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Kinetic studies on the interaction of gold (III) with nucleic acids. IV. RNA-Au (III) system
Chem.-BioL Interactions, 21 (1978) 125--134 © Elsevier/North-Holland Scientific Publishers Ltd.
125
KINETIC STUDIES ON THE INTERACTION OF GOLD (III) WITH NUCLEIC ACIDS. IV. RNA-Au (III) SYSTEM
CHHABINATH MANDAL and UMA SANKAR NANDI
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore560 012 (India) (Received July 8th, 1977) (Accepted January 14th, 1978)
SUMMARY
The kinetics of the interaction of Au(III) with whole yeast RNA has been studied using UV-spectrophotometry. The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. The effects of initial composition, temperature, ionic strength, pH and chloride ion on the kinetics have been studied. Activation energy is found to be 11.5 kcal/mol. Effect of ionic strength indicates that both the positively charged and neutral species o f Au(III) take part in the rate limiting step, the former being dominant at low ionic strength. A plausible mechanism has been proposed which involves the interaction of two nucleotide units of RNA with one species of Au(III) in the rate limiting step.
INTRODUCTION
Studies on the interaction of metal ions with nucleic acids have resulted is useful and interesting information on the secondary structures of nucleic acids and have led to a comprehensive understanding of the biological functions of nucleic acids at the molecular level. Metal ions are believed to hold R N A in specific conformation and stablize its ordered structure. This may bear a functional relationship to protein synthesis and the transmission of genetic information. Metal ions mainly interact with the phosphate moiety electrostaticaUy or with the bases of DNA. Such studies have already been shown to be useful in the separation and isolation of satellite DNA's, in cancer chemotherapy, in the determination of base sequence, and many others. Pt (II) compounds have been found to have antitumor activity and gold being isoelectronic with platinum is expected to exhibit similar behaviour. Pillai and Nandi studied the interaction of Au(III) with nucleic acids [1,2]. Au(III) forms complexes with native and denatured DNA, and RNA with drastic changes
126 in optical and hydrodynamic properties of the nucleic acids. They observed that the absorption maximum o f RNA shifted to higher wavelengths in complex formation along with hyperchromicity. In viscometric studies they observed a 2.7-fold decrease in nsp/c of RNA in the complex formation. The reaction between Au(III) and R N A was found to be quite slow, though it was the fastest among the nucleic acids studied [ 1 ] and the kinetics could be followed using the conventional techniques for slow reactions. MATERIALS AND METHODS
Material and sample preparation Highly polymerized whole yeast R N A was obtained from BDH, Poole, United Kingdom. The R N A was dissolved in acetate buffer containing sodium perchlorate (0.01 M), sodium acetate (0.01 M) and adequate quantity of acetic acid to adjust to the desired pH. The stock R N A solution was standardized spectrophotometrically using ( P ) m a s = 8.7 X 103 A M -1 • cm -1 [1], where (P) denotes concentration o f the phosphate moeity of RNA. Chloroauric acid, HAuCla • 3 H 2 0 , was supplied b y Johnson Mathey Chemicals Ltd., London. The stock solution was prepared in doubly distilled water and the concentration was determined spectrophotometrically according to Vydra and Celikovsky [3] using e313 = 5.5 X 103 A M-' • cm -1. All other reagents were of the Analytical Reagent Grade and the solutions were prepared in d o u b l y distilled water (pH, 6.6--6.8).
Experimental The UV-spectra were recorded using an UNICAM SP700A double beam automatic recording spectrophotometer equipped with heating and thermostating devices. Two matched quartz cells of path length 1 cm were used for taking samples and reference solutions. Since Au(III) absorbs in the UV region, difference spectra of the RNA-Au(III) solutions were taken against Au(III) solutions under almost identical conditions. The desired volumes o f stock Au(III) solutions were added to the buffer medium 48 h before starting the reaction so as to attain the hydrolytic equilibria of AuC17,. The concentration of R N A was maintained at 1.642 × 10 -4 M (P) unless otherwise stated. For kinetic experiments, transmittances at two wavelengths e.g., 250 and 280 nm, were recorded at the desired intervals of time. From these transmittances the ratios of absorptions at 250 nm to that at 280 nm (A25o/A28o) were calculated and used for calculating the extent of reaction. Time taken to record the transmittance at a given wavelength was about 2 sec. Experiments were carried o u t in acetate buffer at room temperature maintained at 25°C unless otherwise stated. For experiments above room temperature two accessories (SP770 and SP775, constant temperature cell unit and electrical controller) of the s p e c t r o p h o t o m e t e r were used. The temperature could be controlled within + 0.05°C. In the kinetic studies of nucleic acid-metal ion systems using spectrophotometer different parameters e.g., absorbances at
127 260 nm [5] and 290 nm [10] and the absorbance ratio A2so/A27o [7] were used to follow the course of the reactions. For this system we used the ratio A2so/A2ao, because this parameter varies linearly with the extent of reaction.
