A Comparative Study of Calf Thymus DNA Binding to Cr(III) and Cr(VI) Ions
2000; Elsevier BV; Volume: 275; Issue: 14 Linguagem: Inglês
10.1074/jbc.275.14.10150
ISSN1083-351X
AutoresHirohumi Arakawa, R. Ahmad, M. Naoui, Heidar‐Ali Tajmir‐Riahi,
Tópico(s)Chromium effects and bioremediation
ResumoChromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5–7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r = 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant ofK = 508 m−1. Cr(III) chelates DNA via guanine N-7 and the nearest PO2 group with overall binding constant of K = 3.15 × 103m−1. Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm−1. At low Cr(III) concentration (r = 1:40), the number of Cr(III) ions bound to DNA were 6–7 cations/500 base pairs, and this increased to 30–35 cations/500 base pairs at high metal ion content (r = 1:4). DNA condensation occurred at high cation concentration (r = 1:10). No major alteration of DNA conformation was observed, and the biopolymer remained in the B family structure upon chromium complexation. Chromium(VI) salts are well known to be mutagens and carcinogens and to easily cross the cell membranes. Because they are powerful oxidizing agents, Cr(VI) reacts with intracellular materials to reduce to trivalent form, which binds DNA. This study was designed to investigate the interaction of calf thymus DNA with Cr(VI) and Cr(III) in aqueous solution at pH 6.5–7.5, using Cr(VI)/DNA(P) molar ratios (r) of 1:20 to 2:1 and Cr(III)/DNA(P) molar ratios (r) of 1:80 to 1:2. UV-visible and Fourier transform infrared (FTIR) difference spectroscopic methods were used to determine the metal ion-binding sites, binding constants, and the effect of cation complexation on DNA secondary structure. Spectroscopic results showed no interaction of Cr(VI) with DNA at low anion concentrations (r = 1:20 to 1:1), whereas some perturbations of DNA bases and backbone phosphate were observed at very high Cr(VI) contents (r > 1) with overall binding constant ofK = 508 m−1. Cr(III) chelates DNA via guanine N-7 and the nearest PO2 group with overall binding constant of K = 3.15 × 103m−1. Evidence for cation chelate formation comes from major shiftings and intensity variations of the guanine band at 1717 and the phosphate asymmetric stretching vibration at 1222 cm−1. At low Cr(III) concentration (r = 1:40), the number of Cr(III) ions bound to DNA were 6–7 cations/500 base pairs, and this increased to 30–35 cations/500 base pairs at high metal ion content (r = 1:4). DNA condensation occurred at high cation concentration (r = 1:10). No major alteration of DNA conformation was observed, and the biopolymer remained in the B family structure upon chromium complexation. Chromium(VI) salts are well known to be mutagens and carcinogens and to easily invade the insides of cells (1.De Flora S. Wetterhahn K.E. Life Chem. Rep. 1989; 7: 169-244Google Scholar). Cr(VI) produced DNA cross-links in rat tissues in vivo (2.Tsapakos M.J. Hamton T.H. Wetterhahn K.E. Cancer Res. 1983; 43: 5662-5667PubMed Google Scholar) and in cultured cellsin vitro (3.Tsapakos M.J. Hampton T.H. Sinclair P.R. Sinclair J.F. Bement W.J. Wetterhahn K.E. Carcinogenesis. 1983; 4: 959-966Crossref PubMed Scopus (45) Google Scholar, 4.Fornace Jr., A.J. Seres D.S. Lechner J.R. Harris C.C. Chem.-Biol. Interact. 1981; 36: 345-354Crossref PubMed Scopus (130) Google Scholar). Although Cr(VI) damaged nuclear DNA in whole cells, no reaction of Cr(VI) with isolated DNA occurred in vitro at physiological pH in the absence of a metabolizing system (5.Tsapakos M.J. Wetterhahn K.E. Chem.-Biol. Interact. 