THE MEASUREMENT OF HUMAN THYROXINE-BINDING PROTEINS BY ELECTROPHORESIS ON CELLULOSE ACETATE MEMBRANES: STUDIES ON A SPURIOUS FOURTH THYROXINE-BINDING COMPONENT

Abstract

Electrophoresis of human serum enriched with 131I-labeled L-thyroxine was performed on strips of cellulose acetate in a tris(hydroxymethyl)aminomethane –ethylenediaminetetraacetate – borate buffer, pH 9.0. High-sensitivity scanning of the strips revealed the presence of a poorly defined radioactive component between α2- and γ-globulins. This component was present in all sera in which the affinity of thyroxine-binding prealbumin (TBPA) was less than 30%, and was inversely related to TBPA at thyroxine levels of 0.02,1.0, and 5.0 μg/ml. The α2γ component, like the other three thyroxine-carrying proteins, was found to vary considerably with the volume of serum applied to the strip. With increasing sample volumes, the percentage of radioactivity in the α2γ component decreased but the absolute amount remained constant. Furthermore, the α2γ component contained increasing proportions of the total radioactivity as larger amounts of stable L-thyroxine were added to the serum. Soaking the strips in buffer containing 0.06% bovine albumin before the samples were applied virtually eliminated the α2γ component and substantially increased TBPA. It was concluded that the α2γ component was due to trailing of TBPA across albumin and thyroxine-binding globulin, and that all the electrophoretic data obtained with media where some radioactivity cathodal to thyroxine-binding globulin was present should be cautiously reappraised.