ANALYTICAL
BIOCHEMISTRY
89, 22-30 (1978)
Platinum Determination in DNA- Platinum Complexes by Fluorescence Spectrophotometry JEAN-LUC BUTOUR AND JEAN-PIERRE MACQUET’ Laboratoire
de Pharmacologic et de Toxicologic Fondamentales Narbonne, 31078 Toulouse Cedex, France
du CNRS,
205 route
de
Received August 3, 1977 A new method of platinum determination in a series of DNA-platinum complexes using a fluorescence technique is presented. The interaction between DNA and the platinum compounds able to form either cis- or transbidentate complexes can be quantified. The fixation of these platinum compounds on DNA inhibits the intercalation of ethidium bromide (EtdBr) and induces a linear decrease of the DNA-EtdBr fluorescence up to rb = 0.20. The method has been tested in different ionic media and over a wide range of ionic strength. Similar results were obtained with different DNAs, and no significant dependence on the G + C content was detected. This technique allowed the detection of one platinum atom fixed per 200 nucleotides.
Since Rosenberg et al. discovered the antitumor properties of cis-platinum compounds (1,2), a great deal of work has been concerned with platinum complexation on macromolecules of biological interest. Among the techniques used for platinum analysis, atomic absorption spectrophotometry is the best known. Platinum compounds complexed with biological samples and nucleic acids in vitro were easily determined using flame atomic absorption (3-5). The in vivo platinum distribution in cells, organs, blood, or urine was studied with graphite furnaces (6-8). Several authors have proposed other techniques for platinum determination based on the use of molecules radiolabeled with 14C, 3H or ls5’Vt (9- 11) or on calorimetric assay (12). We propose a new method for platinum determination in DNAZ-platinum complexes based on an interesting observation reported in previous studies (13). We have shown that the fixation of some platinum(I1) compounds inhibits the interaction of intercalating agents such as ethidium bromide with DNA. This inhibition was demonstrated for the platinum 1 To whom reprint requests should be addressed. * Abbreviations used: DNA, deoxyribonucleic acid; EtdBr, ethidium bromide, 3,8-diamino6-phenyl-5-ethylphenanthridinium bromide; en, ethylenediamine, H2N-CHp-CH2-NH*; dien, bis(2-aminoethyl)amine, H2N-CH,-CH,-NH-CH,-CH,-NH2; G + C, guaninecytosine base pair. 0003-2697/78/0891-0022$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
22
SPECTROFLUOROMETRIC
DETERMINATION TABLE
23
OF Pt
1
FORMULATION AND DENOMINATION OF THE PLATINUM COMFQUNDS USED IN THE PRESENT STUDY Platinum compounds
Chemical denomination
[WNHMJICI [Pt(dien)Cl]Cl c~s-P~(NH~)~CI~ cis-Pt(en)Cl* rrans-Pt(NH,),Cl,
cis-[Pt(NH3,(H,O),lcNO,), cis-[Pt(en)(H,O)ll(NO,), rvans-[Pt(NH,),(H,O),](NO,), KVWNHJCl,l
K,[PGI
Chlorotriammineplatinum(I1) chloride Chlorodiethylenetriamineplatinum(I1) chloride cis-Dichlorodiammineplatinum(I1) cis-Dichloroethylenediamineplatinum(II) rrans-Dichlorodiammineplatinum(I1) cis-Diaquodiammineplatinum(I1) nitrate cis-Diaquoethylenediamineplatinum(I1) nitrate trans-Diaquodiammineplatinum(I1) nitrate Potassium trichloromonoammineplatinate(II) Potassium tetrachloroplatinate(I1)
Number of labile sites 1 1 2 2 2” 2 2 2 3 4
a truns-Pt(NH&Cl, was found to react with DNA either by displacement of one (monodentate complex) or two chlorine atoms (nuns-bidentate complex).
