Preparations and Properties of Apo- and Reconstructed Rhus-Laccases

Abstract

1. Conditions for preparation of apo-laccase [EC 1. 10. 3. 2] and reconstruction of the holo-enzyme by treatment with copper ion were examined. The apo-enzyme was prepared by dialysis against 10-20mM cyanide solution containing 20-50mM ascorbate at pH 7.0-7.4. The properties of native laccase, such as its activity, copper content, absorption spectrum and electron spin resonance spectrum, were almost completely regained by treatment of this apo-protein with cuprous ion and the percentage re-construction was 70 to 90%. Some, though much less reconstruction was achieved by treatment of apo-enzyme with cupric ion.
2. The relationships between the copper content, the absorbancy at 615mμ and the activity were studied using “partial apo-enzymes, ” derivatives of native enzyme containing various amounts of copper obtained during cyanide-treatment. The de-crease in activity of the “partial apo-enzymes” was much larger than the decrease in their copper content. Thus, removal of half the initial amount of copper resulted in loss of more than 90% of the original activity. The cooperative action of copper atoms in the laccase reaction is suggested.