The reactions of Zn-, Zn,Cd-, and Cd-thioneins with EDTA and apo-carbonic anhydrase have been studied. The ligand substitution reaction of zinc with EDTA is multiphasic, having both associative and dissociative components in the rate expression. The cadmium sites are about 2 orders of magnitude less reactive. In contrast, apo-carbonic anhydrase abstracts zinc from Zn-thionein and ZnCd-thionein in second-order processes that are 2-3 orders of magnitude more rapid than those involving EDTA and approach the rate for unligated Zn2+ with the apo-protein. In comparison with other zinc proteins, Znthionein contains unusually reactive metal sites, suggesting that this protein may be a physiological zinc transfer protein, able to donate zinc to zinc-requiring apo macromolecules.Although metallothioneins have been known for more than 20 years, insight into their molecular function is only gradually being developed. Metallothionein was discovered as the principal cadmium-binding protein in the cytosol of horse kidney cortex (1). Chemical studies of metallothionein reactions have not progressed far beyond the first report after its identification in cells (2). Hence, although metallothionein binds cadmium after exposure of the host to this toxic metal and recently has been shown to bind zinc and copper under various physiological conditions, no bioinorganic studies have provided a foundation for reasonable hypotheses as to the meaning of these results (3)(4)(5)(6)(7)(8). Such studies are difficult because there are seven sites that may be occupied by a combination of Zn and Cd ions.Recently, we have begun to investigate the metal-sulfhydryl sites of metallothionein to explore possible roles they may play in the metallobiochemistry of cells. Independently, Udom and Brady (9) have published biochemical results consistent with the hypothesis that Zn-thionein may be a physiological donor of zinc to zinc-apometalloproteins. In this communication, data are presented in ligand substitution reactions of various thioneins with EDTA and apo-carbonic anhydrase, which support this thesis. MATERIALSBovine carbonic anhydrase B was purchased from Worthington; 5,5'-dithio-bis(2-nitrobenzoate), phenylmercuric acetate, pnitrophenyl acetate, and pyridine-2,6-dicarboxylic acid were from Sigma; EDTA (gold label) was from Aldrich; 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene was from Eastman. The metal salts and other chemicals were reagent grade purity. Horse kidney metallothionein was prepared as described (10).Preparation of apo-Carbonic Anhydrase. Bovine carbonic anhydrase B was treated with pyridine-2,6-dicarboxylic acid to remove zinc (11).Analytical Methods. Metal contents of metallothionein and metal-EDTA complexes were measured with a Perkin-Elmer 360 spectrophotometer. The thiol content of metallothionein was measured either colormetrically by using 5,5'-dithiobis(2-nitrobenzoate) or amperometrically by using phenylmercuric acetate as the titrant (12, 13). Disulfide content was determined amperometrically after cleavage of the ...

Rat liver and horse kidney metallothioneins react with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) to release 5-thio-2-nitrobenzoate and metal ions. The reactions are slow and exhibit biphasic kinetics with each process having an empirical rate law of the form:where RSM represents metal-bound thiolate groups.The pseudo-first-order rates are insensitive to pH but are modified in guanidine hydrochloride solution. Rat liver metallothioneins of variable zinc, copper and cadmium composition react similarly and give observable thiol/total metal ratios in good agreement with stoichiometries of SH/(Cd + Zn) of 3 and SH/Cu of 1. A model complex cadmium-2,3-dimercaptopropanol, resembles the protein in its reaction with Nbs2'