom reprint requests and further correspondence should be directed.

378 two nucleic acid complementary bases and their biologically important derivatives could be compared. EXPERIMENTAL Ch em icals

Thymine and its deoxynucleoside (thymidine) and deoxynucleotides were obtained from Calbiochem. All compounds were A grade and were used without further purification. The borate buffer system, pH 9, utilized in this investigation was constituted as follows: 8.75 g Na2B407 • 10 H20, 33.85 g KC1 and 8.34 ml of 1 M HC1 diluted to 1 1 with deionized water. This buffer has an ionic strength of 0.5 and samples of thymine or its derivatives were weighed directly into this solution. All solutions were deaerated with water-saturated nitrogen before study. Apparatus

Differential capacitance measurements were obtained by a phase-selective a.c. polarographic method described previously [1]. A Princeton Applied Research Corporation (PAR) Model 174 polarographic analyzer, coupled with a PAR Model 174/50 a.c. polarographic analyzer interface and a PAR Model 121 lockin amplifier/phase detector were employed for differential capacitance measurements. Complete i.r. compensation was employed throughout this study. A phase angle of close to 90 ° with respect to the applied alternating voltage was employed. It should be noted, however, that no t h y m i n e species studied here is electrochemically reducible. The dropping mercury electrode (DME) was siliconized [3] and was equipped with a mechanical drop dislodger. A pool of mercury inserted at the b o t t o m of a thermostated 5 ml capacity cell served as the counter electrode. A saturated calomel reference electrode (SCE) was used employing a fine Luggin capillary positioned close to the tip of the DME. All potentials are referred to the SCE at 25°C. In the case of all thymine derivatives studied the measured value of the differential capacitance per unit area at fixed a.c. frequency at different times in the drop life was found to be constant at times between < 1--10 s over the entire range of potentials studied, even with the most dilute solutions employed; i.e., d.c. equilibrium was rapidly established. For convenience, all subsequent measurements were made (without damping on the Model 174) with a controlled droptime of 2.00 s. Utilizing a.c. signals of 10 mV peak-to-peak amplitude, it was also f o u n d that between 10--250 Hz the measured capacitance was independent of frequency even at potentials corresponding to the broad, low cathodic desorption peaks. It was not possible to measure differential capacitance at frequencies much greater than 250 Hz because the a.c. polarographic m e t h o d for measurement of differential capacitance is restricted to relatively low frequencies [4]. Because of the absence of any significant frequency dispersion all capacity data reported here were measured at 100 Hz and 10 mV peak-to-peak.

379

The d.c. potential was scanned at a sweep rate of 0.005 V S- 1 . Alternating current vs. potential curves and alternating current vs. time curves were recorded on a Hewlett-Packard Model 7001A XY recorder. The electrocapillary m axi m um potential of the pH 9 borate buffer (--0.500 V) was measured with a capillary el ect r om et er similar in design to t hat of Broadhead et al. [5]; pressures were determined with a fused quartz high-precision pressure gauge (Mensor Corporation). The assumption is made t h r o u g h o u t this study that the solute activity can be replaced by its molar c onc e nt r at i on in the very dilute solutions employed. RESULTS AND DISCUSSION

Analysis of capacitance data Capacitance versus potential (C vs. E) curves were obtained with a b o u t 10 concentrations of the t h y m i n e species over a potential range from --0.2 V to --1.9 V. At a potential of --1.6 V or m or e negative, C vs. E curves for aqueous solutions o f the organic adsorbate coincided with the background electrolyte C vs. E curve. Therefore, t he back-integration m e t h o d of Grahame et al. [6] was utilized to calculate the charge at the mercury-electrolyte solution interface. Thus, E

q - - q * = f , CdE

(1)

were q is the charge relative to q*, the charge at the starting potential E* (E* was normally --1.8 V). By measurement of an electrocapillary curve for the background electrolyte solution the value of the electrocapillary maximum potential (ECM) was obtained (--0.500 V). At the ECM in the background electrolyte solution q = 0. It is then simple to calculate the absolute charge at E". Since at E" (and at potentials ca. 0.2 V more positive than E*) background and background plus thymine species C vs. E curves were coincident, the charge values for all solutions are the same. Hence the values of q -- q* are readily converted to the absolute charge values as a function of both E and a, the activity of the organic solute. A second integration of charge was then carried out to obtain interfacial tension 7; i.e., E