Treatment of kinetic data The experimental data were subjected to fit the classical rate equations for a number o f combinations o f stoichiometry (2 : 1, 1 : I and 1 : 2) and orders (first and second orders with respect to each) of nucleotide unit of RNA and Au(III) using a F O R T R A N IV c o m p u t e r programme. For all the combinations, the functions, f(x), of the extent o f reaction (x) were derived and the standard deviations o f the plots of f(x) vs. time were calculated. The minimum standard deviation indicated the best fit of the experimental data to the corresponding equation. From the slope of this best fitted plot the rate constant was obtained. More details about the functioning and listing of the programme are available elsewhere [4]. The e x t e n t of reaction was calculated using the following equation. P0 - Pt x = P0 - P0o
"
CRNA(P)
where P0, Pt and P0o are the values of the time
Equilibrium results In order to examine the nature of changes in the spectral properties of RNA in complex formation a set of equilibrium experiments were carried out. The reaction mixtures were prepared with a constant RNA concentration (= 0.821 × 10 -4 M(P)) and different amounts of Au(III) to get r-values between 0.0 and 2.0 (r = number of moles of Au(III)/number of moles of RNA(P)). After 24 h UV-spectra were recorded in the range 200 nm to 3 0 0 nm followed by another set of recordings after 48 h; no appreciable change was observed indicating t h a t 24 h was sufficient to reach equilibrium. The absorption m a x i m u m shifted to a higher wavelength along with hyperchromicity as reported earlier [1]. Absorptions at both the wavelengths e.g. 250 nm and 280 nm increase with the increase in r-value but at lower r-values (0.5) the increase in the latter wavelength is more compared to that at the former. Consequently the ratio A2so/A2ao decreases sharply in going from r = 0.0 to r = 0.5 as shown in Fig. 1. The plot of A:so/A28o vs. r-value shows a break near r = 0.5 and the decrease in A2so/A2ao with r-value is much less in going from r = 0.5 to r =
128 1.8
°16 c~c° "1.4 O t~ o4
1.2 1.0 0
1 0.4.
I 0,8
L 1.2 r-VaLue
I 1.6
I 2.0
Fig. 1. Plot o f A2so/A28 o vs. r-value. C o n d i t i o n : R N A c o n c e n t r a t i o n , 0 . 8 2 1 X 10 -4 M(P); pH, 5.0; ionic s t r e n g t h , 0.02 M; R o o m t e m p e r a t u r e , 25°--26°C.
2.0 comprising only 15% o f the total change. This indicates that the sharp change in A2so/A28o characterizes a complex of stoichiometry 2 : 1 with respect to nucleotide unit of R N A and Au(III) respectively. In the kinetic studies of the nucleic acid-metal ion systems using spectrap h o t o m e t r y various parameters e.g. absorptions at 260 nm [5] and at 290 nm [6] and the ratio o f absorptions (A2so/A2~o) [7] have been used. For the present system we used the ratio A2so/A28o for calculating the extent of reaction because it is the only parameter varying linearly with the extent of reaction (Fig. 1 ).
Kinetic results For all the experimental sets with varying r-values, temperature, ionic strength, pH etc. the kinetic data gave the best fit to the rate equations carresponding to second order with respect to nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. Fig. 2 shows the plot of f(x) vs. time for the above mentioned combination of 16 i._: 12
8
'o
0
0
10
20
30 4.0 Time (rain.)
50
60
Fig. 2. Plot o f f(x) vs. time. C o n d i t i o n : R N A c o n c e n t r a t i o n , 1.642 × 10 4 M (P); pH, 5.0; ionic s t r e n g t h , 0.02 M; R o o m t e m p e r a t u r e , 25 ° - 2 6 ° C .
129 TABLE I DEPENDENCE
OF RATE CONSTANTS
Condition: RNA concentration, temperature, 25”-26°C.
R-value Rate constant X 10e6 (I’ - molmz . min-’ )
1.642
x
ON r-VALUE. 10m4 M(P); pH, 5.0;ionic
strength, 0.02 M; Room
0.50
0.75
1.00
1.25
1.50
2.717
1.925
1.407
1.224
1.106
stoichiometry and orders. Only 50-6096 of the total change in the spectral variable, A250/A280, fitted a straight line. At higher level of reaction the plot deviates to give a lower slope. Effect of r-value. A set of experiments was carried out with a constant initial concentration of RNA and variable concentrations of Au(III), the range of the r-values being between 0.5 and 1.5. The results of a typical set of experiments are presented in Table I. The rate constants are found to decrease with the increase in r-value. Effect of temperature. The effect of temperature was studied in the range 25”-50°C at six different temperatures. The components of both the sample and reference solutions were preheated to the desired temperature and placed in the thermostated chamber of the spectrophotometer after rapid mixing. Arrhenius plots were obtained by plotting the logarithm of rate constants against the inverse of absolute temperature as shown in Fig. 3. The activation energy derived from the slope of the straight line is 11.5 kcal/mol. Effect of ionic strength. The ionic strength of the medium was varied by adding concentrated solution of NaC104. Account of NaC104 and sodium acetate already present in the medium was taken when calculating ionic strength. The concentration of acetic acid added to adjust the pH was not added to the ionic strength as most of it remained undissociated contributing very few ions to the solution. The rate constants decreased with increase in
103/ T”K
Fig. 3. Plot of log k vs. l/T”K. pH, 5.0; ionic strength, 0.02 M.
