CHARACTERIZATION OF CALF LIVER CU,ZN-METALLOTHIONEIN - NATURALLY VARIABLE CU AND ZN STOICHIOMETRIES

Cu,Zn-metallothioneins were purified from bovine calf liver in order t o examine the stoichiometry of metal binding to the protein. Copper an d zinc analyses were carried out by atomic absorption spectrophotometr y. Consistent quantitative thiolate analyses were obtained spectrophot ometrically with Ellman's reagent and amperometrically with phenylmerc uric acetate. These were used to define protein concentration. A compl ementary method to assess the sum of the thiol and Cu(I) content of me tallothionein involved titration of the reducing equivalents of the pr otein with ferricyanide. The stoichiometry of reaction was consistent with the oxidation of all the sulphydryl groups to disulphides and all of the bound Cu from the cuprous to the cupric oxidation state. Accor ding to these methods, total numbers of zinc plus copper ions bound to metallothionein isolated from a number of calf livers centred on abou t 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 7-phenylsulphonyl-2,9-dimethyl-1,10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assesse d. Zinc metallothionein reacted almost entirely in two slow steps with ferricyanide. As the Cu content of the protein increased, the fractio n of reaction occurring in the time of mixing increased in parallel. B CS was able to remove 70-80 % of metallothionein-bound Cu as Cu(I). Th e rest was resistant to reaction.