Comparative characterization of methods for removal of Cu(II) from the active sites of fungal laccases

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