Biochemical Pharmacology. Vol. 33, No. 22. pp. 3689-3697. 1984 Primed in Great Britain. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA KINETIC STUDY AND INTERMEDIATES IDENTIFICATION OF NORADRENALINE OXIDATION BY TYROSINASE ,MERCEDES JIMENEZ, FRANCISCOGARCIA-CA~OVAS, FRANCISCOGARCIA-CS~R~~ONA, Josi A. LOZANO and Jo& L. IBORRA* Department0 Interfacultativo de Bioquimica. Universidad de Murcia. Murcia. Spain (Receioed 3 January 1984; accepted 16 March 1984) Abstract-Characterization of intermediates formed in the noradrenaline oxidation by mushroom tyrosinase and sodium periodate has been performed by rapid scanning spectrophotometry and graphical analysis of obtained spectra. In a pH range from 5.0 to 6.0, it has been possible to detect o-nor- adrenalinequinone-H+ as the first intermediate in these oxidations. The following steps for noradren- aline transformation into noradrenochrome would be: noradrenaline -+ o-noradrenalinequinone-H + -+ o-noradrenalinequinone + leukonoradrenochrome --* noradrenochrome. It has been also verified that o-noradrenalinequinone-H’ is transformed into noradrenochrome at a constant ratio. The stoi- chiometry for this converstion followed the equation: 2-noradrenalinequinone-H’ + noradrenaline + noradrenochrome. The pathway between noradrenaline and noradrenochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction. We have denominated this type of mechanism as an enzymatic-chemical-chemical mechanism, (E,CC). Whole rate constants for the implicated chemical steps at different pH and temperature values have been evaluated from measurement of the lag period arising from the accumulation of noradrenochrome that takes place when noradrenaline was oxidized at pIf 5-6. The lag period was independent on enzyme concentration, but was increased when pH and/ or temperature were increased. Rate constants pH independent for the deprotonation of nor- adrenalinequinone-H* into noradrenahnequinone and for the internal cyclization of noradren- alinequinone into leukonoradrenochrome have been obtained. We conclude that this minor pathway of noradrenaline oxidation by tyrosinase follows a scheme similar to that established for L-dopa. Tn recent years, several examples of adrenergic drugs oxidation metabolism have been described. Thus, there is strong evidence suggesting that dopamine, noradrenaline and also adrenaline can lead to melanin pigments similar to those obtained from dopa oxidation by tyrosinase [l, 21. Harrison et zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH al. [3] have shown in some in uiua experiments that L- cY-metildopa and D.L-isoproterenol are incorporated into melanin and non-melanin components of the hair of albino and black pigmented guinea pigs; they have also suggested a minor pathway for these compounds that should account for 0.1% of the given dose. Evidence for the involvement of catechol- amines in melanin formation by means of peroxidase oxidation has also been reported [4,5]. Melanin accumulation in skin, brain, liver and lung of schizo- phrenic patients on chlorpromazine therapy has been shown [6,7J; chlorpromazine, a commonly used anti- psychotic agent, should increase the rate of cate- cholamine oxidation through a free radical mech- anism [8]. All this experimental evidence seems to suggest that catecholamines could be involved, through minor pathways, in melanin formation. In preceding papers, we have reported the kinetics of L-dopa oxidation to dopachrome, catalyzed by tyrosinase 191, and the spectrophotometric and volt- ammetric characterization of intermediates of this pathway [lo]. On the other hand, Harrison [ll] has published the detection of fluorescent products in the oxidation of noradrenaline by tyrosinase at reaction times greater than 15 min. This author did not study * To whom correspondence should be addressed. the reaction kinetics at short times and then failed to detect the o-quinone and the corresponding leuko- aminochrome as possible intermediates originated in reaction mechanism. The present paper deais with the characterization study of the intermediates of the enzymic oxidation of noradrenaline by tyrosinase, as we11 as with the kinetics of those reactions that proceed from the formation of noradrenaline a-quinone to that of aminochrome. We conclude that the oxidation path- way is parallel to that of dopa oxidation by tyrosin- ase, that we have previously described [9]. MATERIALSAND METHODS Materials. Mushroom tyrosinase (o-diphenol:Oz oxido-reductase, EC 1.14.18.1, 2230 units/mg) and noradrenaline (L-arterenol) were from Sigma Chemical Co., Saint Louis, MO. Sodium periodate and all other chemicals were of analytical grade from the Merck Company. Intermediate identification. Aminochrome accumulation was spectrophotometrically followed at 490 nm (~90 = 3.58 x lo3 M-’ cm-i at pH 5.9) [12]. At this wavelength neither noradrenaIine nor any other possible intermediate present absorbance. The reaction medium was: 10 mM acetate buffer (pH as indicated in each case), 1.8mM noradrenaline (i.e. in saturated conditions) and such an enzyme amount that once tw was reached (tgg is the time necessary to attain the rate 0.99 Vo/2). In these con- ditions, absorbance increase was approx. 0.150. Determination of intermediate quinone with absorp-