7 - - ~ " = - - f qdE

(2)

E*

The value of 7" was determined directly from electrocapillary measurements on the background electrolyte; moreover, it also does not change with addition of adsorbate since all C vs. E curves were coincident at E*. Thus, eqn. (2) was used to obtain the interfacial tension as a function of both E and a. Finally, the surface spreading pressure, It, at any solute activity and electrode potential was obtained from eqn. (3) 7r = ~,w(E) -- ~,(E)

(3)

380 where ~w is the value o f ~/for t h e b a c k g r o u n d e l e c t r o l y t e solution at a = 0. In the case o f t h y m i n e at c o n c e n t r a t i o n s greater t h a n 16 mM a sharply d e f i n e d pit or well c e n t e r e d at - - 0 . 6 5 V was observed in C vs. E curves. It was n o t possible t o calculate charge and interfacial tension d a t a in this pit region b y integration o f c a p a c i t a n c e data. By analogy with adenine, which exhibits similar c a p a c i t a n c e pits [1,2], it is p r o b a b l e t h a t the a p p e a r a n c e o f this pit indicates a surface reorient a t i o n process. T h e n a t u r e o f this process is c u r r e n t l y being investigated in some detail. Plots o f surface spreading pressure, ~, versus the l o g a r i t h m of activity o f t h y m ine and its d e o x y n u c l e o s i d e and various d e o x y n u c l e o t i d e s were readily superimposable b y abscissa translation (Fig. 1). T h e calculated curves in Fig. 1 r e p r e s e n t least square fits o f the c o m p o s i t e lr, a, E d a t a to an empirical e q u a t i o n ~= {A[lnl+B(E)]]

I I + ( I + B ( ~a E)a)

2 + ( I + B~a2 ( E ) a ) 3 + . "'" 1

(4)

This e q u a t i o n is equivalent to the L a n g m u i r e q u a t i o n at sufficiently low concent r a t i o n s (or if ~ = ~ = ... = 0). A is equal to P m R T (where Fm is the value o f the surface excess of a d s o r b a t e at ~ = 1). Regardless w h a t values are d e t e r m i n e d for ~,/3 .... in eqn. (4), d~/dlna a p p r o a c h e s F ~ R T as a -* ~ . T h e p a r a m e t e r B(E) is d e p e n d e n t o n potential, so t h a t if d a t a are to be fitted s i m u l t a n e o u s l y at several potentials, separate B values m u s t b e inferred for each potential. A non-linear least squares p r o c e d u r e is e m p l o y e d to obtain o p t i m u m values o f all t h e parameters. In practice, d a t a are f i t t e d initially b y assuming ~ = fl = .... = 0, and the least squares values o f A, B ( E ) , and the r o o t m e a n square deviation in ~ (rmsd) are d e t e r m i n e d . T h e n , the process is r e p e a t e d with ~ i n t r o d u c e d as an additional p a r a m e t e r , and least squares values o f c~, A, B ( E ) , and rmsd are inferred. If the rmsd is significantly smaller t h a n the rmsd for the previous fit, the process is rep e a t e d again, with/3 included as a p a r a m e t e r . In n o n e of t h e systems studied did an i m p r o v e m e n t in the goodness o f fit result w h e n n o n - z e r o values o f ~ or of t h e higher-order p a r a m e t e r s were utilized. The t h r e e n u c l e o t i d e systems were, in fact, a d e q u a t e l y f i t t e d b y the e q u a t i o n ~ = A ln[1 + B ( E ) • a]. It was n o t e d t h a t values o f F R T valculated b y analytical d i f f e r e n t i a t i o n o f eqn. (4) using the Gibbs a d s o r p t i o n e q u a t i o n (eqn. 5) r -

