Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

O. V. Koroleva^1*, E. V. Stepanova¹, V. P. Gavrilova², V. I. Biniukov¹, and A. M. Pronin¹

¹Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia; fax: (095) 954-2732; E-mail: alena_koroleva@hotmail.com

²Komarov Botanical Institute, Russian Academy of Sciences, ul. Popova 2, St. Petersburg, 197022 Russia; fax: (812) 234-4512; E-mail: Valeria@VG2438.spb.edu

^* To whom correspondence should be addressed.

Received March 23, 2000; Revision received May 10, 2001

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Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.

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KEY WORDS: laccase, copper sites, EPR spectroscopy, removal of type 2 copper, bathocuproine, reconstitution of laccase

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