527

Biochimica et Biophysica Acta, 563 (1979) 527--533 © Elsevier/North-Holland Biomedical Press

BBA 99488

INTERACTION OF RHODIUM(III) WITH DNA

R. SASI and U.S. NANDI Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012 (India)

(Received July 18th, 1978) (Revised manuscript received February 13th, 1979) Key words: DNA structure; Rhodium-DNA binding

Summary The nature of interaction o f Rh(III) with DNA was studied using viscometry and ultraviolet, visible and infrared spectroscopy. The rate of interaction was found to be very slow at room temperature taking several days for completion. The time needed to attain equilibrium is dependent on the concentrations of metal ion, higher the concentration shorter the period required for equilibration. Visible spectra o f Rh(III) were found to alter considerably in the presence o f DNA. An increase in absorbance and a red shift were observed in the ultraviolet spectra of DNA in the presence of Rh(III). The specific viscosity of DNA solution was found to decrease asymptotically with time and concentrations of metal ion. The melting temperature of DNA was f o u n d to increase at lower metal ion concentrations, whereas at higher values a decrease was obtained. At still higher metal ion concentrations (metal ion/DNA-P > 3) a 'nonmeltable state' of DNA was observed. These results seem to indicate that Rh(III) binds both with the phosphate and the bases of the DNA.

Introduction Following the report by Rosenberg et al. of the anti-tumour and antiviral properties of platinum group metals, considerable interest has developed in the biological and medicinal properties of these c o m p o u n d s [1--3]. Heavy metals like Hg(II) [4,5], Ag(I) [6l, Pt(II) [7,8], Au(III) [9--11] and Pd(II} [12] have been studied in detail, but work on Rh(III) has hardly been reported. Platinum metal co-ordination complexes form a new class of anti-tumour drugs and a series of these c o m p o u n d s have successfully undergone phase I and phase II clinical trials in humans and animals [13--16]. The base specific inter-

528 action has been exploited recently in the sequencing of DNA by electron microscopy where DNA is labelled with heavy metal ions like Pt and Au [17]. Although platinum metal complexes have been found to be effective as drugs for more than 28 types of tumours, they are extremely toxic and can have major pathological effects in humans [18--19]. It is reported that rhodium c o m p o u n d s have antiviral, antibacterial and anticarcinogenic activities similar to platinum c o m p o u n d s [20--24]. Moreover, rhodium c o m p o u n d s are less toxic and in certain cases more effective [24]. Since rhodium c o m p o u n d s are of great interest in cancer chemotherapy, we thought it worthwhile to investigate the nature of interaction of Rh(III) with DNA. Materials and Methods Calf thymus DNA was obtained from Swarch Bioresearch Inc. and was used w i t h o u t further purification. D N A was dissolved by adding small amounts of buffer at intervals of 3--4 h for 2--3 days at 4°C with occasional gentle stirring. The stock solution was diluted to the desired concentration before use. The concentration of DNA solution was determined by measuring the absorbance (A) at 258 nm. RhCl3 . x H 2 0 was purchased from Johnson Matheys Chemicals Ltd. (London). Analytical grade NaC104 • 2H20 (E. Merck, F.R.G.) was used as salt medium. All other chemicals used were of analytical grade. Double-distilled de-ionised water was used in all experiments. All measurements were done at pH 5 using acetic acid/sodium acetate buffer (0.01 M) containing 0.01 M NaC104. Acetate buffer was f o u n d to be most suitable for the present studies. R h o d i u m was estimated spectrophotometricaUy by developing colour with stannous chloride [25]. Since RhC13 solution absorbs in the ultraviolet region b e y o n d 240 nm, an equal a m o u n t of RhC13 solution was added to the reference solution also to cut off its absorption and the difference spectra were taken [26]. A Beckman Double Beam S p e c t r o p h o t o m e t e r Model 25 was used for the ultraviolet and visible spectral studies. For melting temperature (Tin) measurements, UNICAM. S.P. 700, double beam s p e c t r o p h o t o m e t e r with two accessories, contact temperature cell unit (S.P. 770) and electrical controller (S.P. 775) were used. The cell temperature could be controlled to within _+0.2°C. Viscosity measurements were carried o u t at 30°C using Ubbelhode viscometer. Results and Discussion The interaction of Rh(III) with D N A was found to be very slow at room temperature taking weeks for completion. In order to accelerate the reaction, solutions were mixed and kept at 37°C in a thermostat for 3--4 days. It was found that at 37°C it takes about 96 h for equilibrium t o be reached. A number o f chloro-aquo complexes o f Rh(III) are reported to exist in solution, depending upon the chloride ion concentration and pH o f the solution [27]. Spectra of Rh(HI) ~ g e d considerably on addition of chloride ions. Rhodium trichloride w ~ dissolved in the acetate buffer at pH 5 and kept

