CHEMICAL AND ENZYMATIC INTERMEDIATES IN THE PEROXIDATION OF ORTHO-DIANISIDINE BY HORSERADISH-PEROXIDASE .1. SPECTRAL PROPERTIES OF THE PRODUCTS OF DIANISIDINE OXIDATION

Studies of the optical spectra of the products formed during peroxidation of o-dianisidine by horseradish peroxidase [HRP] indicate at least 3 distinct species. At pH 3.7 and 4.degree. C, peroxidation of dianisidine at low concentrations yields the free dianisidine quinonediimine (the 2-equivalent oxidized form) with .lambda.max 452 and 514 nm. At higher concentrations, the first detectable product is not the free quinonediimine but an intermolecular complex (meriquinone or charge-transfer complex) consisting of quinonediimine and parental diamine. This complex is freely reversible and is sensitive to simple dilution or acidification, either of which restores the spectrum of the free quinonediimine. Furthermore, at near-neutral pH, the quinonediimine appears to undergo irreversible self-coupling, yielding yet a different optical spectrum presumably characteristic of the bisazobiphenyl structure proposed by K. M. Moller and P. Ottolenghi. Butylated hydroxyanisole was shown to react in the presence of peroxidase.sbd.H2O2 and dianisidine to yield a spectrum (.lambda.max 575 nm) nearly identical with that obtained when Gibbs reagent (2,6-dichloroquinone 4-chloroimine) was incubated with butylated hydroxyanisole, thus suggesting that the free quinonediimine itself couples with the phenolic antioxidant. Continuous-flow EPR studies of dianisidine oxidation both with HRP.sbd.H2O2 and with ceric sulfate were unable to detect any free dianisidine semiquinone radical in the steady state; apparently oxidation of dianisidine occurs in a rapid 2-electron process in both the HRP.sbd.H2O2 and Ce(IV) systems.