Condition:
RNA concentration,
1.642
X
10e4 M(P);
130
6.2
.6.05,8
~""
~
~
5.6 i 1 L J 0.12 0.18 0.24 0.30 0.36 0.42 0.46 i1/2 Fig. 4. Plot of log k vs. I ~/2. Condition: RNA concentration, 1.642 X 10 -4 M(P); pH, 5.0; Room temperature, 25°--26°C.
ionic strength. The plot o f logarithm o f rate constant against square r o o t o f ionic strength (I 1/2) is shown in Fig. 4. The plot deviates from a straight line at higher ionic strength. The initial slope of the plot is a b o u t -2. Effect of pH. The p H o f the reaction medium was varied from 4.4 to 5.6. The rate constants decreased with increase in pH and the plot o f the logarithm o f rate constant against pH is a straight line with a slope equal to -0.8 as shown in Fig. 5. Effect of chloride ion. The rate shows a drastic decrease with the addition o f C1- ions which was much more than that expected from the effect o f ionic strength. A set of experiments was carried o u t b y adding different quantities o f NaC1. The concentration o f free C1- ions present in the medium due to the hydrolysis o f HAuCI4 was taken as two equivalents o f Au(III) as an approximation (see Discussion) and was taken into consideration when calculating pC1-. The plot o f logarithm of rate constant against pC1- is a straight line with a slope o f +0.32 {Fig. 6).
6.8
..J
6.0
5.6 J 4.4 4,,6
1
4.8
i
5.0 pH
l
5.2
L
5.4
5.6
Fig. 5. Plot of log k vs pH. Conditions R N A concentration, 1.642 x 10 -4 M(P); ionic strength, 0.02M; Room temperature 25°--26°C.
131
.~ 6.4 =.J
6.2
6.0 1.6
2.0
2.4
2.8 pCt-
3.2
3.6
Fig. 6. Plot of log k vs. pCl-. Condition: RNA concentration, 1.642 X 10 -4 M(P); pH, 4.6; ionic strength, 0.02 M (excluding NaC1); Room temperature 25°--26°C. PROPOSED
MECHANISM
AND
DISCUSSION
From the results presented above the following points can be made. (1) The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III). This indicates that two units of nucleotides are involved in binding with one Au(III) in the rate limiting step. (2) The stoichiometry of nucleotide to Au(III) is 2 : 1, which implies t h a t the overall ratio o f nucleotide to Au(III) is 2 : 1 and is achieved in the rate limiting step itself indicating that any fast step, if it occurs, does n o t alter the ratio o f nucleotide to Au(III). (3) Rate constants decrease with the increase in r-value, which is unexpected as the rate constants were calculated f r o m proper rate equations. Similar variation was also observed in our laboratory for native DNA-Au(III) and denatured DNA-Au(III) systems [4,8,9] as well as for other nucleic acidmetal ion systems [5,10]. The sharp decrease in rate constants in higher r-values for the Au(III)-nucleic acid systems has been attributed to two factors. Firstly, there may exist a number of reaction sites on the nucleic acids and different reacting species of Au(III) giving shght difference in reaction rates. At higher levels of reaction the number of reaction sites and reacting species with higher rates becomes less, resulting in an overall decrease in rate constant. This may also be the cause for the deviation of the f(x) vs. time plot beyond 60% reaction. Secondly, an important contribution to this sharp decrease comes from the effect of free C1- ions. In the reaction medium Au(III) exists mainly as the (AuCI2 (OH)2)-ion which is produced from (AuC14)- as follows [11].
(AuCL)- +20H-
(AuCI2(OH)2)- +2CI-
This process adds two equivalents of C1- ions to the medium. Thus the reaction mixtures with higher r-values contain more C1- ions. As the presence of C1- ions largely reduces the rate constants (see the effect of C1-ion) the
132 rate constant decreases with the increase in r-value. The effect of C1- ion has been confirmed in the case of native DNA-Au(III} system [4,8] with some additional experiments in which the concentration o f the free C1- ion was balanced. {4) An application of the Bjerrum-Bronsted equation (log k/ko = A • ZAZBI'P , where k0 and k are the rate constants at zero and at a finite ionic strength (I) respectively; ZA and ZB are the charges on the reacting species and A is a constant depending on dielectric contant and temperature and equals 1.02 for dilute aqueous solutions at 25°C) to the initial slope of the log k vs. I ' P plot (Fig. 4) indicates that the product of charges on the reacting species is - v e at least at lower ionic strengths. Considering the negative charge on nucleotide units due to phosphate, the charge on Au(III) comes to be +1. The major existing species of Au(III) in the medium is (AuC12 (OH)2which is negatively charged. But it can give rise to a positively charged species after dissociation as follows: (AuC12 ( O H ) 2 - a q u -.
" A u C I 2 ( H 2 0 ) 2+ + 2 0 H -
The formation of other positively charged species e.g., (AuCI(OH)) (H20)2* and (Au(OH)2) (H20)2 ÷ is also possible and these species m a y take part in the reaction giving slight differences in rates. These positively charged species have also been identified as the reacting species in the native DNA-Au(III) system [4,8]. In the case of n-DNA-Au(III) system the log k vs. 1lp plot is a straight line over a fair range of ionic strength, but in the case of the RNA-Au(III) system the plot is a curved one as shown b y the broken line in Fig. 4. This deviation may be due to the participation of the neutral species (AuC12(OH)) and (AuCI(OH)2), which are produced at the first step of dissociation o f (AuC12(OH)(, in the rate limiting step. These neutral species have been shown to participate in the reaction of Au(III) with denatured D N A [ 4 , 9 ] . It can be seen from Fig. 4. (broken line) that as the ionic strength increases the rate constants tend to be unaffected by ionic strength. This can be explained by the variation in the relative participation of the neutral and ionic species with the ionic strength. At lower ionic strength the positively charged species may be dominant reactants, b u t as the ionic strength increases the reaction rate o f the charged species decreases. Hence the relative participation of the neutral species increases, as a result, the rate constants tend to be unaffected by the increase of ionic strength and the log k vs. 1lp plot becomes almost parallel to the I~P-axis at high ionic strength. (5) The plot of the logarithm of the rate constant against pH is a straight line with a slope equal to - 0 . 8 . From the effect of pH only on the dissociation of (AuC12(OH)~)- to give (AuC12(H20)ff one expects a linear variation of log k with pH with a slope equal to - 2 . The lower observed slope may be attributed to an additional effect of pH on the rate limiting step (Fig. 7) in the opposite direction. (6) The decrease in rate constant with the addition of NaC1 can be ex-
133
E
AuCI2(OH)21- ~ - 2 O H +20H-
..~
Step-1 ( fast ) (RNA) Step-2
( Slow )
Comptexes with higher Au (II[) Content
Au ([[[)
Fig. 7. Schematic representation of the proposed mechanism.
