Polydisulfides of Substituted Phenols as Effective Protectors of Peroxidase against Inactivation by Ultrasonic Cavitation

E. E. Grintsevich¹, I. E. Adzerikho², A. G. Mrochek², and D. I. Metelitza^1*

¹Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, ul. Kuprevicha 5/2, Minsk, 220141 Belarus; fax: (375)-(172) 63-7274; E-mail: enzyme@ns.iboch.ac.by

²Belorussian State Institute of Physician Improvement, ul. P. Brovki 3, Minsk, 220714 Belarus; fax: (375)-(172) 32-2533; E-mail: chekan@srpmi.minsk.by

^* To whom correspondence should be addressed.

Received November 21, 2000; Revision received March 27, 2001

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Kinetics of inactivation of horseradish peroxidase (HP) induced by low-frequency ultrasonic (US) treatment (27 kHz) with the specific power of 60 W/cm² were studied in phosphate (pH 7.4) and acetate (pH 5.2) buffers within the temperature range of 36.0 to 50.0°C and characterized by effective first-order rate constants of US inactivation k_in(us) in min^-1. Values of k_in(us) depend on the specific ultrasonic power within the range of 20-60 W/cm², on the concentration of HP, and on pH and temperature of the solutions. The activation energy of US inactivation of HP is 9.4 kcal/mole. Scavengers of HO^· radicals, mannitol and dimethylformamide, significantly inhibit the US inactivation of HP at 36.0°C, whereas micromolar concentrations of polydisulfide of gallic acid (poly(DSG)) and of poly(2-aminodisulfide-4-nitrophenol) (poly(ADSNP)) virtually completely suppress the US inactivation of peroxidase at the ultrasonic power of 60 W/cm² on the sonication of the enzyme solutions for more than 1 h at pH 5.2. Various complexes of poly(DSG) with human serum albumin effectively protect HP against the US inactivation in phosphate buffer (pH 7.4). The findings unambiguously confirm a free radical mechanism of the US inactivation of HP in aqueous solutions. Polydisulfides of substituted phenols are very effective protectors of peroxidase against inactivation caused by US cavitation.

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KEY WORDS: horseradish peroxidase, ultrasonic inactivation, inactivation kinetics, phenol polydisulfides, polydisulfide of gallic acid, poly(2-aminodisulfide-4-nitrophenol), free radicals

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