Factors Affecting the Binding of Cortisol by Transcortin

Abstract

By means of a gel filtration on Sephadex columns the plasma corticoids can be separated into two fractions: namely a fraction bound to a macromolecular agent and an unbound fraction. When plasma is overloaded with cortisol before gel filtration, the binding capacity of the macromolecular agent can be evaluated. The bound corticoids can be measured quantitatively either fluorimetrically or colorimetrically. The macromolecular binding agent behaves like transcortin in a series of experiments. Various exogenous and endogenous steroids are known to influence transcortin activity in vivo. Methylestrenolone and lynestrenol administered orally to normal males or to women with various gynaecological conditions did not significantly increase the cortisol binding capacity. Intravenous administration of Metopirone lowered the cortisol binding capacity in normal subjects but not in a patient with hypophysial insufficiency. Attempts were made to remove steroids from plasma without destroying transcortin, in order to rule out any possible interference with the cortisol binding process by other steroids. Extraction of plasma with organic solvents either at room temperature or at minus 25° C irreversibly destroyed most of the transcortin. Dialysis of plasma against a 1% solution of bovine albumin (40 volumes) for 12 h at 40° C decreased the unconjugated corticoid level by 63%. This procedure did not alter transcortin activity in the plasma of normal adults, in umbilical cord blood, in pregnancy plasma or in the plasma of obese patients with a low cortisol binding capacity. However, the lowering in C.B.C. observed in the plasma of patients treated with Metopirone could be partly abolished by this procedure. Although the extraction experiments at low temperature suggested the existence of a lipid-link in the cortisol binding system of human plasma, this could not be demonstrated.

Studies of human transcortin at different pHs: Circular dichroism, polymerisation and binding affinity

“…If we measure the percentage inhibition with respect to the binding capacity at pH 8.0, it can be seen that 93% of the binding power is lost at pH 4.0. This result is similar to the appearance of heat-inactivated trans- These binding affinities determined on pure human transcortin corroborate the results obtained on whole serum [7,14] but the polymerization at low pH has never been observed as impure human transcortin does not polymerise [4]. see four dichroic bands at 320,290,236 and 200 nm.…”

“…Inactivated transcortin was obtained by incubating a solution of 10 mg of transcortin/l ml of distillated water at 60°C for 30 min. Indeed, De Moor et al [7] have shown that human transcortin was irreversibly inactivated at this temperature.…”

“…Since it is well-known [7] that the binding affinity of serum decreases with the pH and becomes zero when the pH = 2, we have recorded the dichroic spectra of the CBG-cortisol complex between pH 7.9 and 4. Below pH 4, CBG precipitates.…”

A Combined Adsorption-Gel Filtration Technique for the Determination of the Cortisol-Binding Capacity of Transcortin

“…The introduction of the adsorption step provides for complete removal of endogenous steroids from plasma without damaging transcortin. Other stripping procedures seem to be less or not at all suitable: Extraction with organic solvents leads to an irreversible denaturation of transcortin, whereas dialysis against bovine albumine at 40 °C fails to remove the endogenous steroids sufficiently (15). Gel filtration at 46 °C (16; own experiments) or at pH 5.0-5.5 (17) brings about the desired stripping effect, yet it lacks the technical simplicity of the adsorption technique.…”