Oxidation of Phenolic Compounds by Peroxidase in the Presence of Soluble Polymers

I. Bratkovskaja^*, R. Vidziunaite, and J. Kulys

Institute of Biochemistry, ul. Mokslininku 12, LT-08662, Vilnius, Lithuania; fax: (8-370) 5-272-9196; E-mail: bratkovskaja@bchi.lt

^* To whom correspondence should be addressed.

Received January 14, 2004; Revision received March 1, 2004

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The kinetics of Coprinus cinereus peroxidase-catalyzed 1-naphthol, 2-naphthol, and 4-hydroxybiphenyl oxidation was investigated. The initial rates of the naphthols' and 4-hydroxybiphenyl oxidations were linearly dependent on enzyme concentration. The rates depended on substrate concentration and saturated at concentrations above 100 µM of hydrogen peroxide, 25-50 µM of naphthols, and 10 µM of 4-hydroxybiphenyl. At the peroxide concentration 100 µM calculated K_m and the maximal rate (V_max) were 74.7 µM and 0.53 µM/sec or 175 µM and 2.0 µM/sec for 1- or 2-naphthol, respectively, and 29.68 µM and 0.42 µM/sec for 4-hydroxybiphenyl. Kinetic measurements of exhaustive naphthol and 4-hydroxybiphenyl oxidation showed that peroxidase is inactivated during the oxidation of the substrates. Different factors and additives, water soluble polymers and albumins (PEG, PEI, PL, BSA, HSA), influenced the initial naphthols and 4-hydroxybiphenyl oxidation rates, peroxidase inactivation rates, and the degree of the substrate conversion. Addition of albumin increased turnover number of naphthols oxidation 1.5-4 times. Light scattering increase was observed when peroxidase-catalyzed oxidation reaction was investigated and suggested that insoluble particles were formed during the process. The addition of polymers, change of concentration and ionic strength of the solution as well as the number of other factors influenced the observed light scattering. The number of particles formed during peroxidase-catalyzed naphthols' and 4-hydroxybiphenyl oxidation and their distribution according to size in the interval 2.5-300 µm were detected by particle counting in solutions.

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KEY WORDS: fungal peroxidase, naphthol, 4-hydroxybiphenyl, albumin, polyethylene glycol, polyethyleneimine, poly-L-lysine, turnover number, light scattering, microparticles

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