Initiation and Inhibition of Free Radical Processes in H₂O₂-Metmyoglobin (Methemoglobin)-2,2´-Azino-bis-(3-Ethylbenzthiazoline-6-Sulfonic Acid) Systems

D. I. Metelitza^1*, A. N. Eryomin¹, D. O. Sviridov², and V. S. Kamyshnikov²

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

^* To whom correspondence should be addressed.

Received July 3, 2000; Revision received December 25, 2000

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Rates of free radical initiation were determined at 20°C in 10 mM phosphate buffer (pH 7.4) in the systems metmyoglobin (methemoglobin)-H₂O₂ using 2,2´-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the diammonium salt (ABTS). The catalytic activity of MetMb was 2-3-fold higher than that of MetHb. The process can be described by the Michaelis-Menten equation, from which effective values of K_m and V_max were calculated. Comparative kinetic studies on the inhibition of ABTS oxidation were carried out using Trolox, propylgallate (PG), polydisulfide of gallic acid (poly(DSG)), polydisulfide of (2-amino-4-nitrophenol) (poly(ADSNP)), and its conjugate with human serum albumin (HSA-poly(ADSNP)). The inhibitors were characterized by inhibition constants K_i and stoichiometric inhibition coefficients f (the number of radicals terminated by a single molecule of inhibitor). The minimum K_i and the maximum f values were obtained for poly(DSG), and in the system of MetHb-H₂O₂-ABTS they were 0.08 µM and 27.5, respectively. According to their antiradical activities, the inhibitors can be arranged as follows: poly(DSG) > poly(ADSNP) > PG > Trolox. PG, poly(DSG), poly (ADSNP), and its conjugate with HSA are suggested as “calibrators”, i.e., inhibition standards for evaluation of antioxidant status of biological fluids in possible test systems based on the free radical-generating pair MetMb-H₂O₂ with ABTS as the acceptor of the active radicals.

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KEY WORDS: metmyoglobin, methemoglobin, hydrogen peroxide, free radical initiation, inhibitors, Trolox, gallic acid polydisulfide, propylgallate, antioxidant status, inhibition constants, 2,2´-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)

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