Identification and purification of a high-affinity thyroxine binding protein that is distinct from albumin and prealbumin in the blood of a turtle, Trachemys scripta

Abstract

Fractionation of plasma proteins in the turtle, Trachemys scripta, confirmed the presence of a high-affinity thyroxine (T 4) binding protein (TBP) that was distinct from albumin (ALB) and prealbumin (PA). The TBP was isolated by adsorption on a T 4-affinity column and a high degree of purification was achieved by gel filtration and preparative electrophoresis. Analysis by reversed-phase HPLC showed a single peak of protein with T 4 binding activity. The electrophoretic mobility of the TBP, based on staining and binding to [ 125I]T 4 on nondenaturing polyacrylamide gels (PAGE), corresponded to that of the major T 4 binding activity previously identified in plasma (ca. 60 kDa). PA was fractionated as a complex with retinol binding protein (PA-RBP) based on retinol associated fluorescence using ion exchange chromatography on DEAE-Sephacel and gel filtration. This complex behaved as a larger and more highly charged molecule than TBP; it was partially dissociated in low ionic strength basic solution. SDS-PAGE of the PA-RBP-enriched fraction revealed a major component of about 48 kDa (possibly free PA), with smaller components corresponding to those expected for free RBP (ca. 22 kDa) and subunits of PA (e.g., 14 and 28 kDa). ALB was purified by ion exchange chromatography on DEAE and gel filtration; it behaved as less basic than PA with MW ≈ 67 kDa. TBP accounted for virtually all the T 4 binding activity of whole plasma: TBP was about 100 times as active and PA and ALB were <1% as active as plasma. The binding affinity of purified TBP was similar to that of whole plasma from turtle and human (e.g., approx. 10 9 M −1 on Sephadex G-25).