1 RT

dTr d lna

(5)

d o n o t d e p e n d significantly on h o w m a n y p a r a m e t e r s (~, ~ .... ) are used in addition to A and B ( E ) . H o w e v e r , P m R T values do vary s o m e w h a t (±10 t o 15%) depending o n w h e r e the series is t e r m i n a t e d . In all cases, the empirical fit selected to r e p r e s e n t d a t a was t h a t which gave a statistically satisfactory rmsd using the m i n i m u m n u m b e r o f parameters. Once a c o m p o s i t e fit had b e e n o b t a i n e d (for d a t a at all potentials) the same f u n c t i o n a l f o r m was used to fit d a t a at fixed p o t e n t i a l s b e t w e e n --0.3 V and --1.0 V. Relatively few d a t a points are available at a given potential; t h e r e f o r e , in o r d e r t o r e d u c e the n u m b e r o f p a r a m e t e r s required to fit d a t a at a given potential, the value o f F m R T d e t e r m i n e d f r o m the c o m p o s i t e fit was assumed to apply at each potential. This a s s u m p t i o n will be valid if t h e g e o m e t r i c a l arrangem e n t o f molecules in t h e s a t u r a t e d m o n o l a y e r is nearly the same t h r o u g h o u t t h e

381 15

A 14

13 I

12 11 10

¥ -2

0

-1

1

10

-,=

2

3

-4

/

_~

o~

-i

;

~

-3

-2

-1

0

1

2

3

4

°

2,

~

-a

-2

-I

o

~

~

a

10

9

E

•

-1.0V

•

-0.9V

*

-0.8V

•

-0.7V

•

-0.6V

6 -0.5V

-3

-2

-1

0

1

2

3

iRa

Fig. 1. Composite ~" versus In a plots for (A) thymine, (B) thymidine, (C) TMP, (D) TDP and (E) TTP in borate buffer pH 9. The rms deviation in n from the calculated curve (continuous line) for (A) is 0.129, (B) 0.265, (C) 0.265, (D) 0.129, and (E) 0.098 mN m - 1 .

382 potential range. The assumption does not require that interactions of the adsorbate with the electrode surface or between adsorbate molecules be independent of potential. The individual 7r--ln a curves were differentiated analytically to yield values of F R T (or F, mol cm -2) corresponding to each concentration and potential. The fact that 7r vs. In a plots are superimposable by abscissa translation has been widely used to prove that the adsorption isotherms are congruent with respect to potential. However, Parsons [7] has shown that superimposability of 7r vs. In a plots is not sufficiently sensitive to provide adequate proof of the congruence of electrosorption isotherms with respect to potential. Accordingly, the first step in interpretation of the interfacial behavior of thymine and its derivatives was to decide by a more sensitive m e t h o d whether the adsorption isotherms were congruent with respect to potential or charge. If the adsorption isotherm is congruent with respect to potential, it follows from Parsons' [8] proof that at any fixed value of electrode potential the charge density, q, in the presence of the adsorbed organic molecules should be a linear function of the fractional surface coverage, 0; i.e., q = qw(1 + 0) + q'O

(6)

Here, qw and q' denote the values of charge at the same electrode potential in the presence of pure supporting electrolyte and when the surface is saturated (0 = 1), respectively. If the adsorption isotherm is congruent with respect to charge, it follows [8] that at any constant value of q, the electrode potential in the presence of adsorbed organic molecules should be a linear function of 0, i.e., E =Ew(1 - - 0 ) +E'O

(7)

Here E w and E' are the values of electrode potential at the same q in the presence of pure supporting electrolyte and when the surface is saturated (0 = 1), respectively. Isotherms (0 vs. concentration or activity) may be obtained by using eqn. (6) at fixed potentials or eqn. (7) at fixed charges to calculate 0. If the isotherms at different potentials with 0 values calculated from eqn. (6) have the same geometrical shape, i.e., t h e y may be superimposed by abscissa translation, then the isotherms are congruent with respect to potential. Alternatively, if the isotherms at different charges with 0 values calculated from eqn. (7) have identical geometrical shapes then the isotherms are congruent with respect to charge. These tests were first proposed by Damaskin [9l and coworkers [10] and recently have been slightly modified by Doblhofer and Mohilner [ 11]. Both of these tests, however, rely on a fairly precise knowledge of C', the capacitance of a completely monolayer covered electrode surface (0 = 1) which is normally assumed to be constant and independent of charge and potential [12]. Accordingly, we have tested for congruence of the adsorption isotherms by preparing plots of q vs. FR T at fixed potentials as a test of eqn. (6) and plots of E vs. F R T at fixed charges as a test of eqn. (7). The values of F R T were calculated by differentiation of individual ~ vs. In a curves, as described earlier, and hence are independent of any adsorption model. Some typical q vs. F R T data for t h y m i n e between - 0 . 3 to --1.0 V are shown in Fig. 2. Between - 0 . 5 V and --1.00 V there is clearly an excellent linear relationship between q and FRT. At more positive potentials, considerable

383 9

7 6 5

3

1 0

•

~

4.

