Thyroxine-Protein Interactions: V. ISOLATION AND CHARACTERIZATION OF A THYROXINE-BINDING GLOBULIN FROM HUMAN PLASMA

Abstract

A thyroxine-binding globulin has been isolated in highly purified form from human plasma. The purification procedure consisted of the following steps: (a) fractionation of plasma with ammonium sulfate, (b) zone electrophoresis on an LKB Porath cellulose column, (c) re-electrophoresis on the Porath column, (d) chromatography on DEAE-cellulose, (e) gel filtration on Sephadex G-150 or G-200, and (f) preparative electrophoresis in polyacrylamide gel. The isolated thyroxine-binding protein was homogeneous when analyzed by electrophoresis in starch gel, immunoelectrophoresis, and disc electrophoresis. The protein sedimented as a single component in the ultracentrifuge when analyzed by the methods of sedimentation equilibrium and sedimentation velocity. The weight average molecular weight as determined by sedimentation equilibrium is 58,000; the sedimentation constant, s20, w0, is 3.92 × 10−13 sec. The thyroxine-binding globulin is a glycoprotein containing 6.70% neutral hexose, 2.38% sialic acid, 3.31% glucosamine, and 0.70% galactosamine. Based on the results of electrophoretic analysis on paper, the final product of thyroxinebinding globulin exhibited a high affinity for thyroxine, with a binding constant of the order of 109M−1. However, the maximal binding capacity of the protein for thyroxine appears to be reduced to about 25% of the expected value if binding is assumed to take place at a single strong binding site on the protein. The isolated thyroxine-binding protein was indistinguishable from endogenous thyroxine-binding α-globulin of serum when analyzed by electrophoresis in starch gel, in polyacrylamide gel, and on paper.