The oxidation of
adrenaline by
ferrylmyoglobin, the product formed by the oxidation of
myoglobin with H2O2, was examined by absorption, fluorescence, and EPR spectroscopy in terms of the formation of intermediate
free radicals and stable molecular products and the binding of
adrenaline oxidation products to the
apoprotein. The reaction of
adrenaline with
ferrylmyoglobin resulted in reduction of the hemoprotein to
metmyoglobin and consumption of
adrenaline. Quantification of
metmyoglobin formed per
adrenaline yielded a ratio of 1.66. The reaction was found first order on
adrenaline concentration and second order on
ferrylmyoglobin concentration. This, together with the above ratio, suggested a mechanism by which two oxoferryl moieties (
ferrylmyoglobin) were reduced by
adrenaline yielding
metmyoglobin and the o-semiquinone state of
adrenaline. The decay of the o-semiquinone to
adrenochrome was confirmed by an increase in absorbance at 485 nm. The product was nonfluorescent; alkalinization of the reaction mixture resulted in a strong fluorescence 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
adrenaline by
ferrylmyoglobin. Semiquinones formed during the
adrenaline/
ferrylmyoglobin interaction were detected by EPR, spin stabilizing these species with Mg2+. The six-line EPR spectrum observed (aN=4.5 G, aN(CH3)=5.1, and a2H=0.91; g=2.0040) may be assigned to the semiquinone forms of
adrenochrome and/or
adrenolutin or a composite of these 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 concentration.
Heme destruction and
lysine loss, inherent in the reaction of
metmyoglobin with H2O2, were prevented 80 and 34% by
adrenaline, respectively. The low protection exerted by
adrenaline against
lysine loss was possibly due to the formation of
Schiff bases between the epsilon-NH2 group of
lysine and the o-
quinone oxidation product(s) of
adrenaline. The yield of
Schiff base formation was 20-25%. The autoxidation of
adrenaline at physiological pH is extremely slow or nonexistent. These data provide a rationale for the primary oxidation of
adrenaline by the pseudoperoxidatic activity of
ferrylmyoglobin and suggest implications of the
free radicals thereby formed for the oxidative damage in
reperfusion injury.