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Fig. 1. E f f e c t o f Calf t h y m u s D N A on t h e visible s p e c t r a o f R h ( I I I ) a f t e r r e a c h i n g e q u i l i b r i u m . D N A conc e n t r a t i o n , 1 . 0 7 4 • 10 -4 M(P). A c e t a t e b u f f e r , p H 5 (0.01 M) c o n t a i n i n g 0.01 M N a C 1 0 4 . r = gaol of R h ( I I I ) a d d e d / t o o l of D N A (P). 1, r - - O; 2, r = 1 3 . 1 ; 3, r = 9.1. Fig. 2. E f f e c t o f R h ( I I I ) o n t h e u l t r a v i o l e t s p e c t r a of Calf t h y m u s D N A w i t h tL'ne at r = 2.5. C o n c e n t r a t i o n of D N A , 1 . 4 7 7 • 1 0 -4 M(P). C o n c e n t r a t i o n of R h ( I I I ) , 3.69 • 10 -`4 M. A c e t a t e b u f f e r , p H 5 (0.01 M) c o n t a i n i n g 0.01 M N a C 1 0 4 , 1, r = 0 ( D N A a l o n e ) ; 2, 0 h; 3, 6 h ; 4, 10 h; 5, 24 h; 6, 4 8 h; 7, 6 0 h; 8, 72 h; 9,84h;lO, 97h.

for 6--7 days to reach equilibrium. The visible spectra of Rh(III) in acetate buffer at pH 5 (0.01 M} containing (0.01 M} NaC104 had two peaks at 380 nm and 475 nm. Wolsey et al. [27] have isolated all the species o f Rh(III) in aqueous solution and have reported the absorption spectra. The predominant species present were found to be [RhC12(H~O)4] ~! and [RhC13(H20)3] under these conditions. The effect of DNA on the visible spectrum of Rh(III) is as shown in Fig. 1. On addition of DNA to the Rh(III) solution, the peaks were found to shift to 395 and 490 nm, respectively. The ultraviolet spectra of DNA were found to be greatly altered on addition o f RhC13 to DNA. The interaction seems to be a slow process and at 37°C takes 96 h to attain equilibrium. With increasing metal ion concentrations the equilibrium was attained faster. There was an increase in the absorbance and a shift in the )~max of DNA on addition of Rh(III). Both the hyperchromicity as well as the e x t e n t of shift were progressive in nature and f o u n d to be dependant on the concentration of the metal ion. Thus a shift in the )~max of DNA from 258 nm to 272 nm after reaching equilibrium occurs at r = 2.5 (Fig. 2) (r = mol o f RhCl3 added/mol o f DNA-P) It was found that there was no change in the absorbance, if DNA was kept alone for even more than five days w i t h o u t the addition of Rh(III), thereby indicating that denaturation is n o t operative. Since purines and pyrimidines are responsible for the absorption

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at 258 nm, the spectra indicate the binding o f metal to the base moieties of DNA. Fig. 2 represents a typical run showing the change in the ultraviolet spectra at r = 2.5 with time. Fig. 3 represents ultraviolet spectra at different r values after equilibrium is attained. U p o n addition of Rh(III) the viscosity of DNA solution decreases considerably with time reaching an almost constant value after 72 h (Figs. 4 and 5). This decrease resembles those of Pd(II)-DNA and Au(III)-DNA systems [9,12, 30]. In the case of Rh(III) the decrease in viscosity is very slow compared to the above systems [9,12]. Figs. 4 and 5 show the effect of Rh(III) on 778p o f DNA solution. ~sp is found to decrease asymptotically with increase in the r values, as shown in Fig. 5. Both the spectral and the viscosity changes at low r values are reversible on adding potassium oxalate, potassium thiocyanate and ethyienediammine maintained at the same pH and ionic strength [30,34]. This probably indicates, as has been reported earlier, that the binding involves the bases thereby, bridging the two strands in the place of H-bonds. pH titration studies indicated the release of H ÷ ion, b u t quantitatively n o t equivalent to 2 (also f o u n d in other studies) [30,34]. Since at the present stage we cannot definitely say a b o u t the binding site, we would refrain from any conclusion on this aspect. The melting temperature (Tin) measurements o f DNA and its complexes with Rh(III) in acetate buffer at pH 5 (0.01 M) containing NaC104 (0.01 M) were carried o u t next (Fig. 6). This was done using the t w o accessories S.P. 770 and S.P. 775 in the UNICAM S.P. 700 s p e c t r o p h o t o m e t e r where the sample and the