compounds which are able to form a cis- or truns-bidentate complex with the DNA bases. The following platinum series was found to modify the DNA secondary structure (13,14): cis-Pt(NH,),Cl,, cis-[Pt(NH,),(H,O),] (NO,),, cis-Pt(en)Cl,, cis-[Pt(en)(HzO),](NO&, trans-Pt(NH,)&, frans[Pt(NH,),(H,O),](NO,),, K[Pt(NH,)CI,], and K,[PtCl,I. The DNA modification was interpreted in terms of local denaturations due to platinum fixation. The complexation of these platinum compounds induces a fluorescence decrease in DNA-EtdBr complexes. A linear relationship was found between the fluorescence decrease and rb (number of platinum atoms fixed per nucleotide) up to 0.20. We propose the use of this fluorescence decrease as an analytical tool for platinum determination in DNA-platinum complexes studied either in vitro or in viva. MATERIALS
AND METHODS
Platinum compounds. Potassium tetrachloroplatinate was bought from “La compagnie des metaux precieux” (Paris, France) and was the starting salt for the other platinum compounds which were synthesized as previously reported (15-20). The hydrolyzed platinum compounds cis- and trans-[Pt(NH,),(H,O),1(NO,), and cis-[Pt(en)(H,O),](NO& were prepared overnight in the dark at 50°C by addition of two equivalents of silver nitrate to the corresponding chloro species. The silver chloride formed was eliminated by filtration. All the platinum compounds were recrystallized at least twice before use and are presented in Table 1. Suits. The different salts, NaCl, KN03, NaNO,, KCl, NaClO,, and
24
BUTOUR
AND
MACQUET
Na$O,, were purchased from Prolabo (Paris, France), Fluka (Buchs, Switzerland), and Merck (Darmstadt, Germany). Ethidium bromide (3 &diamino-6-phenyl-5-ethylphenanthridinium bromide) was bought from Sigma Chemical Co. (St. Louis, MO., U.S.A.), its molar absorption coefficient was 5450 M-‘cm-’ at A = 480 nm in water (21). Nucleic acids. Clostridium perfringens (32% G + C; Ebb,, = 6100), Escherichia coli (50% G + C; Earn = 6100), and Micrococcus lysodeikticm (72% G + C; E(P)~~~= 6270) DNAs were purchased from Sigma Chemical Co. (St. Louis, MO., U.S.A.) and salmon sperm DNA (41% G + C; 4Q266 = 6300) from Worthington Biochemical Corp. (Freehold, New Jersey, U.S.A.). Protein and RNA content were determined as previously reported (22,23) and were found to be less than 1%. DNA -platinum complexes. DNA solutions (approximately 1 mg/ml) were prepared by gentle stirring of DNA in an aqueous 10e2 M NaClO, solution at 4°C for 48 hr. The platinum compounds were weighed and dissolved in a lop2 M NaClO, solution just before use, and aliquots of these mother solutions were added to DNA (0.250 mg/ml as final concentration). DNA-F? complexes (final volume of 1 ml) were prepared by stirring at room temperature in the dark for 2 days. The pH values of the different DNA-F? solutions varied from 5.8 to 6.2. Platinum content in each complex was analyzed by atomic absorption spectrophotometry (4). Spectrophotometric measurements. Ultraviolet spectrophotometric and spectrofluorometric measurements were performed with a Zeiss PMQII spectrophotometer equipped with a ZFM4 fluorescence attachment. Excitation light (546 nm) was provided by a mercury lamp and selected with a M 546 filter. Emitted fluorescence was measured at 590 nm. Fluorescence measurements were expressed by the ratio Z1/ZO,where I, = fluorescence intensity of the DNA-Pt-EtdBr complex minus fluorescence intensity of pure EtdBr and I0 = fluorescence intensity of the DNA-EtdBr complex minus fluorescence intensity of pure EtdBr. The accuracy on Z,lI,, is better than 3%. These measurements were performed at 25°C in 0.4 M KN03 to avoid the nonfluorescent fixation site of EtdBr to DNA (21). In the standard conditions the final volume was 2.5 ml, and the concentrations were 0.01 mg/ml for DNA and 0.04 mg/ml for EtdBr corresponding to the saturation of all the intercalation sites in DNA. It should be noted that the above standard procedure may be performed using a smaller volume (less than 1 ml) with a DNA concentration ten times lower (lOea g/ml). The amount of chlorine ions liberated during the complexation of transPt(NH&C12 with DNA was measured by potentiometric determination (24). Two Ag/AgCl electrodes were immersed into two cells (one with the DNA-platinum complex, the other with DNA alone as a reference) connected with a salt bridge. The potential between the two cells was determined with a digital Minisis 5000 voltmeter (Tacussel) with an accuracy of 5 0.1 mV.
SPECTROFLUOROMETRIC
DETERMINATION
25
OF Pt
RESULTS DNA and DNA-Pt
Saturation with EtdBr
A saturation curve is obtained [ref. (13), Fig. 1 ] when increased EtdBr concentrations are added to a constant concentration of salmon sperm DNA (0.01 mgiml). DNA or DNA-Et saturation with EtdBr is achieved when fluorescence intensity is constant. The complexation of one platinum atom per 10 nucleotides (rb = 0.10) leads to a fluorescence decrease of about 50%. Ultraviolet spectrophotometric studies have shown that this fluorescence decrease was not due to quenching by heavy atom effect (13). Fluorescence Decrease
The percent of fluorescence decrease (II/Z,) for a series of DNA-EtEtdBr complexes synthesized with salmon sperm DNA was plotted against the number of platinum atoms (rb) bound per nucleotide [ref. (13) Figs. 2, 3, and 41. I, is the fluorescence value obtained on each plateau for the different DNA-Et-EtdBr complexes, I0 being the fluorescence of the DNA-EtdBr complex. The results obtained for salmon sperm DNA complexed with cis-
- 0.025 - 0.050 0.75
- 0.075
.