1983; 46: 265-277Crossref PubMed Scopus (176) Google Scholar). The Cr(VI) that is taken up is considered to be reduced by glutathione, cysteine, or ascorbic acid into Cr(III) (6.Connett P.H. Wetterhahn K.E. J. Am. Chem. Soc. 1985; 107: 4282-4288Crossref Scopus (202) Google Scholar), and the resulting cation reacts with DNA to form Cr(III)-DNA adducts. Because Cr(III) is a final form of chromium within the cell, the interaction of Cr(III) with DNA may play crucial role in the carcinogenetic action of Cr(VI) salts. The conversion of B form into Z form in the purine-pyrimidine sequence of DNA has been considered to be a factor in the transcriptional activity of genes (7.Razin A. Riggs A.D. Science. 1980; 210: 604-610Crossref PubMed Scopus (1541) Google Scholar). Cr(III) is found to interact with the poly(dG-dC) at low concentration and change B form to Z form in the presence of ethanol (8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar). However, Cr(III) at high concentration causes DNA condensation, inhibiting the alteration of B to Z structure (8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar). Moreover, the study on the effect of Cr(III) on DNA replication with single-stranded DNA template and micromolar concentration of Cr(III) revealed that Cr(III) bound in a dose-dependent manner to the template DNA and prevents DNA replication (9.Snow E.T. Xu L.S. Biochemistry. 1991; 30: 11238-11245Crossref PubMed Scopus (86) Google Scholar). However, if the unbound chromium was removed from the system by gel filtration, the rate of DNA replication by polymerase I (Klenow fragment) on the chromium-bound template increased more than 6-fold relative to control. This increase was paralleled by as much as a 4-fold increase in processivity and a 2-fold decrease in replication fidelity. When the concentration of Cr(III) increased further, DNA-DNA cross-links occurred to inhibit the polymerase activity. Trivalent chromium can bind purified DNA and form lesions capable of obstructing DNA replication in vitro (10.Bridgewater L.C. Manning F.C. Woo E.S. Patierno S.R. Mol. Carcinog. 1994; 9: 122-133Crossref PubMed Scopus (114) Google Scholar, 11.Bridgewater L.C. Manning F.C. Patierno S.R. Carcinogenesis. 1994; 15: 2421-2427Crossref PubMed Scopus (109) Google Scholar). It has also been observed that intact Novikoff ascites hepatoma cells exposed to potassium chromate formed cross-linking of nuclear proteins to DNA (12.Wedrychowski A. Ward W.S. Schmidt W.N. Hnilica L.S. J. Biol. Chem. 1985; 260: 7150-7155Abstract Full Text PDF PubMed Google Scholar). Recently, Cr(III) was shown to cause mutational spectrum in shuttle vector systems replicated in human cells (13.Tsou T.C. Lin R.J. Yang J.L. Chem. Res. Toxicol. 1997; 10: 962-970Crossref PubMed Scopus (58) Google Scholar). Thus, the interaction of Cr(III) with DNA may be responsible for carcinogenic activity of chromium. There are many agents that are specific for guanine alkylation in the O-6, N-7, or C-8 position. Several of these are highly active carcinogens, such asN-acetoxy-N-2-acetylaminofluorene, which alkylates in the C-8 position, and nitrosoamines, nitrogen mustards, nitrourea, and aflatoxin, which alkylate on the N-7 position (14.$$$$$$ ref data missingGoogle Scholar). The action of certain carcinogens, e.g. modification of guanine by N-7 methylation or by alkylation at C-8 withN-acetoxy-N-2-acetylaminofluorene facilitated the B to Z transition of poly(dG-dC) (15.Moller A. Nordheim A. Nichols S.R. Rich A Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4777-4781Crossref PubMed Scopus (66) Google Scholar, 16.Santella R.M. Grunberger D. Weinstein I.B. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1451-1455Crossref PubMed Scopus (126) Google Scholar, 17.Sage E. Leng M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4597-4601Crossref PubMed Scopus (125) Google Scholar). On the contrary, modification of poly(dG-dC) with the antitumore drugcis-diamine-dicholoplatinum (II) (a cross-linking agent) prevented the B to Z conversion (18.Malfoy B. Hartmann B. Leng M. Nucleic Acids Res. 1981; 9: 5659-5669Crossref PubMed Scopus (59) Google Scholar, 19.Ushay H.M. Santella R.M. Caradonna J.P. Grunberger D. Lippard S.J. Nucleic Acids Res. 1982; 10: 3573-3587Crossref PubMed Scopus (43) Google Scholar). It was found that Cr(III) preferentially binds guanine-containing DNAs (5.Tsapakos M.J. Wetterhahn K.E. Chem.-Biol. Interact. 1983; 46: 265-277Crossref PubMed Scopus (176) Google Scholar, 20.Wolf T. Kasemann R. Ottenwalder H. Carcinogenesis. 1989; 10: 655-659Crossref PubMed Scopus (51) Google Scholar). The results of a study on the DNA replication system showed that most of Cr(III) binding to the single-stranded template DNA is electrostatic because 40% or more of bound cation could be displaced by high salt wash, whereas only 20% or less chromium is chelatable by EDTA (9.Snow E.T. Xu L.S. Biochemistry. 1991; 30: 11238-11245Crossref PubMed Scopus (86) Google Scholar). In the present study, we have investigated the complexation of Cr(III) and Cr(VI) with calf thymus DNA using UV-visible and FTIR 1The abbreviations used are: FTIR, Fourier transform infrared. difference spectroscopy at pH 6.5–7.5 with Cr(III)/DNA(P) of 1:80 to 1:2 and Cr(VI)/DNA(P) of 1:20 to 2:1. Structural analysis regarding the chromium-binding sites, binding constants, and the alteration of DNA secondary structure are reported here. This is a first infrared spectroscopic study regarding the Cr(III)-DNA chelate formation and should help to elucidate the nature of the carcinogenic potential of chromium. Highly polymerized type I calf thymus DNA sodium salt (7% sodium content) was purchased from Sigma and was deproteinated by the addition of CHCl3 and isoamyl alcohol in NaCl solution. Crystalline CrCl3(H2O)6 and K2CrO4 salts were of reagent grade. Sodium DNA was dissolved to 1% w/w (25 mm DNA(phosphate)) in 50 mm NaCl and 1 mm sodium cacodylate (pH 7.30) at 5 °C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The appropriate amount of CrCl3(H2O)6 and K2CrO4 (0.3 to 10 mm) was prepared in distilled water and added dropwise to DNA solution to attain desired Cr(III)/DNA(P) molar ratios of 1:80, 1:40, 1:20, 1:10 1:4, and 1:2 and Cr(VI)/DNA(P) molar ratios of 1:20, 1:10, 1:5, 1:1, and 2:1 at a final DNA concentration of 0.5% w/w or 12.5 mm DNA(phosphate). The pH values of solutions were adjusted to 6.5–7.5, using NaOH solution. The infrared spectra were recorded 2 h after mixing of chromium salt and DNA solutions. The infrared spectra of Cr(III)-DNA complexes with r = 1:2 could not be recorded as solution because of solid gel formation. When UV absorption spectra were recorded, solutions containing DNA at a final concentration of 6.25 mm phosphate and 50 mmNaCl were used. The mixtures containing calf thymus DNA (6.25 mm of phosphate) and Cr(III) at molar ratios of 1:40 to 1:4 in 50 mm NaCl were applied to Sephadex G-25 (0.8 × 4.5 cm) column equilibriated in water, and 20 fractions were collected. The concentrations of Cr(III) and DNA in each fraction were determined by atomic absorption spectroscopy at 357.9 nm and by UV at 260 nm. The concentrations of Cr(III)-DNA complexes in each mixture were analyzed from regions where elution patterns of Cr(III) and DNA overlapped. Infrared spectra were recorded on a Bomem DA3–0.02 FTIR spectrometer equipped with a nitrogen cooled HgCdTe detector and KBr beam splitter. The solution spectra were taken using AgBr windows with resolution of 2–4 cm−1 and 100–500 scans. Each set of infrared spectra were taken (three times) on three identical samples with the same DNA and metal ion concentrations. The water subtraction was carried out with 0.1 m NaCl solution used as a reference at pH 6.5–7.5 (21.Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (202) Google Scholar). A good water subtraction was achieved as shown by a flat base line around 2200 cm−1where the water combination mode is located. This method is a rough estimate but removes the water content in a satisfactory way. The difference spectra ((DNA solution + chromium solution) − (DNA solution)) were produced, using a sharp DNA band at 968 cm−1 as internal reference. This band, because of deoxyribose C-C stretching vibrations, exhibits no spectral changes (shifting or intensity variations) on Cr-DNA complexation, and it was cancelled upon spectral subtraction. The spectra were smoothed with a Savitzky-Golay procedure (21.Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (202) Google Scholar). The intensity ratios of several DNA in-plane vibrations related to A-T and G-C base pairs and the PO2 stretchings were measured (with respect to the reference band at 968 cm−1) as a function of chromium concentration with an error of ±3%. These intensity ratios were used to calculate binding constants of Cr(III) with DNA bases and the backbone phosphate group. UV absorption spectra were recorded on a Hewlett Packard 8452A Diode Array Spectrophotometer. The chromium concentrations were determined using atomic absorption Perkin-Elmer Aanalyst 100 Spectrometer. At r = 1:20 to 1, Cr(VI) does not bind to DNA in aqueous solution. Evidence for this comes from the lack of major spectral changes (intensity or shifting) of the prominent DNA in-plane vibrations (21.Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (202) Google Scholar, 22.Keller P.B. Hartman K.A. Nucleic Acids Res. 1986; 14: 8167-8182Crossref PubMed Scopus (42) Google Scholar, 23.$$$$$$ ref data missingGoogle Scholar, 24.Loprete D.M. Hartman K.A. Biochemistry. 1993; 32: 4077-4082Crossref PubMed Scopus (127) Google Scholar, 25.Starikov E.B. Semenov M.A. Maleev V.Y. Gasan A.I. Biopolymers. 1991; 31: 255-273Crossref Scopus (63) Google Scholar, 26.Brahms S. Brahms J. Pilet J. Isr. J. Chem. 1974; 12: 153-163Crossref Scopus (47) Google Scholar, 27.Prescot B. Steinmetz W. Thomas Jr., G.J. Biopolymers. 1984; 23: 235-256Crossref PubMed Scopus (257) Google Scholar, 28.Taillandier E. Liquier J. Taboury J.A. Clark R.J.H. Hester R.E. Advances in Infrared and Raman Spectroscopy. Wiley Heyden, New York1985: 65-114Google Scholar) at 1717 (mainly guanine), 1663 (mainly thymine), 1609 (mainly adenine), 1492 (mainly cytosine), and 1222 cm−1(asymmetric PO2 stretch) (Figs. 1B and 2). However, at high chromate concentration (r > 1), some perturbations of DNA vibrations were observed. The guanine band at 1717 cm−1shifted toward a lower frequency at 1715, whereas the thymine band at 1663 shifted toward a higher frequency at 1665 cm−1 (Fig.1 A). Similarly, the adenine band at 1609 cm−1was observed at 1614 cm−1 in the presence of chromate anion (Fig. 1 A, r = 2). The phosphate asymmetric stretching vibration at 1222 cm−1 of the uncomplexed DNA was shifted toward a lower frequency at 1220 cm−1 as Cr(VI) concentration increased (Fig.1 A, r = 2). Positive peaks centered at 1653 cm−1 and 1215 cm−1 in the difference spectra