AN ELECTRON-PARAMAGNETIC-RESONANCE METHOD FOR QUANTIFICATION OF COPPER ISOTOPES IN PROTEINS - APPLICATION TO A MIXED-ISOTOPE DERIVATIVE OF LACCASE

We have developed a method of denaturing a copper-containing protein a nd sequestering the copper in the form of the bis(acetylacetonato)copp er(II) complex. Because of the excellent resolution of the hyperfine s tructure in the resulting EPR spectrum, the method is useful for deter mining the Cu-65/Cu-63 isotope ratio in proteins such as tree laccase. Here we analyzed a mixed isotope sample prepared by reconstituting Cu -63-enriched type 2-depleted laccase with Cu-65(I) and found that it c ontained only 25% Cu-65. This proves that reconstitution of the type 2 copper is possible under conditions that permit little or no exchange between resident copper and the external pool. However, exchange does occur under reducing conditions when we expose the holoprotein to a l arge excess of exogenous copper. (C) 1995 Academic Press, Inc.