ISOLATION AND ANALYSIS OF A THIOL-SENSITIVE, HIGH-AFFINITY ZINC-BINDING PROTEIN FROM PLASMA (PROTEINS, ALBUMIN, GLUTATHIONE)

GERALD ROSS MAGNESON, Purdue University

Abstract

Metal-binding studies using a new enzymic assay procedure to measure near picomolar concentrations of free zinc in biological fluids indicate that a thiol-sensitive component in blood plasma, presumably a protein, exhibits high affinity for zinc, binds the majority of exchangeable zinc in plasma, and is neither albumin nor alpha-2-macro-globulin. In contrast to a widely-accepted model that proposes that albumin serves as the major binding protein for the binding of exchangeable zinc in blood plasma (Giroux and Henkin, 1972), these studies indicate that the free zinc concentration in both commercial rabbit serum and fresh horse plasma, about 10('-10) M, is at least an order of magnitude lower than that previously reported (Berthon et al., 1980) and is too low to be rationalized solely in terms of binding to albumin. Under appropriate conditions, the apparent binding constant of a thiol-sensitive, high-affinity zinc-binding component (protein) for zinc, about 2 to 6 x 10('9) M('-1), is about two orders of magnitude greater than that of albumin, about 3 x 10('7) M('-1). A high-affinity zinc-binding component was partially purified from rabbit serum via two different chromatographic protocols and was shown to be similar to a zinc-binding fraction of human serum previously reported (Himmelhoch et al., 1966). Because its molecular weight (i.e., <25 >kDa) is much lower than that expected for the majority of proteins in blood, further attempts to isolate a native form of the high-affinity, zinc-binding component were conducted by using freshly prepared horse plasma that was treated in different ways to minimize potential proteolysis. Studies with fresh horse plasma provide somewhat different results than those with rabbit serum. In these studies, a previously unreported zinc-binding component (protein) present in small amounts relative to albumin was shown to bind zinc much more tenaciously than does albumin, but only in the presence of a reducing agent (e.g., 1 mM glutathione). In addition, the zinc-binding affinity of freshly prepared albumin is not affected by glutathione. This thiol-sensitive, high-affinity zinc-binding component (protein) was partially purified by column chromatography; its subunit M(,r) is about 70 kDa. The differences in the properties of the high-affinity zinc-binding component (protein) in commercial serum and fresh plasma are perplexing and remain to be elucidated.

Degree

Ph.D.

Subject Area

Biochemistry

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