plained by the masking of the reacting species by CI- ions according to the following equation: ( A u C 1 2 ( H 2 0 ) 2 + + CI-
"~ (AuCl3)aqu
All ~the other possible neutral and positive ions may also combine with C1ions to form inactive species. The slope of the plot of log k vs. pCl- (Fig. 6) is 0.32 which is equal to that for the native DNA-Au(III) system in which the positively charged species are the major reactants [4,8]. This strongly supports the concept that the positively charged species (AuC12(H20)2 + (as well as other positively charged species) is the dominant reacting species at low ionic strength (0.02M). Similar masking of reacting species by C1- ions was observed in the case of the cis-Pt(II)-DNA system [7] in which the reacting species are the mono- and di-positive ions. On the basis of all these results a mechanism has been proposed for the reaction of Au(III) with RNA as presented schematically in Fig. 7. Step 1 is the equilibrium dissociation of the major existing species (AuC12(OH)2)to give the reacting species, (AuC12(H20)~) ÷. This step is fast compared to the next step so that the equilibrium concentration of (AuC12(H20)2) + is always maintained in the medium. Had it been the rate limiting step the reaction would be first order with respect to Au(III), and independent of RNA concentration, but that was not observed. The positively charged gold species approaches the negatively charged phosphate of the nucleotide resulting in the formation of a transition complex. Step 2 is the slowest step and involves the reaction of two nucleotides of RNA with one (AuC12(H20)2)+
134 ion. The nucleotides may be the complementary ones in the double stranded region or the adjacent ones in the single stranded region o f RNA. The subsequent steps of the reaction may give rise to the formation of complexes with higher Au(III)-content b u t their kinetics could not be followed by this m e t h o d as the change in the variable parameter, A25o/A28o, is very small b e y o n d r = 0.5. The scheme presented in Fig. 7 is a general representation of a n u m b e r of processes in which the reaction sites and reacting species vary from one to the other. The other positively charged species such as (AuCI(OH) (H20)2)÷ and (Au(OH)2 (H20)2)÷ also participate in step 2 at low ionic strength. But at higher ionic strength the neutral species such as (AuC12(OH)), (AuCI(OH)2) etc. take part predominantly in the rate limiting step 2. All these species may exist in weakly hydrated forms satisfying higher coordination number. CONCLUSIONS
The paper describes the results of the first systematic kinetic studies on the interaction of Au(III) with RNA. A plausible mechanism is proposed which explains almost all the observations. Third order reactions are very unc o m m o n because of the very less probability o f triple collision. But, in this case, as the t w o nucleotide units take part in the reaction always remain very close to one another, the chance of the reactants coming together is quite high, permitting an overall third order reaction. Application of the BjerrumBronsted equation to a reaction involving three reactants requires a triple p r o d u c t of charges considering the reactants individually. Thus an individual consideration of the nucleotides gives the product of charges as - 1 which is not in accordance with the observation. But since the two nucleotides participating in the rate limiting step are held together the positively charged ion approaches towards t w o units o f negative charges in the formation of a transition complex. Hence the product of charges appears to b e - v e as, electrostatically, the process is the interaction between a doubly charged negative unit of nucleotides and a singly charged positive species of Au(III). ACKNOWLEDGEMENTS
The authors are thankful to Dr. V. Kalpagam for her interest in this investigation. This work was supported by the University Grants Commission, India. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
C.K.S. Pillai, Ph.D. Thesis, Indian Institute of Science, Bangalore, India, 1974. C.K.S. Pillai and U.S. Nandi, Biopolymers, 12 (1973) 1431. R. Vydra and J. Celikovsky, Collect. Czech. Chem. Commun., 23 (1958) 539. C. Mandal, Ph.D. Thesis, Indian Institute of Science, Bangalore, India, 1976. D.C. Liebe and J.E. Stuehr, Biopolymers, 11 (1972) 145 and 167. M.N. Williams and D.M. Crothers, Biochemistry, 14 (1975) 1944. P. Horacek and J. Drobnik, Biochim. Biophys. Acta, 254, {1971) 341. C. Mandal and U.S. Nandi, Communicated to Biopolymers. C. Mandal and U.S. Nandi, Communicated to European Journal of Biochemistry. M.N. Williams, Dissertation, Yale University, 1968. D.W. Gibson, Dissertation, John Hopkins University, Baltimore, Marland 1969.