- 0.100 2 >
- 0.125 - 0.150
CL50
2
[SALT] x Y
FIG. 1. Fluorescence changes obtained with a series of DNA-R complexes prepared with salmon sperm DNA and cis-Pt(NH,),Cl, in the presence of different ionic species. The platinum content corresponded to rb = 0.20. The different curves correspond to: NaCI, 0; KCI, W; KN03, A; NaN03, V‘; NaC104, A; and Na$04, V.
26
BUTOUR
AND MACQUET
FIG. 2. Relationship between the fluorescence decrease and the number of platinum atoms fixed per nucleotide in different DNA-Pt complexes prepared with Clostridium perfringens DNA and: Kz[PtC14], A; cisPt(NH,)&I,, n ; cis-Pt(en)Cl*, 0; and trans-Pt(NH,),Cl,, A.
PfW-WX
312, cis-Pt(en)Cl,, and cis-[Et(en) c~~-[P~(NH~)~(H~O),I(NO (H,O),](NO,), were already presented in ref. 13 [(Fig. 3)]. These platinum compounds gave similar results and were found to be fixed to DNA presumably on the same sites. Moreover the ratio Z,/ZO vs rb is linear up to rb = 0.20. A 50% fluorescence decrease is obtained for these cis-chelate DNA-Et complexes when rb equals 0.125. The fluorescence decrease obtained with the DNA-F? complexes prepared from K[A(NH,)Cl,] and K,[PtCLJ was found to be slightly greater than those obtained with the cis-F’t compounds, except in the case of Clostridium perfringens. This was attributed to a more important perturbation of the DNA secondary structure (13,14). trans-Pt(NHJ,Cl,
With the trans-Pt(NH&Cl~ compound, two types of DNA-Pt complexes were characterized (13,14). These two types were differentiated by the number of platinum fixation sites on DNA, either one as in the monodentate DNA-Et complex or two in the trans-bidentate DNA-Et complex. When only one chlorine atom is displaced, the fluorescence decrease of the DNA-Et-EtdBr complexes was about 25% for rb = 0.20 [ref. (13,
SPECTROFLUOROMETRIC
DETERMINATION
OF Pt
27
{Fig. 4})]. The displacement of the second chlorine atom leads, as in the case of the &-platinum compounds, to a fluorescence decrease of 65% (r,, = 0.20). The complexation oftrans-[Pt(NH,),(H,O),](NO,), with DNA also induces a fluorescence decrease of 65% (r,, = 0.20). The different binding sites of cis- and trans-platinum compounds to DNA were already discussed (13,24,25). Salt Effects In Fig. 1 the fluorescence change of the DNA-Pt-EtdBr complexes synthesized between cis-Pt(NH&C& and salmon sperm DNA is presented as a function of concentration for different salts. trans-Pt(NH&& and K2[PtC14] gave the same type of curves. The DNA-Pt complexes were prepared during 1 week at room temperature in the dark. The platinum compounds introduced corresponded at complete fixation to rb = 0.20. The influence of different amounts of KCl, NaCl, NgSO,, NaNO,, NaClO,, and KNOB was studied. Fluorescence measurements were performed as previously described in 0.4 M KN03 (see Materials and Methods). The results show that chlorine ions strongly inhibit the platinum complexation on DNA. A complete platinum fixation was obtained when the chlorine ion content was less than lop3 M for [DNA(P)] = 8. 10e4 M and Yb = 0.20. Just a slight effect was noticed for the other salts.
FIG. 3. Fluorescence Micrococcus
lysodeikricus
decrease for a series of DNA-R complexes prepared DNA (see legend to Fig. 2 for explanation of symbols).