of chromate-DNA complexes is coming from minor increases in the intensity of DNA vibrations (guanine, thymine, and adenine bases and backbone phosphate) (Fig. 1 B). The observed spectral changes are due to indirect anion-DNA interaction though water molecules of hydration on the DNA and hydrogen donor site of the bases. The two negative charges associated with the chromate anion and the presence of a negative charge on the backbone PO2 group are the major factors for a weak chromate-DNA complexation. It should be noted that the presence of a strong band at 893 cm−1 in the infrared spectra, which appears at 877 cm−1 in the difference spectra of the chromate-DNA complexes are due to the CrO42− vibrations (29.Nakamoto K. Infrared Spectra of Inorganic and Coordination Compounds. John Wiley & Sons, New York1963: 103-110Google Scholar) (Fig. 1,A and B, r = 2). Our observations are consistent with other spectroscopic studies that showed no major Cr(VI)-DNA interaction in vitro in the absence of reducing agents (5.Tsapakos M.J. Wetterhahn K.E. Chem.-Biol. Interact. 1983; 46: 265-277Crossref PubMed Scopus (176) Google Scholar, 8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar, 30.Hneihen A.S. Standeven A.M. Wetterhahn K.E. Carcinogenesis. 1993; 14: 1795-1803Crossref PubMed Scopus (45) Google Scholar). However, in the presence of metabolizing system, where Cr(VI) is reduced to Cr(III), major Cr(III)-DNA complexation has been observed (5.Tsapakos M.J. Wetterhahn K.E. Chem.-Biol. Interact. 1983; 46: 265-277Crossref PubMed Scopus (176) Google Scholar, 8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar, 10.Bridgewater L.C. Manning F.C. Woo E.S. Patierno S.R. Mol. Carcinog. 1994; 9: 122-133Crossref PubMed Scopus (114) Google Scholar, 11.Bridgewater L.C. Manning F.C. Patierno S.R. Carcinogenesis. 1994; 15: 2421-2427Crossref PubMed Scopus (109) Google Scholar).Figure 2Intensity ratio variations for several DNA in-plane vibrations at 1717 (G and T), 1663 (T, G, A, and C), 1609 (A), 1494 (C and G), and 1222 cm−1 (PO2 stretch) as a function of Cr(VI) concentration (different Cr(VI)/DNA(P) molar ratios).View Large Image Figure ViewerDownload Hi-res image Download (PPT) At low cation concentrations (r = 1:80 to 1:20), Cr(III) binds DNA through guanine and the backbone PO2 group. Evidence for this comes from major spectral shiftings of the bands at 1717 cm−1 (mainly guanine) and at 1222 cm−1 (PO2 asymmetric stretch) (21.Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (202) Google Scholar, 22.Keller P.B. Hartman K.A. Nucleic Acids Res. 1986; 14: 8167-8182Crossref PubMed Scopus (42) Google Scholar, 23.$$$$$$ ref data missingGoogle Scholar, 24.Loprete D.M. Hartman K.A. Biochemistry. 1993; 32: 4077-4082Crossref PubMed Scopus (127) Google Scholar, 25.Starikov E.B. Semenov M.A. Maleev V.Y. Gasan A.I. Biopolymers. 1991; 31: 255-273Crossref Scopus (63) Google Scholar, 26.Brahms S. Brahms J. Pilet J. Isr. J. Chem. 1974; 12: 153-163Crossref Scopus (47) Google Scholar, 27.Prescot B. Steinmetz W. Thomas Jr., G.J. Biopolymers. 1984; 23: 235-256Crossref PubMed Scopus (257) Google Scholar, 28.Taillandier E. Liquier J. Taboury J.A. Clark R.J.H. Hester R.E. Advances in Infrared and Raman Spectroscopy. Wiley Heyden, New York1985: 65-114Google Scholar). The guanine band at 1717 cm−1 shifted toward a lower frequency at 1709 cm−1 and the phosphate band at 1222 cm−1 was observed at 1216 cm−1, upon Cr(III) complexation (Fig. 1 A). In addition, a major increase in the intensity of guanine band at 1717 cm−1(40%) and PO2 band at 1222 cm−1 (30%) was observed as Cr(III) concentration increased to r = 1:20 (Fig. 3). The weak positive peaks at 1710 (G), 1660 (T), 1611 (A), 1215, and 1088 cm−1 (phosphate) in the difference spectrum of the Cr-DNA complexes are due to a minor increase of the intensity of DNA bases and the PO2vibrations at low Cr(III) concentration (r = 1:80) (Fig. 1 B). However, as cation concentration increases tor = 1:20, the positive features in the difference spectrum of Cr-DNA complexes grow in intensity, particularly for the guanine band at 1709 and the PO2 band at 1214 cm−1 (Fig. 1 B, r = 1:20). In addition, the relative intensity ratios of the νSPO2 (1088 cm−1) and νAsPO2 (1222 cm−1) have changed from 1.7 (DNA-free) to 1.5 (Cr-DNA complexes). The observed spectral shiftings (Δν = 8 to 6 cm−1) and the major intensity variations for the guanine band at 1717 cm−1 (40%) and the PO2 vibration at 1222 cm−1 (30%) are due to the cation chelation via guanine N-7 and the backbone phosphate group (Figs. 1 B and 3, r = 1:20). It has been suggested that the infrared spectral changes related to the DNA in-plane vibrations at 1720–1600 cm−1 are due to the metal interaction through guanine N-7 site (21.Alex S. Dupuis P. Inorg. Chim. Acta. 1989; 157: 271-281Crossref Scopus (202) Google Scholar, 22.Keller P.B. Hartman K.A. Nucleic Acids Res. 1986; 14: 8167-8182Crossref PubMed Scopus (42) Google Scholar). It should be noted that although a minor intensity variations were observed for the thymine band at 1663 (10%) and adenine band at 1609 cm−1(5%), no major spectral shiftings for this vibration occurred upon Cr(III) complexation (Figs. 1 A and 3). Thus, the amount of chromium that directly binds to the A-T base pair is negligible, whereas an indirect cation binding (via H2O) to the A-T bases cannot be excluded. The calculation of the overall binding constants were carried out using UV spectroscopy as reported (31.Klotz I.M. Hunston D.L. Biochemistry. 1971; 16: 3065-3069Crossref Scopus (545) Google Scholar). If the equilibrium for chromium cation and DNA is established as in Equation 1, then the binding constants of chromium cation complexes with DNA can be described as in Equation 2.DNA+Chromium cation=DNA:ChromiumEquation 1 K=[DNA:Chromium][DNA][Chromium]Equation 2 The double reciprocal plot of 1/[complexed chromium]versus 1/[free cation] is linear and the binding constant (K) is calculated from the ratio of the intercept on the vertical coordinate axis to the slope (31.Klotz I.M. Hunston D.L. Biochemistry. 1971; 16: 3065-3069Crossref Scopus (545) Google Scholar). Concentrations of complexed chromium were determined by subtracting absorbance of uncomplexed DNA at 280 nm from those of complexed DNA. Concentrations of free cation were determined by subtracting those of complexed chromium from total chromium used for the experiment. Our data of 1/[complexed Cr] almost proportionally increased as a function of 1/[free Cr], and overall binding constants for Cr(VI)-DNA and Cr(III)-DNA were estimated to beK = 508 m−1 andK = 3.15 × 103m−1, respectively. Because the Cr(III)-DNA interaction mainly occurs through guanine N-7 and the backbone PO2 group, the binding constants of Cr(III) with guanine and backbone PO2 were determined from the intensity ratios of DNA in-plane vibrations related to guanine (1717 cm−1) and backbone phosphate (1222 cm−1) stretchings. Using the data from uncomplexed DNA and Cr(III)-DNA adduct formed at r = 1:10, the binding constants were estimated to be K(G) = 1.80 × 103m−1 andK(P) = 1.25 × 103m−1. The binding constants for thymine and adenine bases were much smaller than those of the guanine and phosphate group. This indicates that the interaction of Cr(III) with