New method for removing type 2 copper from Rhus laccase.

A new procedure is described for preparing tree laccase that is missing the type 2 copper. The derivative has only about 5% of the activity of the native enzyme, and some, or all, of the residual activity could be due to traces of holoprotein. The type 1 copper is fully oxidized in the purified type-2-depleted protein, while the type 3 site is reduced to the extent of at least 85%. However, the type 3 coppers can be reoxidized by treatment with excess H2O2. Reconstitution is achieved by incubation with Cu(I), and the remetalated protein exhibits the activity and the spectral properties of the native enzyme. The type 2 copper is removed by dialysis against a redox buffer containing ferri- and ferrocyanide ions as well as EDTA. More than 25% of the total copper is removed from laccase during the procedure, but the type-2-depleted fraction is readily isolated by means of an ion-exchange column. The practical advantages of this procedure are described. Finally, the simplicity of the method raises hopes that the mechanism of depletion can be defined.