Isolation and Characterization of a New Zinc-Binding Protein from Albacore Tuna Plasmat

The protein responsible for sequestering high levels of zinc in the plasma of the albacore tuna (Thunnus alalunga) has been isolated by sequential chromatography. The glycoprotein has a molecular weight of 66000. Approximately 8.2% of its amino acid residues are histidines. Equilibrium dialysis experiments show it to bind 3 mol of zinc/mol of protein. The stoichiometric constant for the association of zinc with a binding site containing three histidines was determined to be This protein is different from albumin and represents a previously uncharacterized zinc transport protein. H i g h levels of zinc, relative to other trace elements, in the tissue of fsh have been recognized for a long time (Vinogradov, 1953). Plasma of the albacore tuna (Thunnus alalunga), for example, has a zinc level more than 12 times higher than human serum. It is clear from the work of many investigators [see Vallee (1959)] that in biological systems zinc must exist as complexes with organic ligands rather than as free ions in solution. This paper presents our isolation and investigation of the plasma protein in albacore which is responsible for complexing the large amount of endogenous zinc. Fletcher and Fletcher (1978) described a similar Zn-binding protein(s) in winter flounder responsible for binding almost all plasma zinc. Since. zinc in plasma is chelated to endogenous ligands, only high-affinity sites on a plasma-binding protein are likely to be occupied under biological conditions. We have therefore designed experiments to characterize such sites by chelating the zinc with a well-characterized ligand in order to prevent hydrolysis and to form a mathematical basis with which to quantitate the competition for protein uptake of this metal. Experiments using competitive chelation to modulate zinc binding to proteins have been carried out with some enzymes (Cohen & Wilson, 1966; Billo et al., 1978). We have generalized this approach to multicomponent zinc-binding systems and have adapted the traditional Scatchard analysis of equilibrium binding to investigate the high-affinity Zn-binding sites on the tuna protein. 'This work was supported by USPHS Grant AM-12386. *Corrcspondcnct should be addressed to this author. 0006-2960/87/0426-3228SOI .50/0 MATERIALS AND METHODS Albacore Plasma. Albacore were captured with hookand-line in the Pacific Ocean within 200 miles of San Diego during the months of July and August of 1979-1983 aboard the N.O.A.A. research vessel David Starr Jordan. Blood was collected immediately in syringes by cardiac puncture. Because blood clotting and clot dissolution occur almost simultaneously in this fish, true %erum" was not obtained; rather, plasma was collected within 30 min of bleeding after centrifugation of red cells at approximately 2000g for 15 min at room temperature. Plasma was stored frozen at -18 OC. Radiozinc Solutions. 65ZnCI, ('carrier-free"; New England Nuclear Corp., Boston, MA; specific activity 6-7 mCi/mg) was diluted into 0.1 N HCI prior to use. Radioactivity measurements were made in a Nuclear-Chicago Model 1085 y well counter with the spectrometer calibrated to capture the 1.11-MeV emission (3~10%) of 65Zn. Protein Purification. ( A ) Rationale for Purification. At the beginning of our studies, we were able only to distinguish the tuna plasma Zn-binding protein as one of the two most anodal "albumin-like" bands in sodium dodecyl sulfate (SDS) gel electrophoresis (data not shown). For purification, we therefore explored chromatographic systems that would fractionate these two bands, which seemed similar in size and electrophoretic mobility, on the basis of properties other than charge alone. Metal-chelating resins with iminodiacetate functionalities have been used successfully for affinity chromatography of other metal-binding proteins (Muszynska et ai., 1986). Lectin affinity chromatography has been of value in the separation of various non-glycoprotein plasma albumins