Spectroscopic studies of mercury binding to metallothionein

Abstract

Metallothioneins (MT) are a class of low-molecular weight, sulfur-rich metalloproteins that have been isolated from a wide variety of organisms [1]. Exposure to metal ions, such as Cd, Zn, Cu, Hg, Ag and Bi, result in tissues, especially the liver and kidneys, that contains much higher levels of these proteins. The most widely studied metallothioneins have been ▪ hepatic proteins isolated following induction with CdCl 2 [2–4]. In these proteins there is usually a mix of cadmium and zinc with a very little copper. Early reports have described some similar induction properties for mercury from which a metallothionein-like protein was obtained [5]. Despite this very wide interest in the metallothioneins there are very few papers describing mercury binding in vitro [6, 7]. Mercury has been shown to displace zinc and cadmium from metallothionein in vitro [6, 7]. Changes in the metal composition within Cd,ZnMT are readily followed in the UV absorption, CD and MCD spectra as the prominent shoulder at 250 nm is a good indicator of the presence of cadmium [2, 3]. In titrations with mercury the 250 nm band is replaced by a broad band near 300 nm [6]. In this paper we present a detailed description of mercury binding to metallothionein. Titrations were carried out using horse kidney and rat liver Cd,ZnMT and rat liver Bi,ZnMT. Metal binding was followed by monitoring the absorption, CD and MCD spectra in the 210–350 nm region. Rat liver Cd,ZnMT 2, and Bi, ZnMT 2 rat kidney Hg,CuMT 1 were induced in rat tissues by injections of CdCl 2, BiCl 3 or HgCl 2, respectively [8]. Equine metallothionein was isolated from horse kidney [9]. Optical measurements were carried out as previously reported [2]. Figure 1 shows an example of the optical changes that occur when mercury is added in vitro to metallothionein. In these experiments horse kidney. Cd,ZnMT 1 was titrated with aliquots of an aqueous solution of Hg(NO 3) 2. The metal concentrations in the Cd, ZnMT 1 were determined by atomic absorption spectroscopy; the values measured were Cd: 4.0, Zn: 1,2 and Cu: 0.2 (each expressed as moles of metal/mole of protein). Line #1 in Fig. 1 shows the spectra obtained by all three techniques for the native protein; it is characteristics of Cd,ZnMT data that derivative-shaped Cd and MCD envelopes are observed under the 250 nm shoulder in the absorption spectrum [2, 3]. As mercury is slowly added, we observe first a slight increase in absorbance at 250 nm, however, after the addition of 1.50 mol eq (line #5) the absorbance at 250 nm gradually decreases. Throughout the whole titration the absorbance at 300 nm, and both the CD and MCD spectra show continuous changes that mark the loss of the cadmium and the binding of mercury to the protein. The final traces in Fig. 1 (line #9) show that the cadmium has been displaced by the mercury: there is no longer a shoulder at 250 nm in the absorption spectrum, and the derivative-shaped envelopes in both CD and MCD spectra have collapsed. The new absorption, CD and MCD intensity at 300 nm is assigned as the sulfur to mercury charge transfer band. We observe similar spectra for the Hg,CuMT from rat kidney and Hg complexes of BAL that serve as models of the binding sites in metallothioneins.