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reference solutions were heated, and absorbances at 260 nm were measured at different temperatures. Adopting the standard procedure, the Tm was determined from the plot of absorbance against temperature [28,29,31]. The Tm of native DNA was found to be 65°C, while for DNA-Rh(III) complexes at low r values (r = 0.4) it was higher. As the values of r increased b e y o n d 0.4 there was a decrease in Tm till at r = 1.8 the value became 59°C. This indicates that at lower concentrations, Rh(III) stabilizes the DNA helix by interacting with the phosphate backbone of DNA as has been shown by many authors for other metals in particular Eichhorn et al. [28--30]. The Tm decrease indicates the destabilization of the helix, involving binding mainly with the bases of the D N A [29]. As is now well k n o w n [28--30] at high ionic strength say 0.2 M, the phosphate binding can be completely eliminated and one can pinpoint the base binding by using higher ionic strength. We have therefore c o n d u c t e d Tm studies o f D N A in acetate buffer, pH 5 (0.01 M) containing 0.2 M NaC104. At this ionic strength the Tm measurements indicate that it decreases even at low r values also (r < 0.5) and there was no increase in Tm even for r value of 0.05. This result is a clear indication of the absence of binding o f phosphate with Rh(III) at high ionic strength. In fact decrease in Tm possibly indicates binding o f Rh(III) to the bases and this is also supported by ultraviolet spectral data. The infrared spectra of Rh(III)-DNA complex at r = 0.3 obtained by precipitating with alcohol showed a decrease in the intensity of the band at 1242 cm -1 due to phosphate of DNA with a shift to 1224 cm -1. According to Sutherland and Tsuboi [32], the bands at 1242 cm -1 of DNA is mainly due to antisymmetric stretching vibrations of the phosphate, and the change in the position o f the band at 1242 cm -1 is a direct indication of the phosphate binding [32,33].

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Combining all these results from ultraviolet and visible spectra, viscosity, pH titrations, reversibility of the interaction Tm measurements and indication of infrared spectra, we feel it would not be unreasonable to conclude that Rh(III) binds to the phosphate and to the bases, the former binding being more predominant at low r values. As observed in Fig. 6 that for r > 3 the melting profile becomes fiat showing no change in the absorbance with temperature which has been termed by some authors in the literature as 'non-meltable state' [30]. This probably indicates, that there exist, extensive metal binding throughout the DNA-helix replacing H-bond by metal bonds rendering the complex 'non-meltable' as has been observed with Pt, Pd and Au [9,10,12,30]. Condusion Ultraviolet spectral studies of Rh(III)-DNA system indicated the binding of DNA with Rh(III). Since purines and pyrimidines are responsible for the absorption at 258 nm, the red shift in the spectra show that the binding occurs with the bases. The disruption of the hydrogen bonds between base pairs and consequent binding of Rh(III) to the bases might have given rise to a shift in the positions of kmax. However, the increase in Tm at lower r values ( r less than 0.4) suggest that the double helical structure of DNA is stabilised by binding of Rh(III) with phosphate of DNA. These results are similar to those of Au(III).DNA [30] and Cu(H)-DNA [28] systems and indicate that Rh(III) binds with the phosphate and the bases of the DNA.

