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Methylene Blue, Janus Green B and Nile Blue sulfate. Introduction. We had shown earlier that several redox reactions of eoenzymes 1 and pigment for-.
Journal of Radioanalytical and Nuclear Chemistry, Articles, Vol. 131, No. 1 (1989) 95-!03

REDUCTION OF METHYLENE BLUE AND RELATED DYES BY GAMMA-IRRADIATED ALKALI HALIDES H. J. ARNIKAR, S. NILEGAONKAR, S. B. BHOSALE, A. H. KAPADI Maharashtra Association for the Cultivation ~ofScience, Law College Road, Pune 411 004 (India)

(Received October 13, 1988) A spectrophotometric study is reported of the reduction of some dyes having the methylene blue structure used in bacteriological staining. The reduction is effected by the species liberated during the dissolution of ~-irradiated sodium chloride, in the same way as by direct low ~,-dose. The G values for the two modes of the reduction are compared and the effects of radical scavengers on the reactions are studied. Results are found to be similar to the chemically and biologically induced reductions. The dyes studied include besides Methylene Blue, Janus Green B and Nile Blue sulfate.

Introduction We had shown earlier that several redox reactions of eoenzymes 1 and pigment formation from phenolic compounds, 2 which are normally catalyzed by specific enzymes, can also be brought about by the species 'liberated during the dissolution of y-irradiated alkali halide crystals) We had attributed these reactions to the radiation energy stored in the crystals in the form of F and hole centers. As an extension of this work, we report here our results on the reduction of Methylene Blue and Janus Green B, induced by "y-irradiated sodium chloride, lhese dyes are used as stains in bacteriology and as redox indicators. Methylene Blue especially acts as hydrogen acceptor and can be reduced ar~aerobically to a colourless compound, Leucomethylene Blue. 4 The reduction reaction is studied spectrophotometrically and the results compared with the reduction effected by other means, as by zinc dust and by the reduced form of nicotinamide adenine dinucleotide (NADH).

Experiment~ Methylene Blue (MB), and Nile Blue sulfate 'were obtained from Loba Chemicals while Janus Green B (JGB) and NADH were supplied by E. M e r c k a n d ~gma Chemicals respectively. Sodium chloride and potassium chloride used were of, BDI:I (AnalaR grade). Elsevier Sequoia S. A., Lausanne A kad dm iai K iad6, Budapest

H. J. ARNIKAR et al.: REDUCTIONOF METHYLENEBLUE 2 ml of the aqueous solution of the dyes of gM concentrations were taken in a cuvette and different amounts of NaC1 which had been y-irradiated for 75 hours at a dose rate of 0.037 kGy/min were added. In another set of experiments, aqueous solutions of the dyes of the same concentrations were directly irradiated to different doses in a 6~ y-chamber. The reactions of MB and JGB were monitored spectrophotometrically at 668 nm and 600 rim, respectively. From the change in the absoxbante, the amount (micromol) of dye converted was calculated, the applicability of Beer's law having been verified earlier over this concentration range. To confirm that the reaction occurring was just the reduction of MB and JGB, the dye (MB) waS directly reduced chemically by zinc dust in an acidic medium and biologically by adding NADH under anaerobic conditions provided by an atmosphere of 02-free nitrogen. The same results were observed in all the cases namely a decrease in the absorption maximum at 668 nm proportional to the amount of reductant added. The dose rate of the 6~ source at the position of irradiation was determined by Fricke dosimetry and found'to be 0.037 kGy/min. Since all the solutions irra9diated were very dilute, being less than micromolar in concentration, their ( Z ~ ) values were taken to be the same as for water viz. 0.556. The corresponding value for the Fricke solution also being nearly of the same magnitude, the dose rates for all the solutions irradiated were taken to be same as for the Fricke solution. The effects of adding small amounts of scavengers of hydrated electrons and .OH radicals were studied in the case of MB.

Results Figure 1 shows the changes in the absorption spectrum of MB in the UV-visible region before and after its reduction by y-irradiated NaC1 and KC1 in the acidic medium of pH 2.0: The figure also shows a smaller decrease in the absorption of the dye due to the same quantity of unirradiated NaC1 as a salting out effect. Also plotted in the same figure is the total reduction of the dye by zinc dust in acidic medium. It may be seen that the effect due to y-irradiated NaC1 is slightly less than that by zinc. Figure 2 shows the changes in the absorption spectrum of MB due to its reduction by NADH in aqueous medium, at pH 7. For comparison, the absorption spectra of MB reduced by NADH and ordinary NaC1 are shown in the same figure. The effect, it may be noticed, is less compared to that b y irradiated NaC1. Figure 3 shows similar changes in the absorption spectrum of JGB before and after its reduction by y-irradiated sodium chloride and by zinc dust in acidic medium. It may be Seen that the effect due to y-irradiated NaC1 is comparatively higher than the reduction by zinc dust, unlike in the case of MB. 96

H. J. A R N I K A R et al.: R E D U C T I O N OF M E T H Y L E N E BLUE

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H.. J. ARNIKAR et al.: REDUCTION OF METHYLENE BLUE A

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colour after exposure to air, or on the addition of ammonium hydroxide, further confirms the mech~aism suggested. The reduction of MB, and Janus Green B (JGB) having a similar structure is shown in Fig. 5. Yet another dye, Blue Nile sulfate, having a similar basic structure also showed similar reduction reactions.

References 1. H. J. ARNIKAR, S. NILEGAONKAR, S. B. BHOSALE, A. H. KAPADI, J. Radioanal. Nucl. Chem., 108 (1986) 229. 2. H. J. ARNIKAR, S. NILEGAONKAR, S. B. BHOSALE, A. H. KAPADI, J. Radioanal. Nucl. Chem., 125 (1988)57.

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H. J. ARNIKAR et al.: REDUCTION OF METHYLENE BLUE 3. H. J. ARNIKAR, B. S. M. RAO, M. J. BEDEKAR, Radiochem. Radioanal. Lett., 36 (1978) 73. 4. B. L. OSER, Hawk's Physiological Biochemistry, McGraw-Hill, Inc., New York, 1954. 5. J. W. T. SPINKS, R. J. WOODS, An Introduction to Radiation Chemistry, Wiley, New York, 1964. 6. H. J'. ARNIKAR, Essentials of Nuclear Chemistry, Wiley Eastern Ltd., New Delhi, (II ED), 1987. 7. G. AHNSTROM, Acta Chem. Scand., 19 (1965) 300. 8. H. J. ARNIKAR, P. S. DAMMLE, B. D. CHAURE, J. Chem. Phys., 55 (1971) 3668. 9. E. J. HART, M. ANBAR, The Hydrated Electron, Wiley Interscience, New York, 1970.

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