125
KINETIC STUDIES ON THE INTERACTION OF GOLD (III) WITH NUCLEIC ACIDS. IV. RNA-Au (III) SYSTEM
CHHABINATH MANDAL and UMA SANKAR NANDI
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore560 012 (India) (Received July 8th, 1977) (Accepted January 14th, 1978)
SUMMARY
The kinetics of the interaction of Au(III) with whole yeast RNA has been studied using UV-spectrophotometry. The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. The effects of initial composition, temperature, ionic strength, pH and chloride ion on the kinetics have been studied. Activation energy is found to be 11.5 kcal/mol. Effect of ionic strength indicates that both the positively charged and neutral species o f Au(III) take part in the rate limiting step, the former being dominant at low ionic strength. A plausible mechanism has been proposed which involves the interaction of two nucleotide units of RNA with one species of Au(III) in the rate limiting step.
INTRODUCTION
Studies on the interaction of metal ions with nucleic acids have resulted is useful and interesting information on the secondary structures of nucleic acids and have led to a comprehensive understanding of the biological functions of nucleic acids at the molecular level. Metal ions are believed to hold R N A in specific conformation and stablize its ordered structure. This may bear a functional relationship to protein synthesis and the transmission of genetic information. Metal ions mainly interact with the phosphate moiety electrostaticaUy or with the bases of DNA. Such studies have already been shown to be useful in the separation and isolation of satellite DNA's, in cancer chemotherapy, in the determination of base sequence, and many others. Pt (II) compounds have been found to have antitumor activity and gold being isoelectronic with platinum is expected to exhibit similar behaviour. Pillai and Nandi studied the interaction of Au(III) with nucleic acids [1,2]. Au(III) forms complexes with native and denatured DNA, and RNA with drastic changes
126 in optical and hydrodynamic properties of the nucleic acids. They observed that the absorption maximum o f RNA shifted to higher wavelengths in complex formation along with hyperchromicity. In viscometric studies they observed a 2.7-fold decrease in nsp/c of RNA in the complex formation. The reaction between Au(III) and R N A was found to be quite slow, though it was the fastest among the nucleic acids studied [ 1 ] and the kinetics could be followed using the conventional techniques for slow reactions. MATERIALS AND METHODS
Material and sample preparation Highly polymerized whole yeast R N A was obtained from BDH, Poole, United Kingdom. The R N A was dissolved in acetate buffer containing sodium perchlorate (0.01 M), sodium acetate (0.01 M) and adequate quantity of acetic acid to adjust to the desired pH. The stock R N A solution was standardized spectrophotometrically using ( P ) m a s = 8.7 X 103 A M -1 • cm -1 [1], where (P) denotes concentration o f the phosphate moeity of RNA. Chloroauric acid, HAuCla • 3 H 2 0 , was supplied b y Johnson Mathey Chemicals Ltd., London. The stock solution was prepared in doubly distilled water and the concentration was determined spectrophotometrically according to Vydra and Celikovsky [3] using e313 = 5.5 X 103 A M-' • cm -1. All other reagents were of the Analytical Reagent Grade and the solutions were prepared in d o u b l y distilled water (pH, 6.6--6.8).
Experimental The UV-spectra were recorded using an UNICAM SP700A double beam automatic recording spectrophotometer equipped with heating and thermostating devices. Two matched quartz cells of path length 1 cm were used for taking samples and reference solutions. Since Au(III) absorbs in the UV region, difference spectra of the RNA-Au(III) solutions were taken against Au(III) solutions under almost identical conditions. The desired volumes o f stock Au(III) solutions were added to the buffer medium 48 h before starting the reaction so as to attain the hydrolytic equilibria of AuC17,. The concentration of R N A was maintained at 1.642 × 10 -4 M (P) unless otherwise stated. For kinetic experiments, transmittances at two wavelengths e.g., 250 and 280 nm, were recorded at the desired intervals of time. From these transmittances the ratios of absorptions at 250 nm to that at 280 nm (A25o/A28o) were calculated and used for calculating the extent of reaction. Time taken to record the transmittance at a given wavelength was about 2 sec. Experiments were carried o u t in acetate buffer at room temperature maintained at 25°C unless otherwise stated. For experiments above room temperature two accessories (SP770 and SP775, constant temperature cell unit and electrical controller) of the s p e c t r o p h o t o m e t e r were used. The temperature could be controlled within + 0.05°C. In the kinetic studies of nucleic acid-metal ion systems using spectrophotometer different parameters e.g., absorbances at
127 260 nm [5] and 290 nm [10] and the absorbance ratio A2so/A27o [7] were used to follow the course of the reactions. For this system we used the ratio A2so/A2ao, because this parameter varies linearly with the extent of reaction.