with
28
BUTOURANDMACQUET
G + C Content Effect To test the influence of the G + C content in DNA on the fluorescence decrease, a series of DNA-Et complexes were prepared with four different sources of DNA: Clostridium perfringens (32% G + C), salmon sperm (41% G + C), Escherichia co/i (50% G + C), and Micrococcus lysodeikticus (72% G + C). In Figs. 2 and 3 it can be seen that the results are similar with the complexes prepared from cis-Pt(NH,),Clz, cis-Pt(en)Cl,, and truns-Pt(NH,),Cl, and that there is no influence of the G + C content. In each case an rb of 0.125 gave a fluorescence decrease of about 50%. The DNA-Et complexes prepared with K,[PtCl.J corresponding to rb = 0.125 gave a fluorescence decrease of about 70%, except in the case of Clostridium perfringens (Fig. 2) where the decrease is only 55%. In this later case the difference could be due to a greater reactivity and a lower specificity of the K,[ACl,] compound as previously reported (13), and it has also been noted in a circular dichroism study (25). DISCUSSION
The analytical method described in this paper allows the determination of platinum(I1) compounds having at least two labile sites and able to form a cis- or trans-bidentate complex with DNA. This new method of platinum assay is very simple to achieve and does not require expensive materials. The results are reproducible and accurate (0 to 4%). The detection limit (taken as twice the background) was found to be 5.10~* g/ml of platinum which is quite similar to the results obtained by flame atomic absorption spectrophotometry (5). This value corresponds to the fixation of one platinum atom per 200 to 250 nucleotides. The interaction of platinum compounds with DNA having only one labile [Pt(NH,),Cl]Cl, site has also been studied. This is the case for [F’t(dien)Cl]Cl, and trans-Pt(NH&Cl, (under restrictive conditions for this compound) which make one DNA-Pt bond. The DNA secondary structure is not strongly altered, and EtdBr can intercalate quite normaly in the DNA-Et complexes synthesized with these platinum compounds (13,14). In ref. (13, [Figs 2 and 41) one can see that the complexation of these salts gave a weak fluorescence decrease, less than 20% at saturation (rb = 0.20). Consequently, it is not possible to calculate accurately the quantity of platinum atoms fixed to DNA using this technique. This new analytical method for platinum determination in DNA-platinum complexes using the DNA secondary structure perturbations induced by the platinum compounds does not imply a purification of the complexes. This is a very interesting positive point for this technique compared with atomic absorption spectrophotometry. If no significant DNA secondary structure perturbations are detected, however, the use of atomic
SPECTROFLUOROMETRIC
DETERMINATION
29
OF Pt 0 - 0.025 - 0.050 - 0.075 - 0.100 . 0.125 . 0.150 2 - 0.200
. 0.300
0
1
2
3
4 TIME
5
6
(hours)
FIG. 4. Kinetics of DNA-platinum complexation. The reactions were run at 37°C in lo-* M NaClO, (10 ml), and the platinum compounds introduced corresponded to 0.40 Pt atom per nucleotide. At the starting point, platinum solutions (in lo-* M NaClO,) were added to salmon sperm DNA (0.25 mgiml). Aliquots (0.10 ml) were taken at different times and added to 2.4 ml of EtdBr (0.04 mgiml) in 0.4 M KNO,. Fluorescence measurements were performed as previously described, and platinum bound to DNA was calculated from ref. (13, [Figs. 3 and 41).
absorption spectrophotometry is required. An other application of this technique was carried out and is given in Fig. 4 where the kinetics of cis-Pt(NH,),Cl, and ~runs-F’t(NH~)~Cl~ with salmon sperm DNA were directly followed. From this figure it can be seen that these two platinum isomers react differently with DNA, the tran~-Pt(NH~)~Cl~ compound reacting more rapidly than the ciS-Et(NH3)2C12 which seemed to exhibit a cooperative binding. In conclusion, this study clearly demonstrates that EtdBr can be used as an analytical probe for platinum analysis in bidentate DNA-Et complexes. Moreover, analysis of the kinetics of platinum fixation on DNA is very easy to achieve using this fluorescence technique but is also limited to cis- or trans-bidentate platinum compounds. ACKNOWLEDGMENTS The authors want to thank Dr. J. Hubert (Montreal, Canada) and Dr. P. Poilblanc (Toulouse, France) for critical readings of the manuscript.
30
BUTOUR
AND
MACQUET
REFERENCES 1. Rosenberg, B., Van Camp, L., and Krigas, T. (1965) Nature (London) 205, 698-699. 2. Rosenberg, B., Van Camp, L., Trosko, J. E., and Mansour, V. H. (1%9) Nature (London) 222, 385-386. 3.
4. 5. 6. 7.
Miller, R. G., and Doerger, J. U. (1975) Af. Absorption Newsle?t. 14, 66-67. Macquet, J. P., and Theophanides, T. (1975) Biopolymers 14, 781-799. Macquet, J. P., Hubert, J., and Theophanides, T. (1974)Anal. Chim. Acta 72,251-259. Pascoe, J. M., and Roberts, J. J. (1974) Biochem. Pharmacol. 23, 1345-1357. Litterst, C. L., Gram, T. E., Dedrick, R. L., Leroy, A. F., and Guarino, A. M. (1976) Cancer
Res.
36, 2340-2344.
Jones, A. H. (1976) Anal. Chem. 48, 1472-1474. 9. Wolf, W., and Manaka, R. C. (1977) J. Clin. Hematol. 8.
Oncol.
Wadley
Med.
Bull.
7,
79-95.
10. Howle, J. A., Gale, G. R., and Smith, A. B. (1972)Biochem. Pharmacol. 21, 1465-1475. 11. Gale, G. R., Rosenblum, M. G., Atkins, L. M., Walker, E. M., Jr., Smith, A. B., and Meischen, S. J. (1973) J. Nat. Cancer Inst. 51, 1227-1234. 12. Whiting, R. F., and Ottensmeyer, F. P. (1977) Biochim. Biophys. Acta 474, 334-348. 13. Butour, J. L., and Macquet, J. P. (1977) Eur. J. Biochem. 78, 455-463. 14. Macquet, J. P., and Butour, J. L. (1977) J. Clin. Hematol. Oncol. Wadley Med. Bull. 7, 469-486.