6

~=

~

• ~

~&

-0.5\

-1 -0.hV (j

•

e-

-6 -7 -8 -9 -10 -11 -12 -13 I'RT

Fig. 2. Test of congruence of adsorption of thymine with respect to potential at a dropping mercury electrode in borate buffer pH 9, Potential values are indicated in the Figure.

scatter o f t h e p o i n t s is n o t e d . It was c o n c l u d e d t h e r e f o r e t h a t t h e i s o t h e r m s were c o n g r u e n t w i t h r e s p e c t t o p o t e n t i a l at p o t e n t i a l s o f - - 0 . 5 V and m o r e negative. Similar linear r e l a t i o n s h i p s b e t w e e n q a n d F R Y at p o t e n t i a l s o f - - 0 . 5 V a n d m o r e negative w e r e o b s e r v e d f o r t h y m i d i n e , a n d t h y m i d i n e m o n o n u c l e o t i d e s . I n c o n t r a s t t o t h e linear q vs. F R Y p l o t s at fixed p o t e n t i a l s s h o w n in Fig. 2, p l o t s o f E vs. F R T a t f i x e d charges f o r t h y m i n e e x h i b i t e d m a r k e d d e v i a t i o n s

384 -1.1

j

-10.78,uc

-1.0 S

~

-9,38,uc

~ -0,9

® -0.8

-0.7y

-0.6

0

~ I

1

I

2

378pc I

3

4

I

rRT

i

5

6

i

7

Fig. 3. Test o f c o n g r u e n c e o f a d s o r p t i o n of t h y m i n e w i t h respect to charge at a d r o p p i n g m e r c u r y e l e c t r o d e in b o r a t e b u f f e r pH 9. Charge values are i n d i c a t e d in t h e Figure.

from linearity (Fig. 3). All other t h y m i n e derivatives showed similar behavior. It was concluded therefore that the adsorption isotherms are not congruent with respect to charge. In view of the fact that the isotherms are clearly congruent with respect to potential, it was decided to fit ~, a and E data to the Frumkin isotherm equation [13]. In the fixed potential form of the Frumkin equation (eqn. 8), B and 0/(1

-- 0) = Ba

exp(2 ~0)

(8)

are constants depending on interactions between the adsorbate molecules and the electrode surface and on lateral intermolecular interactions between the adsorbate molecules, respectively. The constant B is dependent on potential according to eqn. (9) [14] (9)

B = B o exp(--~/FmRT)

where Fm is the limiting surface excess of the solute at full monolayer coverage, in tool cm -2. The function dp = [,),w(O) -- ,),w(E)] +

C'EE

~C E 2

N -- 1

'

(10)

is evaluated in terms of E, the potential relative to the ECM for the electrolyte, EN, the ECM for the mercury-solution interface at 0 = 1, C' and the interfacial tension for pure supporting electrolyte solution ~'w. The term Bo in eqn. (9) is the value of B at the ECM for the pure electrolyte solution. By combining eqns.

385 (8) and (9) one obtains the generalized Frumkin equation 0/(1 -- 0) = B o a exp(2 s 0 )

exp(--(P/FmRT)

(11)

The electrocapillary maximum potential and 7w(0) and 7w(E) values were determined by electrocapillary measurements on the pure supporting electrolyte solution. Hence, there are only two parameters in eqn. (10), C' and EN, and three additional parameters, 0/, Bo and I~m, which must be evaluated in fitting data to the generalized Frumkin equation (eqn. 11). Combination of the Gibbs adsorption equation (eqn. 5) with the Frumkin equation (eqn. 8) and integration yields an expression that directly relates u to 0, 0/and l~m [15]: = FmRT[--In(1 -- 0) - - 010 2]

(12)