the A-T base pair is negligible. In a previous study, based on the intensity ratio variations of the infrared absorption bands, the binding constants of the chlorophyll-DNA complexes were determined, where Mg(II) was coordinated to the backbone PO2 group and the guanine N-7 sites (32.Neault J.F. Tajmir-Riahi H.A. Biophys. J. 1999; 76: 2177-2181Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The somewhat low stabilities (k = ∼103m−1) of the cation chelate complexes are attributed mainly to the ionic nature of the Cr-O-P-O interaction. It has been suggested that most of Cr(III) binding to the single-stranded template DNA is electrostatic because 40% or more of bound cation could be displaced by high salt wash, whereas only 20% or less of the chromium is chelatable by EDTA (9.Snow E.T. Xu L.S. Biochemistry. 1991; 30: 11238-11245Crossref PubMed Scopus (86) Google Scholar). On the other hand, the overall binding constant for Cr(VI)-DNA was estimated to be 508m−1. Such a low stability of the complexes is indicative of no direct Cr(VI)-DNA interaction in aqueous solution. Based on the data from UV absoprtion spectra, numbers of Cr(III) bound to DNA were estimated to be 9 cations/500 base pairs atr = 1:40 and 32 cations/500 base pairs atr = 1:4 (Fig. 4). To compare the numbers of Cr(III) bound to DNA by other method, the mixtures were subjected to Sephadex G-25 column (0.8 × 4.5 cm) equilibrated in aqueous solution, and concentrations of Cr(III)-DNA complexes were determined by atomic absorption and UV absorption spectroscopic methods. The numbers of Cr(III) bound to DNA were calculated to be 6 cations/500 base pairs at r = 1:40 and 30 cations/500 base pairs at 1:4. Another research group (13.Tsou T.C. Lin R.J. Yang J.L. Chem. Res. Toxicol. 1997; 10: 962-970Crossref PubMed Scopus (58) Google Scholar) showed that about 25 Cr(III) cations bound to 1000 nucleotides of a plasmid DNA at a low concentration of chromium(III) chloride in the presence of 10 mm potassium phosphate buffer (pH 7.5), whereas the number increased to about 100 cations/1000 nucleotides in the presence of Tris-HCl and EDTA. Thus, the number of Cr(III) bound to DNA could be affected by the solution compositions including salts, buffers, and chelators. The numbers of Cr(III) bound to calf thymus DNA were less than those bound to a plasmid DNA (13.Tsou T.C. Lin R.J. Yang J.L. Chem. Res. Toxicol. 1997; 10: 962-970Crossref PubMed Scopus (58) Google Scholar). This may be due to the higher NaCl concentration (50 mm) used in our DNA preparation with respect to plasmid DNA solution (10 mmpotassium salt), which increases the double helical stability and therefore limits the Cr(III) access to different DNA-binding sites. At higher cation concentration (r = 1:10 and 1:4), Cr(III) induces DNA condensation. Evidence for this comes from a major decrease in the intensity of the bands at 1717 (guanine), 1663 (thymine), and 1222 cm−1(PO2stretch) (Fig. 3). DNA condensation by Cr(III) ions is well investigated using electron microscopy (33.Osterberg R. Persson A. Bjursell G. J. Biomol. Struct. Dyn. 1984; 2: 285-290Crossref PubMed Scopus (41) Google Scholar). The condensation of DNA begins at r = 1:10 and continues up to r = 3:1 (33.Osterberg R. Persson A. Bjursell G. J. Biomol. Struct. Dyn. 1984; 2: 285-290Crossref PubMed Scopus (41) Google Scholar). However, because of the solid gel formation of DNA solution in the presence of Cr(III) at high cation content (r = 1:2), the infrared spectra of chromium-DNA complexes formed at high cation concentration could not be recorded as solution. It was considered that the condensation of DNA occurrs by charge neutralization of DNA by multivalent cations (34.Manning G.S. Quart. Rev. Biophys. 