Bleomycin-DNA interactions: fluorescence and proton magnetic

Aug 1, 1977 - Steven J. Sucheck, Jeffrey F. Ellena, and Sidney M. Hecht. Journal of the American Chemical Society 1998 120 (30), 7450-7460. Abstract |...
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BLEOMYCIN-DNA INTERACTIONS

Bleomycin-DNA Interactions: Fluorescence and Proton Magnetic Resonance Studied Mingjien Chien,$ Arthur P. Grollman, and Susan B. Horwitz*

ABSTRACT: The interaction of bleomycin A2 with DNA has been examined by fluorescence spectroscopy and proton magnetic resonance techniques. Fluorescence bands observed at 353 and 405 nm in the spectrum of bleomycin were assigned to the bithiazole and 4-aminopyrimidine rings, respectively. Quenching of bithiazole fluorescence by DNA was used to determine apparent equilibrium constants for the complex which, in 2.5 mM tris(hydroxymethy1)aminomethane buffer, pH 8.4, are 1.2 X lo5 M-I for bleomycin and 1.4 X lo5 M-l

for tripeptide S , a partial acid hydrolysis product of the antibiotic. Under these conditions, one molecule of bleomycin binds for every five to six base pairs in DNA. In the proton magnetic resonance spectrum of bleomycin, resonances emanating from the bithiazole rings and dimethylsulfonium groups are preferentially broadened and reduced in intensity in the presence of DNA, suggesting that these moieties bind most tightly to the polymer.

T h e bieomycins, extracted from cultures of Streptomyces uerticullus by Umezawa and his collaborators, were isolated as their copper chelates. This family of glycopeptide antibiotics exhibits a wide spectrum of antimicrobial activities and is currently used in the therapy of certain neoplasms in man (Umezawa, 1975). The structure of bleomycin A2 is shown in Figure 1 . Bleomycin decreases the melting temperature of isolated DNA and causes single strand scissions in the polymer (Suzuki et al., 1968, 1969; Nagai et al., 1969a,b; Shirakawa et a]., 197 1 ). Fragmentation of DNA is enhanced in the presence of sulfhydryl compounds or hydrogen peroxide (Nagai et a]., 1 9 6 9 ~ )during ; this process, free bases are released (Haidle, 1971 ; Muller et al., 1972; Haidle et al., 1972; Kuo and Haidle, 1974). Bleomycin inhibits replication of vaccinia virus in HeLa cells, protects mice against the effects of vaccinia infection, and induces fragmentation of vaccinia DNA (Takeshita et al., 1974, 1976). The extent of bleomycin-induced degradation of DNA is determined by the concentrations of bleomycin, ATP, metal ions, and sulfhydryl agents present; however, the precise mechanism by which this effect occurs is unclear (Sausville et al., 1976; Takeshita et a]., 1977). The fluorescent properties of bleomycin have not previously been reported. In the present report, we use fluorescence spectroscopy and proton magnetic resonance techniques to examine the binding of bleomycin and its hydrolysis products to DNA. These experiments allow us to propose certain features of the bleomycin-DNA complex.

material contained several components (instability of bleomycin has been reported (Umezawa, 1971)) and was purified by column chromatography at 4 "C on a CMI-Sephadex C-25 column. The column was eluted using a pH gradient with 25 mM ammonium formate, pH 6.4, and 25 mM ammonium bicarbonate, pH 9.5, as initial and final buffer solutions, respectively. The major peak eluting at pH 8.4-8.6 was lyophilized and used for all experiments. Calf thymus DNA, purchased from Worthington Biochemical Corp., was dissolved in 0.1 M NaCl and dialyzed exhaustively, first against 0.1 M EDTA and then against 0.1 M NaCl at 4 "C. The dialyzed solution of DNA (approximately 2 mg/mL) was stored at 4 "C and the final concentration determined by phosphate analysis (Ames, 1966). Hydrolysis of Bleomycin. Partial acid hydrolysis of bleomycin was conducted according to the procedure of Umezawa (1973). Ten milligrams of bleomycin was dissolved in 25 mL of 6 N HCI, incubated with mild stirring at 37 "C for 4 days, and then evaporated to dryness at 25 OC. The hydrolysis products were adsorbed on a column of CM-Sephadex C-25 and eluted at 4 "C with a linear gradient (0.025 to 0.5 M ) of ammonium formate, pH 6.4. A major component was eluted at 0.26 M ammonium formate. The properties of this compound, as tested by paper chromatography (1-butanol-acetic acid-water, 4: 1:2) and paper electrophoresis (formic acid-acetic acid-water, pH 1.7 (3:1:6), and 0.065 M potassium phosphate buffer, pH 8.4), compared closely with an authentic sample of tripeptide S (Figure 1) provided by Professor Umezawa. Hydrolysis of this compound in 12 N HCI for 24 h at 100 "C yielded three spots with Rf values identical with the hydrolysis products of tripeptide S . Material excluded by the CM-Sephadex C-25 column was purified on a column of DEAE-Sephadex A-25. The compound eluting at 0.1 M ammonium formate showed an ultraviolet absorption spectra similar to that of 4-aminopyrimidine. A pK, of 5.3 was demonstrated by plotting intensities of fluorescence emission at 405 nm against pH in the presence of DNA. Thus, this compound, referred to in the Results section as compound