15. Kong, P. C., and Rochon, F. D. (1975) Chem. Commun. 15, 599-600. 16. Kauffman, G. B., and Cowan, D. 0. (1963) Inorganic Syntheses, Vol. 7, pp. 239-245, McGraw-Hill, New York. 17. Johnson, G. L. (1966) Inorganic Syntheses, Vol. 8, pp. 242-244, McGraw-Hill, New York. 18. Mann, F. G. (1934) J. Chem. Sot., 466-474. 19. Tschugaev, L. A. (1915) J. Chem. Sot., 1247-1250. 20. Keller, R. N. (1946) Inorganic Syntheses, Vol. 2, pp. 250-253, McGraw-Hill, New York. 21. Le Pecq, J. B. (1971) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 20, pp. 41-86, Wiley, New York. 22. Lowry, 0. H., Rosebrough, M. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem.
193, 265-275.
23. Defiance, P., and Delesdain, N. (1%2) Pathol. Biol. 10, 153-155. 24. Macquet, J. P., and Theophanides, T. (1975) Bioinorg. Chem. 5, 59-66. 25. Macquet, J. P., and Butour, J. L. (1978) Eur. J. Biochem. 83, 375-387.
BIOCHEMISTRY
89, 22-30 (1978)
Platinum Determination in DNA- Platinum Complexes by Fluorescence Spectrophotometry JEAN-LUC BUTOUR AND JEAN-PIERRE MACQUET’ Laboratoire
de Pharmacologic et de Toxicologic Fondamentales Narbonne, 31078 Toulouse Cedex, France
du CNRS,
205 route
de
Received August 3, 1977 A new method of platinum determination in a series of DNA-platinum complexes using a fluorescence technique is presented. The interaction between DNA and the platinum compounds able to form either cis- or transbidentate complexes can be quantified. The fixation of these platinum compounds on DNA inhibits the intercalation of ethidium bromide (EtdBr) and induces a linear decrease of the DNA-EtdBr fluorescence up to rb = 0.20. The method has been tested in different ionic media and over a wide range of ionic strength. Similar results were obtained with different DNAs, and no significant dependence on the G + C content was detected. This technique allowed the detection of one platinum atom fixed per 200 nucleotides.
Since Rosenberg et al. discovered the antitumor properties of cis-platinum compounds (1,2), a great deal of work has been concerned with platinum complexation on macromolecules of biological interest. Among the techniques used for platinum analysis, atomic absorption spectrophotometry is the best known. Platinum compounds complexed with biological samples and nucleic acids in vitro were easily determined using flame atomic absorption (3-5). The in vivo platinum distribution in cells, organs, blood, or urine was studied with graphite furnaces (6-8). Several authors have proposed other techniques for platinum determination based on the use of molecules radiolabeled with 14C, 3H or ls5’Vt (9- 11) or on calorimetric assay (12). We propose a new method for platinum determination in DNAZ-platinum complexes based on an interesting observation reported in previous studies (13). We have shown that the fixation of some platinum(I1) compounds inhibits the interaction of intercalating agents such as ethidium bromide with DNA. This inhibition was demonstrated for the platinum 1 To whom reprint requests should be addressed. * Abbreviations used: DNA, deoxyribonucleic acid; EtdBr, ethidium bromide, 3,8-diamino6-phenyl-5-ethylphenanthridinium bromide; en, ethylenediamine, H2N-CHp-CH2-NH*; dien, bis(2-aminoethyl)amine, H2N-CH,-CH,-NH-CH,-CH,-NH2; G + C, guaninecytosine base pair. 0003-2697/78/0891-0022$02.00/O Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
22
SPECTROFLUOROMETRIC
DETERMINATION TABLE
23
OF Pt
1
FORMULATION AND DENOMINATION OF THE PLATINUM COMFQUNDS USED IN THE PRESENT STUDY Platinum compounds
Chemical denomination
[WNHMJICI [Pt(dien)Cl]Cl c~s-P~(NH~)~CI~ cis-Pt(en)Cl* rrans-Pt(NH,),Cl,
cis-[Pt(NH3,(H,O),lcNO,), cis-[Pt(en)(H,O)ll(NO,), rvans-[Pt(NH,),(H,O),](NO,), KVWNHJCl,l
K,[PGI
Chlorotriammineplatinum(I1) chloride Chlorodiethylenetriamineplatinum(I1) chloride cis-Dichlorodiammineplatinum(I1) cis-Dichloroethylenediamineplatinum(II) rrans-Dichlorodiammineplatinum(I1) cis-Diaquodiammineplatinum(I1) nitrate cis-Diaquoethylenediamineplatinum(I1) nitrate trans-Diaquodiammineplatinum(I1) nitrate Potassium trichloromonoammineplatinate(II) Potassium tetrachloroplatinate(I1)
Number of labile sites 1 1 2 2 2” 2 2 2 3 4
a truns-Pt(NH&Cl, was found to react with DNA either by displacement of one (monodentate complex) or two chlorine atoms (nuns-bidentate complex).