1978; 11: 179-246Crossref PubMed Scopus (2671) Google Scholar). Another study with cobalt(III)-hexamine suggested that the binding of the cation to the backbone phosphate group causes DNA condensation (35.Widom J. Baldwin R.L. J. Mol. Biol. 1980; 144: 431-453Crossref PubMed Scopus (494) Google Scholar). Our infrared spectroscopic study with chromium(III) chloride clearly showed that direct binding of the cation to guanine N-7 site, and the backbone phosphate that forms chelation may provide driving force for DNA condensation. Our infrared spectroscopic results showed that Cr(III) and Cr(VI) cations do not induce DNA conformational changes from B to A or B to Z structure. Evidence for this comes from no major displacement of the DNA conformational indicator at 836 cm−1 (deoxyribose-phosphate stretching mode) toward a lower frequency (Fig. 1 A). In a B to A transition, the DNA infrared marker bands at 836 and 1717 cm−1 were shifted toward lower frequencies at 810 and 1700 cm−1, respectively, whereas the backbone PO2 asymmetric stretching at 1222 cm−1 shifted toward a higher frequency at 1240 cm−1 (24.Loprete D.M. Hartman K.A. Biochemistry. 1993; 32: 4077-4082Crossref PubMed Scopus (127) Google Scholar, 28.Taillandier E. Liquier J. Taboury J.A. Clark R.J.H. Hester R.E. Advances in Infrared and Raman Spectroscopy. Wiley Heyden, New York1985: 65-114Google Scholar, 36.Tajmir-Riahi H.A. Neault J.F. Naoui M. FEBS Lett. 1995; 370: 105-108Crossref PubMed Scopus (82) Google Scholar). In a B to Z transition, the marker infrared bands at 1717 and 836 cm−1 were observed at 1690 and 800 cm−1, respectively, whereas the PO2 band at 1222 cm−1 was displaced toward a lower frequency at 1215 cm−1 (24.Loprete D.M. Hartman K.A. Biochemistry. 1993; 32: 4077-4082Crossref PubMed Scopus (127) Google Scholar, 28.Taillandier E. Liquier J. Taboury J.A. Clark R.J.H. Hester R.E. Advances in Infrared and Raman Spectroscopy. Wiley Heyden, New York1985: 65-114Google Scholar, 36.Tajmir-Riahi H.A. Neault J.F. Naoui M. FEBS Lett. 1995; 370: 105-108Crossref PubMed Scopus (82) Google Scholar). Such spectral changes did not occur for DNA marker bands in the presence of Cr(III) and Cr(VI) cations. (Fig. 1 A). The major shifting of the bands at 1717 (guanine) to 1709 cm−1 and 1222 (phosphate) to 1216 cm−1 in the spectra of the Cr(III)-DNA complexes are due to the direct Cr(III) coordination to both guanine N-7 and backbone phosphate group (Fig. 1 A). Other spectroscopic studies also showed that chromium cations inhibit B to A or B to Z conformational changes upon DNA complexation (8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar). However, at low cation concentration, Cr(III) can induce B to Z conformational transition for synthetic polynucleotides in the presence of ethanol (8.Floro N.A. Wetterhahn K.E. Biochem. Biophys. Res Commun. 1984; 124: 106-113Crossref PubMed Scopus (12) Google Scholar). On the basis of our spectroscopic results of calf thymus DNA in the presence of Cr(VI) and Cr(III) metal ions in aqueous solution, the following remarks can be made: (a) No direct Cr(VI)-DNA interaction was observed in vitro, whereas Cr(III) chelates DNA through guanine N-7 and the backbone phosphate group; (b) The somewhat low stability of the Cr(III)-DNA complex is mainly due to the ionic nature of the Cr(III)-phosphate interaction; (c) Cr(III) induces DNA condensation at high cation concentration; and (d) Cr(VI) and Cr(III) ions do not induce DNA conformational changes at low or high cation concentration.
Referência(s)