ISOLATION, IDENTIFICATION, AND PROPERTIES OF A NEW THYROXINE-BINDING PROTEIN - HUMAN BLOOD-SERUM APOLIPOPROTEIN-A-I

A protein with molecular weight approximately 25 kD, which differs in properties from the known T4-binding proteins, was isolated from human blood serum by affinity chromatography on thyroxin (T4)-Sepharose. Using immunodiffusion, radioimmunoassay, ultracentrifugation, determination of lipid composition, differential precipitation, and electrophoresis it was shown that the protein isolated is contained in the high-density lipoprotein (HDL) particles and represents apolipoprotein All (apoA-1). Chromatography on cholate-Sepharose separated apoA-1 from the lipid component and protein impurities and was effective in the isolation of apoA-1 directly from blood serum. The apoA-1-HDL and apoA-1 obtained by affinity chromatography, as well as the HDL3 fraction isolated by ultracentrifugation according to the standard procedure, possessed T4-binding activity; their affinity for the hormone was of the same order of magnitude. The interaction of T4 with these preparations induced differential UV absorption signals, changed the characteristics of the intrinsic fluorescence of apoA-1, but did not affect the circular dichroism of the hormone-protein system. The binding of spin-labeled T4 to apoA-1, apoA-1-HDL3 led to a substantial change in the shape of the ESR spectrum and an increase in the effective rotational correlation time. The mobility of the radical fragment of spin-labeled T4 depended on the composition and properties of the protein preparation. The electron spectroscopic data suggest that the interaction of T4 with HDL proceeds according to specific mechanisms, and the molecular structures of the complexes formed are not characteristic of any other known T4-binding proteins.