Steroid-protein interaction: from past to present.

Structure of human α1-acid glycoprotein and its high-affinity binding site

Steroid-protein interactions. XXXIV. Chemical modification of α1-acid glycoprotein for characterization of the progesterone binding site

Kinetic and equilibrium studies on the interaction of steroids with human corticosteroid-binding globulin (CBG, transcortin) were performed with pH, temperature, and steroid structure as variables. Dissociation rate constants were determined fluorometrically; the values for cortisol, corticosterone, deoxycorticosterone, and progesterone are 0.031, 0.047, 0.10, and 0.16 s-1, respectively, at 20 degrees C, pH 7.4. The pH dependence of the dissociation rate constant for the corticosterone complex below pH 10.5 at 20 degrees C is given by koff = 0.043 (1 + [H+]/10(-6.50)) s-1; above pH 11, koff = 0.030 (1 + 10(-12.15/[H+] s-1. A temperature-dependence study of koff for the cortisol and progesterone complexes gave values of 0.0028 s-1 and 0.012 s-1 at 4 degrees C, respectively, and 0.88 s-1 and 4.5 s-1 at 37 degrees C, with progesterone dissociating about four to five times faster over the entire temperature range. The affinity constants, determined by equilibrium dialysis, for the binding of cortisol, corticosterone, and progesterone at 4 degrees C were 7.9, 7.2, and 7.0 X 10(8) M-1; values of 0.40 and 0.26 X 10(8) M-1 were determined at 37 degrees C for cortisol and progesterone. The close similarity of the affinity constants of the three steroids combined with differing dissociation rates implies that the association rate changes with steroid structure, in contrast to our earlier findings with progesterone-binding globulin.

Kinetic and equilibrium studies on steroid interaction with human corticosteroid-binding globulin.

Progesterone is bound in the blood serum of humans and most other mammalian species to three proteins: albumin; corticosteroid-binding globulin (CBG), or transcortin; and a,-acid glycoprotein (AAG), or orosomucoid. Whereas human serum albumin (HSA) consists of a folded polypeptide chain free of carbohydrate, AAG and CBG are glycoproteins with relatively high sugar content (TABLE I ) . The binding of the steroid hormones to the serum proteins is mediated essentially by hydrophobic forces and hydrogen bonds; noncovalent complexes are formed that are dissociable. The rate of dissociation increases with rising temperature. An inverse relationship exists between the serum concentrations of the three proteins and their affinity for progesterone. The association constant of CBG for progesterone at 37°C is approximately 500 times higher than that of HSA, but there are about 800 HSA molecules for each molecule of CBG in normal blood serum. This is the reason why the participation of HSA in the binding of steroid hormones is substantial'J; about half of the progesterone in pregnancy serum is associated with albumin (TABLE 2). A smaller percentage is albumin bound in the case of cortisol, a steroid of more hydrophilic nature than is progesterone. The biologic significance of protein binding of the steroid hormones3 is seen in several ways. The steroids are being transported by the serum proteins. This function does not include, however, the need for solubilization. because the maximal levels4 of even the least soluble progesterone are within the limits of aqueous solub i l i t ~ . ~ An important aspect of the protein complex formation is the protection of the steroid hormones from chemical or enzymatic attack, resulting in decreased metabolic clearance. This steroid-conserving mechanism has been emphasized recently6 for the progesterone-binding globulin (PBG) of the pregnant guinea pig, a serum glycoprotein to be discussed below. The steroids are biologically inactive as long as they are associated with protein; they can be \"activated\" by dissociation to the unbound hormone. In this manner, a relatively large amount of steroid can be carried in an indifferent storage form and can be made available immediately at the target tissue. For progesterone, nearly all of the hormone circulates in the body as a biologically inactive species (TABLE 2). The most desirable objective of the study of steroid hormone interaction with proteins would be the investigation of steroid receptor Unfortunately, none of these receptors has been available in purified form in sufficient amounts to make such study practical. Therefore, we have concentrated our efforts on the exploration of the chemical basis for the interaction of steroid hormones with serum proteins, preferably those of high affinity and specificity. Earlier results from our and other laboratories on the binding of progesterone to the three human proteins

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Papers

Steroid-protein interactions. Fluorescence quenching of progesterone-binding globulin and alpha1-acid glycoprotein upon binding of steroids.