ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 229, No. 1, February 15, pp, 220-225, 1984 Excitation of Micelle-Solubilized Chlorophyll during the Peroxidase- Catalyzed Aerobic Oxidation of lsonicotinic Acid Hydrazide LILIAN NASSI AND GIUSEPPE CILENTOl Department of Biochemistry, Institute de Q&mica. Universidade de S&o Paula, Caixa Postal 20.780, 01498--S&o Paula SP, Brazil Received July 8, 1983, and in revised form October 20, 1983 Addition of micelle (hexadecyl-trimethylammoniumbromide)-solubilized chlorophyll a to the isoniazid/peroxidase/Mn2+/Oz system promotes light emission, identified as chlorophyll fluorescence. Based on O2 consumption, the quantum yield of chlorophyll excitation to the S1state exceeds 6 X 10m6. At least part of the excitation has its origin in the conversion of an intermediate-presumably a diazene-to pyridine-4-carbox- aldehyde. On the basis of the present and earlier results [K. Zinner, C. C. C. Vidigal, N. Duran, and G. Cilento (1977) Arch. Biochem. Biophys. 180, 452458.1, it is inferred that isoniazid, an important chemotherapic and also a carcinogenic agent, can lead to a substantial generation of electronically excited states. In earlier papers (1,2), indications were presented that excited species may be formed when the tuberculostatic and car- cinogenic drug isonicotinic acid hydrazide (“Isoniazid”)2 is oxidized to pyridine-4- carboxaldehyde by the horseradish per- oxidase/Mn2+/02 system. Chlorophyll solubilized in micelles (3) or present as chloroplasts (4, 5) is able to accept energy from enzyme-generated triplet species. Chl may also become ex- cited through interaction with appropriate “high-energy” intermediates. To obtain additional information, we have studied the oxidation of INH in the presence of micelle-solubilized Chl. MATERIALS AND METHODS INH (Ely Lilly), Chl a and b, HRP type VI, histidine, catalase, superoxide dismutase (Sigma Chemical Co.); 1To whom correspondence should be addressed. ‘Abbreviations used: Chl a, chlorophyll a; CTAB, hexadecyl-trimethylammoniumbromide; DMPO, 5,5- dimethylpyrroline-N-oxide; HRP, horseradish per- oxidase; INH, isonicotinic acid hydrazide; PBN, a- phenyl-teti-butylnitrone; POPOP, 1,4-bis[2-(5-phen- yloxazole)]benzene. DMPO and PBN (Aldrich Chemical Co.); and sodium benzoate (BHD) are commercially available. CTAB (Merck, p.a.) was recrystallized from acetone-meth- anol (85:15, v:v). Unless otherwise stated, the reaction mixture consisted of 1.2 X 10m4 M INH, 3.2 PM HRP, 7.2 X 10e5 M Mn’+, 1.6 mM CTAB, and 3.5 PM Chl a in 0.1 M phosphate-38.5 mM pyrophosphate buffer, pH 7.4. Chl a dissolved in ethanol was added to aqueous solutions of the detergent. Chl content was determined spectrophotometrically at 436 nm (6 = 1.12 X lo5 MS’ cm-‘) (6). The final ethanol content was 0.13 M. In all experiments, the temperature was 35°C and the final volume was 3 ml. Oxygen consumption was followed in a Yellow Springs Instruments Model 53 oxygen monitor. Che- miluminescence and emission spectra (counts per 5 s) were measured in a Hamamatsu TVC-76’7 pho- tocounter with a Hamamatsu R-562 photomultiplier tube. Absorption and fluorescence spectra were re- corded on a Zeiss DMR-10 spectrophotometer and a Perkin-Elmer MPF-4 spectrofluorimeter, respectively. The fluorescence quantum yield of micelle-solubi- lized Chl a was determined at 25°C by comparative measurements relative to Chl a in acetone (7); a value of 0.15 was obtained for CTAB-solubilized Chl a in the presence of INH and Mn2+ ions (standard con- ditions). The efficiency of energy transfer was calculated as the integrated photon emission (with a POPOP emis- sion standard) per mole of oxygen consumed. Ap- 0003-9861/84 $3.00 Copyright 0 1984 by Academic Press, Inc. All rights of reproduction in any form reserved. 220