Treatment of kinetic data The experimental data were subjected to fit the classical rate equations for a number o f combinations o f stoichiometry (2 : 1, 1 : I and 1 : 2) and orders (first and second orders with respect to each) of nucleotide unit of RNA and Au(III) using a F O R T R A N IV c o m p u t e r programme. For all the combinations, the functions, f(x), of the extent o f reaction (x) were derived and the standard deviations o f the plots of f(x) vs. time were calculated. The minimum standard deviation indicated the best fit of the experimental data to the corresponding equation. From the slope of this best fitted plot the rate constant was obtained. More details about the functioning and listing of the programme are available elsewhere [4]. The e x t e n t of reaction was calculated using the following equation. P0 - Pt x = P0 - P0o
"
CRNA(P)
where P0, Pt and P0o are the values of the time
Equilibrium results In order to examine the nature of changes in the spectral properties of RNA in complex formation a set of equilibrium experiments were carried out. The reaction mixtures were prepared with a constant RNA concentration (= 0.821 × 10 -4 M(P)) and different amounts of Au(III) to get r-values between 0.0 and 2.0 (r = number of moles of Au(III)/number of moles of RNA(P)). After 24 h UV-spectra were recorded in the range 200 nm to 3 0 0 nm followed by another set of recordings after 48 h; no appreciable change was observed indicating t h a t 24 h was sufficient to reach equilibrium. The absorption m a x i m u m shifted to a higher wavelength along with hyperchromicity as reported earlier [1]. Absorptions at both the wavelengths e.g. 250 nm and 280 nm increase with the increase in r-value but at lower r-values (0.5) the increase in the latter wavelength is more compared to that at the former. Consequently the ratio A2so/A2ao decreases sharply in going from r = 0.0 to r = 0.5 as shown in Fig. 1. The plot of A:so/A28o vs. r-value shows a break near r = 0.5 and the decrease in A2so/A2ao with r-value is much less in going from r = 0.5 to r =
128 1.8
°16 c~c° "1.4 O t~ o4
1.2 1.0 0
1 0.4.
I 0,8
L 1.2 r-VaLue
I 1.6
I 2.0
Fig. 1. Plot o f A2so/A28 o vs. r-value. C o n d i t i o n : R N A c o n c e n t r a t i o n , 0 . 8 2 1 X 10 -4 M(P); pH, 5.0; ionic s t r e n g t h , 0.02 M; R o o m t e m p e r a t u r e , 25°--26°C.
2.0 comprising only 15% o f the total change. This indicates that the sharp change in A2so/A28o characterizes a complex of stoichiometry 2 : 1 with respect to nucleotide unit of R N A and Au(III) respectively. In the kinetic studies of the nucleic acid-metal ion systems using spectrap h o t o m e t r y various parameters e.g. absorptions at 260 nm [5] and at 290 nm [6] and the ratio o f absorptions (A2so/A2~o) [7] have been used. For the present system we used the ratio A2so/A28o for calculating the extent of reaction because it is the only parameter varying linearly with the extent of reaction (Fig. 1 ).
Kinetic results For all the experimental sets with varying r-values, temperature, ionic strength, pH etc. the kinetic data gave the best fit to the rate equations carresponding to second order with respect to nucleotide unit of RNA and first order with respect to Au(III) in the respective stoichiometry of 2 : 1. Fig. 2 shows the plot of f(x) vs. time for the above mentioned combination of 16 i._: 12
8
'o
0
0
10
20
30 4.0 Time (rain.)
50
60
Fig. 2. Plot o f f(x) vs. time. C o n d i t i o n : R N A c o n c e n t r a t i o n , 1.642 × 10 4 M (P); pH, 5.0; ionic s t r e n g t h , 0.02 M; R o o m t e m p e r a t u r e , 25 ° - 2 6 ° C .
129 TABLE I DEPENDENCE
OF RATE CONSTANTS
Condition: RNA concentration, temperature, 25”-26°C.
R-value Rate constant X 10e6 (I’ - molmz . min-’ )
1.642
x
ON r-VALUE. 10m4 M(P); pH, 5.0;ionic
strength, 0.02 M; Room
0.50
0.75
1.00
1.25
1.50
2.717
1.925
1.407
1.224
1.106
stoichiometry and orders. Only 50-6096 of the total change in the spectral variable, A250/A280, fitted a straight line. At higher level of reaction the plot deviates to give a lower slope. Effect of r-value. A set of experiments was carried out with a constant initial concentration of RNA and variable concentrations of Au(III), the range of the r-values being between 0.5 and 1.5. The results of a typical set of experiments are presented in Table I. The rate constants are found to decrease with the increase in r-value. Effect of temperature. The effect of temperature was studied in the range 25”-50°C at six different temperatures. The components of both the sample and reference solutions were preheated to the desired temperature and placed in the thermostated chamber of the spectrophotometer after rapid mixing. Arrhenius plots were obtained by plotting the logarithm of rate constants against the inverse of absolute temperature as shown in Fig. 3. The activation energy derived from the slope of the straight line is 11.5 kcal/mol. Effect of ionic strength. The ionic strength of the medium was varied by adding concentrated solution of NaC104. Account of NaC104 and sodium acetate already present in the medium was taken when calculating ionic strength. The concentration of acetic acid added to adjust the pH was not added to the ionic strength as most of it remained undissociated contributing very few ions to the solution. The rate constants decreased with increase in
103/ T”K
Fig. 3. Plot of log k vs. l/T”K. pH, 5.0; ionic strength, 0.02 M.