compounds which are able to form a cis- or truns-bidentate complex with the DNA bases. The following platinum series was found to modify the DNA secondary structure (13,14): cis-Pt(NH,),Cl,, cis-[Pt(NH,),(H,O),] (NO,),, cis-Pt(en)Cl,, cis-[Pt(en)(HzO),](NO&, trans-Pt(NH,)&, frans[Pt(NH,),(H,O),](NO,),, K[Pt(NH,)CI,], and K,[PtCl,I. The DNA modification was interpreted in terms of local denaturations due to platinum fixation. The complexation of these platinum compounds induces a fluorescence decrease in DNA-EtdBr complexes. A linear relationship was found between the fluorescence decrease and rb (number of platinum atoms fixed per nucleotide) up to 0.20. We propose the use of this fluorescence decrease as an analytical tool for platinum determination in DNA-platinum complexes studied either in vitro or in viva. MATERIALS
AND METHODS
Platinum compounds. Potassium tetrachloroplatinate was bought from “La compagnie des metaux precieux” (Paris, France) and was the starting salt for the other platinum compounds which were synthesized as previously reported (15-20). The hydrolyzed platinum compounds cis- and trans-[Pt(NH,),(H,O),1(NO,), and cis-[Pt(en)(H,O),](NO& were prepared overnight in the dark at 50°C by addition of two equivalents of silver nitrate to the corresponding chloro species. The silver chloride formed was eliminated by filtration. All the platinum compounds were recrystallized at least twice before use and are presented in Table 1. Suits. The different salts, NaCl, KN03, NaNO,, KCl, NaClO,, and
24
BUTOUR
AND
MACQUET
Na$O,, were purchased from Prolabo (Paris, France), Fluka (Buchs, Switzerland), and Merck (Darmstadt, Germany). Ethidium bromide (3 &diamino-6-phenyl-5-ethylphenanthridinium bromide) was bought from Sigma Chemical Co. (St. Louis, MO., U.S.A.), its molar absorption coefficient was 5450 M-‘cm-’ at A = 480 nm in water (21). Nucleic acids. Clostridium perfringens (32% G + C; Ebb,, = 6100), Escherichia coli (50% G + C; Earn = 6100), and Micrococcus lysodeikticm (72% G + C; E(P)~~~= 6270) DNAs were purchased from Sigma Chemical Co. (St. Louis, MO., U.S.A.) and salmon sperm DNA (41% G + C; 4Q266 = 6300) from Worthington Biochemical Corp. (Freehold, New Jersey, U.S.A.). Protein and RNA content were determined as previously reported (22,23) and were found to be less than 1%. DNA -platinum complexes. DNA solutions (approximately 1 mg/ml) were prepared by gentle stirring of DNA in an aqueous 10e2 M NaClO, solution at 4°C for 48 hr. The platinum compounds were weighed and dissolved in a lop2 M NaClO, solution just before use, and aliquots of these mother solutions were added to DNA (0.250 mg/ml as final concentration). DNA-F? complexes (final volume of 1 ml) were prepared by stirring at room temperature in the dark for 2 days. The pH values of the different DNA-F? solutions varied from 5.8 to 6.2. Platinum content in each complex was analyzed by atomic absorption spectrophotometry (4). Spectrophotometric measurements. Ultraviolet spectrophotometric and spectrofluorometric measurements were performed with a Zeiss PMQII spectrophotometer equipped with a ZFM4 fluorescence attachment. Excitation light (546 nm) was provided by a mercury lamp and selected with a M 546 filter. Emitted fluorescence was measured at 590 nm. Fluorescence measurements were expressed by the ratio Z1/ZO,where I, = fluorescence intensity of the DNA-Pt-EtdBr complex minus fluorescence intensity of pure EtdBr and I0 = fluorescence intensity of the DNA-EtdBr complex minus fluorescence intensity of pure EtdBr. The accuracy on Z,lI,, is better than 3%. These measurements were performed at 25°C in 0.4 M KN03 to avoid the nonfluorescent fixation site of EtdBr to DNA (21). In the standard conditions the final volume was 2.5 ml, and the concentrations were 0.01 mg/ml for DNA and 0.04 mg/ml for EtdBr corresponding to the saturation of all the intercalation sites in DNA. It should be noted that the above standard procedure may be performed using a smaller volume (less than 1 ml) with a DNA concentration ten times lower (lOea g/ml). The amount of chlorine ions liberated during the complexation of transPt(NH&C12 with DNA was measured by potentiometric determination (24). Two Ag/AgCl electrodes were immersed into two cells (one with the DNA-platinum complex, the other with DNA alone as a reference) connected with a salt bridge. The potential between the two cells was determined with a digital Minisis 5000 voltmeter (Tacussel) with an accuracy of 5 0.1 mV.