533

References 1 Rosenberg, B., Van Camp, L., Trosko, J.E. and Mansour, V.H. (1969) Nature 222, 385--386 2 Rosenberg, B. and Van Camp, L. (1970) Cancer Res. 30, 1 7 9 9 - - 1 8 0 2 3 Roberts, J.J. (1974) in R e c e n t Results in Cancer Research (Connors, T.A. and Roberts, J.J., eds.), Vol. 48, pp. 7 9 - 9 7 . Springer-Verlag, Heidelberg and New Y ork 4 Nandi, U.S., Wang, J.C. and Davidson, N. (1965) Biochemistry 4, 1 6 8 7 - - 1 6 9 6 5 Wang, J.C., Nandi, U.S. and Davidson, N. (1965) Biochemistry 4, 1 6 9 7 - - 1 7 0 2 6 Arya, S.K. and Yang, J.T. (1975) Biopolymers 14, 1847--1861 7 Howe-Grant, M., Wu, K.C., Bauer, W.R. and Lippard, S.J. (1976) Biochemistry 15, 4 3 3 9 - 4 3 4 6 8 MiUard, M.M., Maquet, J.P. and Theophanides, T. (1975) Bioehim. Biophys. Acta 402, 166--170 9 Pinai, C.K.S. and Nandi, U.S. (1973) Biopolymers 12, 1431--1435 10 PiUai, C.K.S. and Nandi, U.S. (1972) I n t e r a c t i o n of Gold(III) w i t h nucleic acids, Abs. p. 114, Convention of Chemists, Allahabad 11 Chatterji, D., Nandi, U.S. and Podder, S.K. (1977) Biopolyme r 16, 1863--1878 12 Pillai, C.K.S. and Nandi, U.S. (1977) Biochim. Biophys. Acta 473, 11--16 13 Rosenberg, B. (1973) Naturwlssenschaften 60, 399---406 14 Burchenal, J.H., O'Toolee, T. Kalaher, K. and Chisholm, J. (1977) Cancer. Res. 37, 4 0 9 8 - - 4 1 0 0 1 5 Thomson, A.J. (1977) Platinum Met. Rev. 21, 2--15 16 Cleate, M.J. (1 974) Co-Ordi. Chem. Rev. 12, 3 4 9 - 4 0 5 17 Agarwal, S.K., Wagner, R.W., McAllister, P.K. and Rosenberg, B. (1975) Proc. Natl. Acad. Sci. U.S. 72, 928--932 18 TothAllen, J. (1970) Ph.D. thesis, Michigan State University, MI, U.S.A. 19 Roasof, A.H., Slayton, R.E. and Pertia, C.P. (1972) Cancer Res. 30, 1 4 5 1 - - 1 4 5 6 20 Bromfield, R.J., Dainty, R.H., Gillard, R.D. and Heaton, B.T. (1969) Nature 223, 735--736 21 Gillard, R.D. (1970) Platinum Met. Rev. 14, 50--53 22 Erick, A., Sherwood, E., Beat, J.L. and Kimbal, A.P. (1976) Cancer Res. 36, 2204--2209 23 Bragadin, C.M., Giraldi, T., Cantini, M., Zassinovich, G. and Mestroni, G. (1974) FEBS Lett. 43, 13--16 24 Giraldi, T., Sava, G., Bestoli, G., Mestroni, G. and Zassinovich, G. (1977) Cancer Res. 37, 2662-2666 25 Kasthuneu, J.O. and Evans, H.B. (1960) Anal. Chem. 32, 917--920 26 Jorgeson, C.K. (1962) Absorption spectra and Chemical bondi ng in Complexes, Pergamon PresS, Oxford 27 Wolsey, W.C., Reynolds, C.A. and Kleinberg, J. (1963) In. Chem. 2, 463---468 28 Eichhorn, G.L. and Shin, Y.A. (1968) J. Am. Chem. Soc. 90, 7323--7328 29 Eichhorn, G.L. and Clark, P. (1965) Proc. Natl. Acad. ScL U.S. 53, 586--593 30 Pillai, C.K.S. and Nandi, U.S. (1978) Biopolymers 17, 7 0 9 - 7 2 9 31 Gruenwedel, D.M. (1974) Biochim. Biophys. Acta 340, 16--30 32 Sutherland, G.B.B.M. and Tsuboi, M. (1957) Proc. R. Soc. London, A 239, 446--463 33 Tsuboi, M. (1957) J. Am. Chem. Soc. 79, 1 3 5 1 - - 1 3 5 4 34 Yamane, T. and Davidson, N. (1961) J. Am. Chem. Soc. 83, 2 5 9 9 - 2 6 0 7