Initiation of Radicals in Biochemical Systems: Ferritin--Hydroperoxides--Aromatic Amines

D. I. Metelitza,^1,2 G. S. Arapova,¹ and A. N. Eryomin¹

¹Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, ul. Zhodinskaya 5/2, Minsk, 220141 Belarus; fax: (0172) 63-72-74; E-mail: ibochbel@eco2.iasnet.com

²To whom correspondence should be addressed.

Submitted November 11, 1996; revision submitted January 9, 1997.

The rates of initiation of free radicals were determined in systems containing horse spleen ferritin, H₂O₂, tert-butyl hydroperoxide (TBHP) or cumene hydroperoxide (CHP) in acetate buffer (pH 4.2) or phosphate buffer (pH 6.0) with 10-15% dimethylformamide (DMF). Benzidine (BD), benzidine sulfate (BDS), o-tolidine (TL), 3,3',5,5'-tetramethylbenzidine (TMB), and o-phenylenediamine (PDA) were used as acceptors. In the system ferritin--H₂O₂ oxidation of amine acceptor follows Michaelis--Menten kinetics. For all aromatic amines k_cat, K_m, and their ratios were determined. Peroxidase efficiency of ferritin in the TMB oxidation by hydrogen peroxide (k_cat/K_m) is characterized by a value 2.82·10³ M^-1·sec^-1 comparable with ferroxidase efficiency of apoferritin in the oxidation of Fe²⁺ by oxygen. Reactivity of aromatic amines in the system ferritin--H₂O₂ is similar to the reactivity registered in their peroxidase oxidation and is maximal for TMB and PDA. Bimolecular rate constants of TMB and PDA oxidation in the reaction with H₂O₂, TBHP, and CHP were compared in acetate buffer (pH 4.2) using 0.5 µM ferritin and 5 mM concentration of each substrate. On the oxidation of both amines activity of oxidants decreased in the following order: H₂O₂ > TBHP > CHP. A scheme of radical initiation in the systems ferritin--ROOH (H₂O₂)--amines and the influence of radical acceptors apoferritin and organic co-solvent on the rate of reactions are discussed.

KEY WORDS: ferritin, hydrogen peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, aromatic amine, amine oxidation, free radical initiation, radical initiation, organic co-solvent, radical acceptor, linear termination of radicals on apoferritin, evaluation of rate of radical initiation.

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