SILVER BINDING TO RABBIT LIVER METALLOTHIONEIN - CIRCULAR-DICHROISM AND EMISSION STUDY OF SILVER-THIOLATE CLUSTER FORMATION WITH APOMETALLOTHIONEIN AND THE ALPHA-FRAGMENTS AND BETA-FRAGMENTS

Abstract

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo.alpha. and -.beta. fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20.degree.C and 55.degree.C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the .alpha. and .beta. fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The .alpha. and .beta. fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3-.alpha. MT 1 and Ag6-.alpha. MT 1 is suggested by the spectral data obtained at 20 and 55.degree.C. Formation of Ag3-.beta. MT 1 is suggested by the spectral data recorded at 20.degree.C for the .beta. fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.