The purification of thyroxine-binding globulin and thyroxine-binding prealbumin

Abstract

Methods have been described for the purification of the two principal thyroxine binding proteins in human serum, thyroxine-binding globulin (TBG) and thyroxine binding prealbumin (TBPA). Cohn Fraction IV-9 was used for the large-scale preparation of TBG. Unfractionated human serum or Cohn Fraction IV-9, containing tracer amounts of 131I-labelled l-thyroxine, were subjected to the following procedures to yield fractions of increasing 131I/protein ratios: column electrophoresis on cellulose powder, starch gel electrophoresis, gel filtration and ion-exchange chromatography. The identity and homogeneity of preparations from different purification steps were established by the following procedures: ultracentrifugal analysis, immunoelectrophoresis, zone electrophoresis on paper, cellulose acetate and starch gel in various buffers, immunodiffusion and autoradiography to locate the radioactive thyroxine. Although not pure, the final TBG and TBPA preparations were not contaminated mutually or by any other thyroxine-binding protein. An unidentified α-globulin ( S 20, w = 7.0−7.2) with moderate thyroxine-binding properties was separated from TBG ( S 20, w = 3.5) by ion-exchange chromatography. The importance of using multiple criteria in establishing the identity and homogeneity of TBG and TBPA is discussed.