Materials and Methods A sample of copper-free bleomycin A2 (lot No. 7 1L489) was kindly provided by Bristol Co. and stored at -20 "C. This + From the Departments of Pharmacology and Cell Biology, Albert Einstein College of Medicine, New York, New York. Received August 4, 1976; revised manuscript received May 2, 1977. This work was supported by United States Public Health Service Grants CA-15714 and CA-10665 from the National Cancer Institute, National Institutes of Health, Department of Health, Education and Welfare. Mingjien Chien is a recipient of a fellowship from the Andrew W. Mellon Foundation. Susan 9. Horwitz is a recipient of an Irma T. Hirschl Career Scientist Award. Present address: Department of Rehabilitation Medicine, New York University Medical Center, New York, N.Y. 10016.

*

I Abbreviations used: CM, carboxymethyl; EDTA, ethylenediaminetetraacetic acid; DEAE, diethylaminoethyl; NMR, nuclear magnetic resonance; Tris, tris( hydroxymethy1)aminomethane.

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1, most likely includes the part of bleomycin that contains the 4-aminopyrimidine and imidazole moieties. Sonication of DNA. A solution of 4.5 mM calf thymus DNA was prepared in a buffer composed of 2.5 mM TrisHCI-30 mM NaCI, pH 8.4. Nitrogen was bubbled through the solution for 5 min which was then sonicated for 5 min at 4 OC with a Branson W 185 sonifier set at full power (Richards and Boyer, 1965). The sonication procedure was repeated once. Spectroscopic Measurements. Proton NMR spectra were recorded at 25 OC on a JEOLCO 100-MHz pulsed Fourier transform spectrometer equipped with a Nicolet 1080 digital computer for signal accumulation and Fourier transformation. Eight-millimeter N M R tubes with coaxial inserts containing hexamethyldisiloxane as an external reference were used for spectral studies. All samples were prepared in 1 mL of D2O and the pD was adjusted by adding small amounts of DCI or NaOD. Uncorrected fluorescence spectra were recorded at 25 "C on an Aminco-Bowman spectrophotofluorometer equipped with an X-Y recorder; corrected spectra were taken on a Perkin-Elmer fluorescence spectrophotometer, Model MPF-3. All measurements were made in a cuvette with a I-cm light path. When the samples contained DNA, absorbance of the added DNA at 300 nm was less than 0.05. Quantum yields were calculated by comparing the absorbance at the excitation wavelength, 300 nm, and the emission spectra of the unknown sample with a standard of known quantum yield. Quinine bisulfate, dissolved in 1 N H2S04 (quantum yield, 0.55), was used as a primary standard (Melhuish, 1961). Calculation of Binding Constants. Quantitative measurements of the binding of bleomycin and tripeptide S to DNA were based on fluorescence studies. As discussed in the Results section of this paper, the fluorescence of bleomycin and tripeptide S is partially quenched by DNA, thereby providing a sensitive method to determine apparent equilibrium constants and stoichiometry of binding. Assuming that all binding sites on DNA are equivalent and noninteracting, i.e., binding of one molecule of bleomycin to DNA does not affect binding of a second molecule, an apparent equilibrium constant for the reaction can be expressed by eq 1

_1 --

1

1 +nK(Do)(B) W o )

(1)

(C) which was derived from a more general expression of equilibrium

and the relation (C) = n[(Do) (2b) In these equations, (B), (C), and (D) represent the concentration of free bleomycin, bound bleomycin, and free DNA, respectively. (Do) represents the total concentration of DNA, n is the number of bound drug molecules per DNA nucleotide, and K is the apparent equilibrium constant. (B) and (C) are obtained from fluorescent measurements as follows: two solutions were prepared, one containing DNA, NaCI, and Tris-HC1 buffer, pH 8.4, and the other containing only the buffer. Small amounts of bleomycin, covering a wide range of concentrations, were added to both solutions and the fluorescent intensities were measured at 355 nm following excitation at 300 nm. The solution lacking DNA gives the fluorescent intensity of free bleomycin ( F b ) . The fluorescent

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BLEOMYCIN A,

CO"2

,,s+ H3C CH3

FH3 FH-OH NHf CH-FO

YH (72

bc;s*-cH~-cH~-cH~-NH-co