OXIDATION OF ADRENALINE BY FERRYLMYOGLOBIN

The oxidation of adrenaline by ferrylmyoglobin, the product formed by the oxidation of myoglobin with H2O2, was examined by absorption, fluo rescence, and EPR spectroscopy in terms of the formation of intermedia te free radicals and stable molecular products and the binding of adre naline oxidation products to the apoprotein. The reaction of adrenalin e with ferrylmyoglobin resulted in reduction of the hemoprotein to met myoglobin and consumption of adrenaline. Quantification of metmyoglobi n formed per adrenaline yielded a ratio of 1.66. The reaction was foun d first order on adrenaline concentration and second order on ferrylmy oglobin concentration. This, together with the above ratio, suggested a mechanism by which two oxoferryl moieties (ferrylmyoglobin) were red uced by adrenaline yielding metmyoglobin and the o-semiquinone state o f adrenaline. The decay of the o-semiquinone to adrenochrome was confi rmed by an increase in absorbance at 485 nm. The product was nonfluore scent; alkalinization of the reaction mixture resulted in a strong flu orescence at 540 nm ascribed to 3,5,6-trihydroxyindol or adrenolutin. Hence, adrenochrome and its alkali-catalyzed product, adrenolutin, are the major molecular products formed during the oxidation of adrenalin e by ferrylmyoglobin. Semiquinones formed during the adrenaline/ferryl myoglobin interaction were detected by EPR, spin stabilizing these spe cies with Mg2+ The six-line EPR spectrum observed (a(N) = 4.5 G, a(N)( CH3) = 5.1, and a(2H) = 0.91; g = 2.0040) may be assigned to the semiq uinone forms of adrenochrome and/or adrenolutin or a composite of thes e species. The intensity of the EPR signal increased with time and its subsequent decay followed a second-order kinetics as inferred by the proportionality of the square of the EPR line intensity with H2O2 conc entration. Heme destruction and lysine loss, inherent in the reaction of metmyoglobin with H2O2, were prevented 80 and 34% by adrenaline, re spectively. The low protection exerted by adrenaline against lysine lo ss was possibly due to the formation of Schiff bases between the E-NH, group of lysine and the o-quinone oxidation product(s) of adrenaline. The yield of Schiff base formation was 20-25%. The autoxidation of ad renaline at physiological pH is extremely slow or nonexistent. These d ata provide a rationale for the primary oxidation of adrenaline by the pseudoperoxidatic activity of ferrylmyoglobin and suggest implication s of the free radicals thereby formed for the oxidative damage in repe rfusion injury. (C) 1998 Elsevier Science Inc.