Condition:
RNA concentration,
1.642
X
10e4 M(P);
130
6.2
.6.05,8
~""
~
~
5.6 i 1 L J 0.12 0.18 0.24 0.30 0.36 0.42 0.46 i1/2 Fig. 4. Plot of log k vs. I ~/2. Condition: RNA concentration, 1.642 X 10 -4 M(P); pH, 5.0; Room temperature, 25°--26°C.
ionic strength. The plot o f logarithm o f rate constant against square r o o t o f ionic strength (I 1/2) is shown in Fig. 4. The plot deviates from a straight line at higher ionic strength. The initial slope of the plot is a b o u t -2. Effect of pH. The p H o f the reaction medium was varied from 4.4 to 5.6. The rate constants decreased with increase in pH and the plot o f the logarithm o f rate constant against pH is a straight line with a slope equal to -0.8 as shown in Fig. 5. Effect of chloride ion. The rate shows a drastic decrease with the addition o f C1- ions which was much more than that expected from the effect o f ionic strength. A set of experiments was carried o u t b y adding different quantities o f NaC1. The concentration o f free C1- ions present in the medium due to the hydrolysis o f HAuCI4 was taken as two equivalents o f Au(III) as an approximation (see Discussion) and was taken into consideration when calculating pC1-. The plot o f logarithm of rate constant against pC1- is a straight line with a slope o f +0.32 {Fig. 6).
6.8
..J
6.0
5.6 J 4.4 4,,6
1
4.8
i
5.0 pH
l
5.2
L
5.4
5.6
Fig. 5. Plot of log k vs pH. Conditions R N A concentration, 1.642 x 10 -4 M(P); ionic strength, 0.02M; Room temperature 25°--26°C.
131
.~ 6.4 =.J
6.2
6.0 1.6
2.0
2.4
2.8 pCt-
3.2
3.6
Fig. 6. Plot of log k vs. pCl-. Condition: RNA concentration, 1.642 X 10 -4 M(P); pH, 4.6; ionic strength, 0.02 M (excluding NaC1); Room temperature 25°--26°C. PROPOSED
MECHANISM
AND
DISCUSSION
From the results presented above the following points can be made. (1) The reaction is second order with respect to the nucleotide unit of RNA and first order with respect to Au(III). This indicates that two units of nucleotides are involved in binding with one Au(III) in the rate limiting step. (2) The stoichiometry of nucleotide to Au(III) is 2 : 1, which implies t h a t the overall ratio o f nucleotide to Au(III) is 2 : 1 and is achieved in the rate limiting step itself indicating that any fast step, if it occurs, does n o t alter the ratio o f nucleotide to Au(III). (3) Rate constants decrease with the increase in r-value, which is unexpected as the rate constants were calculated f r o m proper rate equations. Similar variation was also observed in our laboratory for native DNA-Au(III) and denatured DNA-Au(III) systems [4,8,9] as well as for other nucleic acidmetal ion systems [5,10]. The sharp decrease in rate constants in higher r-values for the Au(III)-nucleic acid systems has been attributed to two factors. Firstly, there may exist a number of reaction sites on the nucleic acids and different reacting species of Au(III) giving shght difference in reaction rates. At higher levels of reaction the number of reaction sites and reacting species with higher rates becomes less, resulting in an overall decrease in rate constant. This may also be the cause for the deviation of the f(x) vs. time plot beyond 60% reaction. Secondly, an important contribution to this sharp decrease comes from the effect of free C1- ions. In the reaction medium Au(III) exists mainly as the (AuCI2 (OH)2)-ion which is produced from (AuC14)- as follows [11].
(AuCL)- +20H-
(AuCI2(OH)2)- +2CI-
This process adds two equivalents of C1- ions to the medium. Thus the reaction mixtures with higher r-values contain more C1- ions. As the presence of C1- ions largely reduces the rate constants (see the effect of C1-ion) the
132 rate constant decreases with the increase in r-value. The effect of C1- ion has been confirmed in the case of native DNA-Au(III} system [4,8] with some additional experiments in which the concentration o f the free C1- ion was balanced. {4) An application of the Bjerrum-Bronsted equation (log k/ko = A • ZAZBI'P , where k0 and k are the rate constants at zero and at a finite ionic strength (I) respectively; ZA and ZB are the charges on the reacting species and A is a constant depending on dielectric contant and temperature and equals 1.02 for dilute aqueous solutions at 25°C) to the initial slope of the log k vs. I ' P plot (Fig. 4) indicates that the product of charges on the reacting species is - v e at least at lower ionic strengths. Considering the negative charge on nucleotide units due to phosphate, the charge on Au(III) comes to be +1. The major existing species of Au(III) in the medium is (AuC12 (OH)2which is negatively charged. But it can give rise to a positively charged species after dissociation as follows: (AuC12 ( O H ) 2 - a q u -.
" A u C I 2 ( H 2 0 ) 2+ + 2 0 H -
The formation of other positively charged species e.g., (AuCI(OH)) (H20)2* and (Au(OH)2) (H20)2 ÷ is also possible and these species m a y take part in the reaction giving slight differences in rates. These positively charged species have also been identified as the reacting species in the native DNA-Au(III) system [4,8]. In the case of n-DNA-Au(III) system the log k vs. 1lp plot is a straight line over a fair range of ionic strength, but in the case of the RNA-Au(III) system the plot is a curved one as shown b y the broken line in Fig. 4. This deviation may be due to the participation of the neutral species (AuC12(OH)) and (AuCI(OH)2), which are produced at the first step of dissociation o f (AuC12(OH)(, in the rate limiting step. These neutral species have been shown to participate in the reaction of Au(III) with denatured D N A [ 4 , 9 ] . It can be seen from Fig. 4. (broken line) that as the ionic strength increases the rate constants tend to be unaffected by ionic strength. This can be explained by the variation in the relative participation of the neutral and ionic species with the ionic strength. At lower ionic strength the positively charged species may be dominant reactants, b u t as the ionic strength increases the reaction rate o f the charged species decreases. Hence the relative participation of the neutral species increases, as a result, the rate constants tend to be unaffected by the increase of ionic strength and the log k vs. 1lp plot becomes almost parallel to the I~P-axis at high ionic strength. (5) The plot of the logarithm of the rate constant against pH is a straight line with a slope equal to - 0 . 8 . From the effect of pH only on the dissociation of (AuC12(OH)~)- to give (AuC12(H20)ff one expects a linear variation of log k with pH with a slope equal to - 2 . The lower observed slope may be attributed to an additional effect of pH on the rate limiting step (Fig. 7) in the opposite direction. (6) The decrease in rate constant with the addition of NaC1 can be ex-
133
E
AuCI2(OH)21- ~ - 2 O H +20H-
..~
Step-1 ( fast ) (RNA) Step-2
( Slow )
Comptexes with higher Au (II[) Content
Au ([[[)
Fig. 7. Schematic representation of the proposed mechanism.