SPECTROFLUOROMETRIC
DETERMINATION
25
OF Pt
RESULTS DNA and DNA-Pt
Saturation with EtdBr
A saturation curve is obtained [ref. (13), Fig. 1 ] when increased EtdBr concentrations are added to a constant concentration of salmon sperm DNA (0.01 mgiml). DNA or DNA-Et saturation with EtdBr is achieved when fluorescence intensity is constant. The complexation of one platinum atom per 10 nucleotides (rb = 0.10) leads to a fluorescence decrease of about 50%. Ultraviolet spectrophotometric studies have shown that this fluorescence decrease was not due to quenching by heavy atom effect (13). Fluorescence Decrease
The percent of fluorescence decrease (II/Z,) for a series of DNA-EtEtdBr complexes synthesized with salmon sperm DNA was plotted against the number of platinum atoms (rb) bound per nucleotide [ref. (13) Figs. 2, 3, and 41. I, is the fluorescence value obtained on each plateau for the different DNA-Et-EtdBr complexes, I0 being the fluorescence of the DNA-EtdBr complex. The results obtained for salmon sperm DNA complexed with cis-
- 0.025 - 0.050 0.75
- 0.075
.
- 0.100 2 >
- 0.125 - 0.150
CL50
2
[SALT] x Y
FIG. 1. Fluorescence changes obtained with a series of DNA-R complexes prepared with salmon sperm DNA and cis-Pt(NH,),Cl, in the presence of different ionic species. The platinum content corresponded to rb = 0.20. The different curves correspond to: NaCI, 0; KCI, W; KN03, A; NaN03, V‘; NaC104, A; and Na$04, V.
26
BUTOUR
AND MACQUET
FIG. 2. Relationship between the fluorescence decrease and the number of platinum atoms fixed per nucleotide in different DNA-Pt complexes prepared with Clostridium perfringens DNA and: Kz[PtC14], A; cisPt(NH,)&I,, n ; cis-Pt(en)Cl*, 0; and trans-Pt(NH,),Cl,, A.
PfW-WX
312, cis-Pt(en)Cl,, and cis-[Et(en) c~~-[P~(NH~)~(H~O),I(NO (H,O),](NO,), were already presented in ref. 13 [(Fig. 3)]. These platinum compounds gave similar results and were found to be fixed to DNA presumably on the same sites. Moreover the ratio Z,/ZO vs rb is linear up to rb = 0.20. A 50% fluorescence decrease is obtained for these cis-chelate DNA-Et complexes when rb equals 0.125. The fluorescence decrease obtained with the DNA-F? complexes prepared from K[A(NH,)Cl,] and K,[PtCLJ was found to be slightly greater than those obtained with the cis-F’t compounds, except in the case of Clostridium perfringens. This was attributed to a more important perturbation of the DNA secondary structure (13,14). trans-Pt(NHJ,Cl,
With the trans-Pt(NH&Cl~ compound, two types of DNA-Pt complexes were characterized (13,14). These two types were differentiated by the number of platinum fixation sites on DNA, either one as in the monodentate DNA-Et complex or two in the trans-bidentate DNA-Et complex. When only one chlorine atom is displaced, the fluorescence decrease of the DNA-Et-EtdBr complexes was about 25% for rb = 0.20 [ref. (13,
SPECTROFLUOROMETRIC
DETERMINATION
OF Pt
27
{Fig. 4})]. The displacement of the second chlorine atom leads, as in the case of the &-platinum compounds, to a fluorescence decrease of 65% (r,, = 0.20). The complexation oftrans-[Pt(NH,),(H,O),](NO,), with DNA also induces a fluorescence decrease of 65% (r,, = 0.20). The different binding sites of cis- and trans-platinum compounds to DNA were already discussed (13,24,25). Salt Effects In Fig. 1 the fluorescence change of the DNA-Pt-EtdBr complexes synthesized between cis-Pt(NH&C& and salmon sperm DNA is presented as a function of concentration for different salts. trans-Pt(NH&& and K2[PtC14] gave the same type of curves. The DNA-Pt complexes were prepared during 1 week at room temperature in the dark. The platinum compounds introduced corresponded at complete fixation to rb = 0.20. The influence of different amounts of KCl, NaCl, NgSO,, NaNO,, NaClO,, and KNOB was studied. Fluorescence measurements were performed as previously described in 0.4 M KN03 (see Materials and Methods). The results show that chlorine ions strongly inhibit the platinum complexation on DNA. A complete platinum fixation was obtained when the chlorine ion content was less than lop3 M for [DNA(P)] = 8. 10e4 M and Yb = 0.20. Just a slight effect was noticed for the other salts.