We have continued to utilize the non-linear least squares method described elsewhere [1] to fit ~, a, E data to obtain the best values of the parameters 0/, Bo, Fro, EN and C' and standard errors in these parameters. Briefly, trial values of the 5 parameters are chosen, and values of 0 are calculated from eqn. (11) for each pair of ai and Ei values by an iterative numerical method. This set of calculated 0i values is used to predict a set of ~i values using eqn. (12). For a given set of parameters, a value of the sum of squares of residuals s = Ei(~i -- ~i calculated)2 is obtained and then minimized with respect to simultaneous variation of all 5 parameters. In each thymine system, convergence was obtained in less than 20 iterative cycles. Surface spreading pressure, u, and activity data were only fit to the Frumkin equation for those potentials where linear q vs. FRT plots were obtained, i.e., at potentials at or more negative than --0.5 V. The experimental data on thymine and thymine derivatives gave very good agreement with the Frumkin isotherm. For example, Fig. 4 shows a reduced adsorption isotherm for thymine. The experimental F values were obtained by differentiation of ~ vs. In a plots at individual potentials. The solid curve is the best fit of all data to the Frumkin model. Clearly the individual experimental points, which are obtained without assuming an adsorption model, are in excellent agreement with the Frumkin isotherm. Similar agreement was found in the case of thymidine and the thymidine mononucleotides. Because of the extremely good fit of the data to the Frumkin adsorption model, no attempt was made to fit the data to other adsorption models. The results of analysis of the capacitance curves of thymine, thymidine and thymidine mononucleotides using the Frumkin adsorption mode] are shown in Table I. These results indicate that there is relatively little variation in the Frumkin attraction coefficient, 0/, within this group of thymine derivatives. Indeed, in most instances, 0/is so small that the adsorption behavior may be regarded as being reasonably well described by the Langmuir isotherm. In the case of thymine, the area occupied by one molecule at surface saturation (0 = 1) is 55 + 2 A2. The crystal structure of thymine [16] has been determined and it may be calculated from such data that the area of one thymine is close to 45--50 A2. This suggests, therefore, that thymine is probably adsorbed flat on the electrode surface. This agrees with the behavior observed for adenine, which in regions of concentration and potential outside the anomalous capacitance pit, is also adsorbed in a flat orientation on the electrode surface. In the case of thymidine and thymidine mononucleotides, the area occupied per molecule is much greater

386

4.8 4'4 t 4.0 3.6 g " 3.2 0

x 2.8 E 2.4 2.0

1.6

• -1.0V • -0.9V * -0.8V • -0.7V A -0.6V 6 -0.5V

1.2

0.8 0.4

o

; C/C1" = 1.7 x

;

;

lO

1 0 "10

Fig. 4. R e d u c e d a d s o r p t i o n i s o t h e r m of t h y m i n e at various p o t e n t i a l s b e t w e e n ---0.5 and - - 1 . 0 V. Solid line is t h e b e s t fit o f t h e data t o t h e F r u m k i n e q u a t i o n w i t h ~ = 0.34, B o = 4.05 × 103, [ ' m R T = 7.476, C' = 1 2 . 0 3 / J F c m - 2 and E N = - - 0 . 4 3 5 V.

Table 1 P a r a m e t e r s o f the generalized F r u m k i n e q u a t i o n for t h y m i n e , t h y m i d i n e and t h y m i d i n e mon o n u c l e o t i d e s at pH 9 a Compound

Thymine Thymidine Thymidine-5'monophosphate Thymidine-5'diphosphate Thymidine-5'triphosphate

a

AG ° b/ kJ

EN c~ V vs. SCE

1010

Area/

× Pro/

A2 per

tool cm--2

molecule

rmsd in 7r/raN m-1

0.34 -+ 0.07 - - 0 . 7 3 + 0.31 - - 0 . 0 4 + 0.3

--14.89 --21.74 --22.30

- - 0 . 4 3 5 ± 0.008 - - 0 . 4 5 0 --I--0.012 - - 0 . 4 8 2 ± 0.006

2.150 1.189 0.588

55 -+ 2 100 -+ 8 202 +- 9

0.125 0.255 0.264

0.14 -+ 0.2

--18.31

---0.469 --- 0.004

0.647

183 + 9

0.131

- - 0 . 0 7 -+ 0.10

--18.24

- - 0 . 4 6 3 --- 0.004

1.372

121 -+ 5

0.099

a Borate b u f f e r p H 9.0. b S t a n d a r d free e n e r g y o f a d s o r p t i o n at t h e electrocapillary m a x i m u m p o t e n t i a l for p u r e b a c k g r o u n d e l e c t r o l y t e solution, based o n infinite d i l u t i o n s t a n d a r d states for t h e a d s o r b a t e , b o t h in s o l u t i o n (at u n i t m o l a r i t y ) and on t h e surface (at 0 = l/Z). AG ° = - - R T In B o w h e r e B o is e x p r e s s e d in 1 m o 1 - 1 units. c Electrocapillary m a x i m u m p o t e n t i a l w h e r e 0 = 1 for each c o m p o u n d .