plained by the masking of the reacting species by CI- ions according to the following equation: ( A u C 1 2 ( H 2 0 ) 2 + + CI-
"~ (AuCl3)aqu
All ~the other possible neutral and positive ions may also combine with C1ions to form inactive species. The slope of the plot of log k vs. pCl- (Fig. 6) is 0.32 which is equal to that for the native DNA-Au(III) system in which the positively charged species are the major reactants [4,8]. This strongly supports the concept that the positively charged species (AuC12(H20)2 + (as well as other positively charged species) is the dominant reacting species at low ionic strength (0.02M). Similar masking of reacting species by C1- ions was observed in the case of the cis-Pt(II)-DNA system [7] in which the reacting species are the mono- and di-positive ions. On the basis of all these results a mechanism has been proposed for the reaction of Au(III) with RNA as presented schematically in Fig. 7. Step 1 is the equilibrium dissociation of the major existing species (AuC12(OH)2)to give the reacting species, (AuC12(H20)~) ÷. This step is fast compared to the next step so that the equilibrium concentration of (AuC12(H20)2) + is always maintained in the medium. Had it been the rate limiting step the reaction would be first order with respect to Au(III), and independent of RNA concentration, but that was not observed. The positively charged gold species approaches the negatively charged phosphate of the nucleotide resulting in the formation of a transition complex. Step 2 is the slowest step and involves the reaction of two nucleotides of RNA with one (AuC12(H20)2)+
134 ion. The nucleotides may be the complementary ones in the double stranded region or the adjacent ones in the single stranded region o f RNA. The subsequent steps of the reaction may give rise to the formation of complexes with higher Au(III)-content b u t their kinetics could not be followed by this m e t h o d as the change in the variable parameter, A25o/A28o, is very small b e y o n d r = 0.5. The scheme presented in Fig. 7 is a general representation of a n u m b e r of processes in which the reaction sites and reacting species vary from one to the other. The other positively charged species such as (AuCI(OH) (H20)2)÷ and (Au(OH)2 (H20)2)÷ also participate in step 2 at low ionic strength. But at higher ionic strength the neutral species such as (AuC12(OH)), (AuCI(OH)2) etc. take part predominantly in the rate limiting step 2. All these species may exist in weakly hydrated forms satisfying higher coordination number. CONCLUSIONS
The paper describes the results of the first systematic kinetic studies on the interaction of Au(III) with RNA. A plausible mechanism is proposed which explains almost all the observations. Third order reactions are very unc o m m o n because of the very less probability o f triple collision. But, in this case, as the t w o nucleotide units take part in the reaction always remain very close to one another, the chance of the reactants coming together is quite high, permitting an overall third order reaction. Application of the BjerrumBronsted equation to a reaction involving three reactants requires a triple p r o d u c t of charges considering the reactants individually. Thus an individual consideration of the nucleotides gives the product of charges as - 1 which is not in accordance with the observation. But since the two nucleotides participating in the rate limiting step are held together the positively charged ion approaches towards t w o units o f negative charges in the formation of a transition complex. Hence the product of charges appears to b e - v e as, electrostatically, the process is the interaction between a doubly charged negative unit of nucleotides and a singly charged positive species of Au(III). ACKNOWLEDGEMENTS
The authors are thankful to Dr. V. Kalpagam for her interest in this investigation. This work was supported by the University Grants Commission, India. REFERENCES 1 2 3 4 5 6 7 8 9 10 11
C.K.S. Pillai, Ph.D. Thesis, Indian Institute of Science, Bangalore, India, 1974. C.K.S. Pillai and U.S. Nandi, Biopolymers, 12 (1973) 1431. R. Vydra and J. Celikovsky, Collect. Czech. Chem. Commun., 23 (1958) 539. C. Mandal, Ph.D. Thesis, Indian Institute of Science, Bangalore, India, 1976. D.C. Liebe and J.E. Stuehr, Biopolymers, 11 (1972) 145 and 167. M.N. Williams and D.M. Crothers, Biochemistry, 14 (1975) 1944. P. Horacek and J. Drobnik, Biochim. Biophys. Acta, 254, {1971) 341. C. Mandal and U.S. Nandi, Communicated to Biopolymers. C. Mandal and U.S. Nandi, Communicated to European Journal of Biochemistry. M.N. Williams, Dissertation, Yale University, 1968. D.W. Gibson, Dissertation, John Hopkins University, Baltimore, Marland 1969.