FIG. 3. Fluorescence Micrococcus
lysodeikricus
decrease for a series of DNA-R complexes prepared DNA (see legend to Fig. 2 for explanation of symbols).
with
28
BUTOURANDMACQUET
G + C Content Effect To test the influence of the G + C content in DNA on the fluorescence decrease, a series of DNA-Et complexes were prepared with four different sources of DNA: Clostridium perfringens (32% G + C), salmon sperm (41% G + C), Escherichia co/i (50% G + C), and Micrococcus lysodeikticus (72% G + C). In Figs. 2 and 3 it can be seen that the results are similar with the complexes prepared from cis-Pt(NH,),Clz, cis-Pt(en)Cl,, and truns-Pt(NH,),Cl, and that there is no influence of the G + C content. In each case an rb of 0.125 gave a fluorescence decrease of about 50%. The DNA-Et complexes prepared with K,[PtCl.J corresponding to rb = 0.125 gave a fluorescence decrease of about 70%, except in the case of Clostridium perfringens (Fig. 2) where the decrease is only 55%. In this later case the difference could be due to a greater reactivity and a lower specificity of the K,[ACl,] compound as previously reported (13), and it has also been noted in a circular dichroism study (25). DISCUSSION
The analytical method described in this paper allows the determination of platinum(I1) compounds having at least two labile sites and able to form a cis- or trans-bidentate complex with DNA. This new method of platinum assay is very simple to achieve and does not require expensive materials. The results are reproducible and accurate (0 to 4%). The detection limit (taken as twice the background) was found to be 5.10~* g/ml of platinum which is quite similar to the results obtained by flame atomic absorption spectrophotometry (5). This value corresponds to the fixation of one platinum atom per 200 to 250 nucleotides. The interaction of platinum compounds with DNA having only one labile [Pt(NH,),Cl]Cl, site has also been studied. This is the case for [F’t(dien)Cl]Cl, and trans-Pt(NH&Cl, (under restrictive conditions for this compound) which make one DNA-Pt bond. The DNA secondary structure is not strongly altered, and EtdBr can intercalate quite normaly in the DNA-Et complexes synthesized with these platinum compounds (13,14). In ref. (13, [Figs 2 and 41) one can see that the complexation of these salts gave a weak fluorescence decrease, less than 20% at saturation (rb = 0.20). Consequently, it is not possible to calculate accurately the quantity of platinum atoms fixed to DNA using this technique. This new analytical method for platinum determination in DNA-platinum complexes using the DNA secondary structure perturbations induced by the platinum compounds does not imply a purification of the complexes. This is a very interesting positive point for this technique compared with atomic absorption spectrophotometry. If no significant DNA secondary structure perturbations are detected, however, the use of atomic
SPECTROFLUOROMETRIC
DETERMINATION
29
OF Pt 0 - 0.025 - 0.050 - 0.075 - 0.100 . 0.125 . 0.150 2 - 0.200
. 0.300
0
1
2
3
4 TIME
5
6
(hours)
FIG. 4. Kinetics of DNA-platinum complexation. The reactions were run at 37°C in lo-* M NaClO, (10 ml), and the platinum compounds introduced corresponded to 0.40 Pt atom per nucleotide. At the starting point, platinum solutions (in lo-* M NaClO,) were added to salmon sperm DNA (0.25 mgiml). Aliquots (0.10 ml) were taken at different times and added to 2.4 ml of EtdBr (0.04 mgiml) in 0.4 M KNO,. Fluorescence measurements were performed as previously described, and platinum bound to DNA was calculated from ref. (13, [Figs. 3 and 41).
absorption spectrophotometry is required. An other application of this technique was carried out and is given in Fig. 4 where the kinetics of cis-Pt(NH,),Cl, and ~runs-F’t(NH~)~Cl~ with salmon sperm DNA were directly followed. From this figure it can be seen that these two platinum isomers react differently with DNA, the tran~-Pt(NH~)~Cl~ compound reacting more rapidly than the ciS-Et(NH3)2C12 which seemed to exhibit a cooperative binding. In conclusion, this study clearly demonstrates that EtdBr can be used as an analytical probe for platinum analysis in bidentate DNA-Et complexes. Moreover, analysis of the kinetics of platinum fixation on DNA is very easy to achieve using this fluorescence technique but is also limited to cis- or trans-bidentate platinum compounds. ACKNOWLEDGMENTS The authors want to thank Dr. J. Hubert (Montreal, Canada) and Dr. P. Poilblanc (Toulouse, France) for critical readings of the manuscript.
30
BUTOUR
AND
MACQUET
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