387 than that occupied by t h y m i n e (Table 1). Clearly, there is no systematic variation in area occupied as the molecules become larger. The largest area is that occupied by TMP at 202 _+9 A 2. Although it is tempting to speculate as to specific surface orientations of these molecules we do not believe that this would serve much purpose at this time. The larger areas occupied by thymidine and the thymidine mononucleotides compared to t h y m i n e suggest, however, that appreciable coverage of the electrode surface by deoxyribose and deoxyribosephosphate groups takes place. This behavior contrasts quite sharply with the behavior observed for adenine, deoxyadenosine and deoxyadenosine mononucleotides where the area occupied by the nucleotides is only 20--30% greater than the area occupied by the parent base which suggests that for these compounds the deoxyribose and deoxyribosephosphate groups are largely tilted away from the electrode surface. It should be noted that self-association effects do not seem to be important in bulk solutions of the t h y m i n e derivatives at concentrations used in this study. Such effects are apparently responsible for deviations from linearity of vs. In c plots at high degrees of surface coverage for the deoxyadenosine nucleotides, particularly dAMP [1]. The standard free energy of adsorption, AG °, at the ECM of the pure supporting electrolyte for thymine (--14.89 kJ) is significantly lower than was observed for adenine (--18.53 kJ) [1], i.e., t h y m i n e is appreciably less surface active than adenine. It is also quite clear in both the adenine and t h y m i n e systems that the nucleosides and nucleotides are more surface active than the free bases, i.e., deoxyribose and deoxyribosephosphate are surface active. The fact that the areas occupied by thymidine and thymidine nucleotides are much larger than the area of t h y m i n e further suggests that deoxyribose and deoxyribosephosphate remain adsorbed to a large extent on the electrode, even at high degrees of surface coverage. In the case of deoxyadenosine and deoxyadenosine nucleotides this is not the case. The most logical explanation of these facts is that adenine is so much more surface active than deoxyribose or deoxyribosephosphate that, particularly at high surface concentrations, it competitively displaces the latter groups from the electrode surface. Thymine, on the other hand, is less strongly adsorbed and cannot so effectively displace deoxyribose and deoxyribosephosphate from the electrode. ACKNOWLEDGMENTS This work was supported by NIH Grant No. GM 21034. We would like to thank Mr. James G. Baker for assistance on certain aspects of this work. REFERENCES 1 H. K i n o s h i t a , S.D. Christian a n d G. D r y h u r s t , J. E l e c t r o a n a l . C h e m . , 83 ( 1 9 7 7 ) 151. 2 H. K i n o s h i t a , S.D. Christian, M.H. K i m , J.G. B a k e r a n d G. D r y h u r s t in D.T. S a w y e r (Ed.), Electroc h e m i c a l S t u d i e s of Biological S y s t e m s , ACS S y m p o s i u m Series, 38 ( 1 9 7 7 ) 113. 3 G. D r y h u r s t a n d P.J. Elving, T a l a n t a , 16 ( 1 9 6 9 ) 8 5 5 . 4 B.B. D a m a s k i n , O.A. Petrii a n d V. B a t r a k o v , A d s o r p t i o n of Organic C o m p o u n d s o n E l e c t r o d e s , P l e n u m , N e w Y o r k , 1 9 7 1 , p. 16. 5 D.E. B r o a d h e a d , R.S. H a n s e n a n d G.W. P o t t e r , J. Colloid I n t e r f a c e Sci., 31 ( 1 9 6 9 ) 61. 6 D.C. G r a h a m e , E.M. Coffin, J.P. C u m m i n g s a n d M.A. P o t h , J. A m e r . C h e m . Soc., 74 ( 1 9 5 2 ) 1 2 0 7 . 7 R. Parsons, J. E l e c t r o a n a l . C h e m . , 8 ( 1 9 6 4 ) 93.

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