Purification and characterization of human transcortin

Abstract

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.

Identification of Trp-371 as the main site of specific photoaffinity labeling of corticosteroid binding globulin using .DELTA.6 derivatives of cortisol, corticosterone, and progesterone as unsubstituted photoreagents

“…These association constants agree in magnitude with those previously reported for the binding of cortisol, corticosterone, or progesterone to purified human CBG, either in the absence or in the presence of reducing agents (Stroupe et al, 1978;Mickelson et al, 1981Mickelson et al, , 1982 and for the binding of these same steroids to unpurified CBG from human plasma (Westphal, 1971). However, it has been reported that the number of binding sites and the affinity of CBG in human plasma may be underestimated, as compared with those of purified CBG, owing to differences in environment effects (Mueller & Potter, 1981.…”