Stephen D. Stroupe

Steroid-protein interactions—XXXVIII. Influence of steroid structure on affinity to the progesterone-binding globulin

Abstract The association constants, K A , of the complexes of more than a hundred steroids with the progesterone-binding globulin (PBG) were determined by fluorescence quenching titration and competitive binding equilibrium dialysis. By comparison of the K A values for individual steroids that differed only by one structural change, the effect of this change on the binding affinity and on the free energy of binding was assessed. The binding to PBG is provided predominantly by hydrophobic forces; hydrogen bonding also occurs, for example, at the 3-oxo group. The progesterone molecule appears to be in almost perfect contact with the binding site of PBG; introduction of most substituents decreases the binding affinity. Testosterone is more loosely associated with PBG, but the affinity constant approaches that of the progesterone complex when the 17β-hydroxy group is acetylated. Changing the steroid conformation to a more planar structure results in stronger binding; this is seen with 5α-pregnane-3,20-dione, with the 4,6-dienes, and with the 19-norsteroids. A synoptic interpretation of the effects of the various structural changes on binding affinity leads to a conceptual image of the binding site.

Steroid-protein interactions: Fluorescence quenching of progesterone-binding globulin and α1-acid glycoprotein upon binding of steroids☆

Abstract The influence of progesterone and four other steroids on the intrinsic fluorescence of progesterone-binding globulin was investigated. The corresponding effect of progesterone on α1-acid glycoprotein was also studied. The intrinsic fluorescence of the progesterone-binding globulin and of α1-acid glycoprotein was quenched by about 60 and 17%, respectively, upon forming stoichiometric complexes with progesterone. Graphical analysis of fluorescence quenching titrations with progesterone gave affinity constants at 23 °C of 2 × 109 m −1 for progesterone-binding globulin and 1 × 106 m −1 for α1-acid glycoprotein. With progesterone-binding globulin, affinity constants of 1 × 109 m −1 were determined for desoxycorticosterone, 1 × 108 m −1 for testosterone, and 2 × 106 m −1 for cortisol. The fluorescence quenching of PBG by 5-pregnen-3β-ol-20-one, 5α-pregnanedione, and 5β-pregnanedione, steroids lacking the Δ4-3-keto grouping, was too small to be evaluated; however, binding of the pregnanediones to progesterone-binding globulins was demonstrated when the progesterone-progesterone-binding globulin complex was “unquenched” as a result of competitive displacement of progesterone by addition of the pregnanediones. The quenching phenomenon is assumed to be mainly due to radiationless transfer from protein to the near uv (n → π∗) absorption band of steroids containing the Δ4-3-keto chromophore.

Steroid interactions with progesterone-binding globulin

Abstract The progesterone-binding globulin (PBG) of the pregnant guinea pig has been purified on sulfopropyl Sephadex and subsequent affinitychroma tography on 19-nortestosterone-Sepharose. A pure glycoprotein of molecular weight 88,000 was obtained that contained about 70% carbohydrate. PBG is polydisperse and has a polypeptide core of about 27,000 daltons. It has one binding site for progesterone ( K a 37 ° = 4 × 10 8 M −1 ). Studies on chemical modification of PBG show the involvement of tryptophan, lysine, and tyrosine in steroid binding. In typical reactions, the binding sites were inactivated by the chemical substitutions; complex formation with progesterone protected the binding site from the modification. Association of progesterone or other steroids with PBG results in conformational changes as seen from circular dichroism and ultraviolet difference spectra. The intrinsic tryptophan fluorescence of PBG is quenched by more than 80% when one mol progesterone is bound. This is the basis for a fluorescence quenching titration method to determine binding site concentrations and association constants. Association and dissociation rate constants were measured by stopped-flow fluorescence techniques; it was found that the affinity of the steroid-PBG complexes is controlled by the rate of dissociation. Steroid complexes of high-affinity binders from serum have generally higher dissociation rates than those of cellular receptor proteins. This is in harmony with the physiological functions of the two types of steroid-binding proteins.

Steroid-protein interactions. Purification of progesterone-binding globulin by affinity chromatography.

The progesterone-binding globulin (PBG) found in the serum of pregnant guinea pigs [l] has been purified by various methods, based mainly on ion exchange and gel chromatographic techniques [2-41. It has been recognized as a polydisperse glycoprotein with the unusually high carbohydrate content of about 70% [4]. Taking advantage of the low pI of 2.8 and favorable acid stability [5] , the isolation procedure of PBG was improved [6] by utilizing chromatography on sulfopropyl (SP) Sephadex, a strong cation exchanger. The purified PBG preparations obtained in our laboratory often had values of n, i.e., number of progesterone-binding sites per protein molecule, of significantly less than unity. Assuming that this discrepancy was caused by contamination with inactive material, we purified the PBG by affinity chromatography on modified Sepharose. The present paper describes these studies.

Steroid–protein interactions Kinetics of binding of cortisol and progesterone to human corticosteroid-binding globulin

Human corticosteroid-binding globulin is the first high-affinity steroid binding protein that has been studied in detail [l--S J . Although its general physicochemical parameters (molecular weight, absorptivity, carbohydrate and amino acid content) have been defined, further information related to steroid binding of CBG” is needed. It is noteworthy that the rather unusual progesterone binding globulin (PGB), obtained from pregnant guinea pig serum, has been more thoroughly characterized with respect to specificity [6-81 and with respect to mechanism of binding [9111 than has human CBG . The sensitive method of stopped-flow fluorescence has been applied to explore the mechanism of binding of steroids to human CBG. The binding of steroids to various proteins results in quenching of the intrinsic fluorescence of the protein [9] . The large decrease in fluorescence which occurs upon the binding of 3-oxopene steroids to PBG has been used to measure the rates of complex formation and dissociation [ 1 I] . We have recently demonstrated the generality of the fluorescence quenching method in determinations of the dissociation rate constants of human CBGsteroid com-

Steroid protein interactions. XXVIII. The isoelectric point and pH stability of the progesterone-binding globulin.

Abstract Isoelectric focusing of progesterone-binding globulin of pregnant guinea pig serum occured at pI = 2.8. This result was obtained when the binding activity of the focused protein fractions was measured by equilibrium dialysis. Reliance on localization of the radioprogesterone alone may lead to erroneous results presumably due to dissociation of the uncharged progesterone molecule from the protein during the electrophoretic migration into the acidic milieu. This assumption was corroborated by binding assays over the pH range from 2 to 11. Almost complete dissociation occurred at pH 4 and below; however, return to neutral pH restored over 80% of the binding activity. The pH gradient of the electrofocusing column was improved by adding aspartic and glutamic acid to the pH 3–5 Ampholines.

Mechanism of steroid binding to serum proteins

Association and dissociation rate constants of steroid complexes with progesterone-binding globulin (PBG) and with corticosteroid-binding globulin have been determined, utilizing the fluorescence quenching phenomenon observed on steroid binding to protein. Stopped-flow techniques were used in most cases. The dissociation rates of the complexes with steroid-binding proteins of serum are much greater than those of steroid-receptor complexes, in accordance with the biological functions of these two types of proteins. Association of steroids with PBG is accompanied by conformational changes in both components of the complexes. Chemical modification of tryptophan, lysine, and tyrosine in PBG results in inactivation of the binding site; complex formation with progesterone protects against this inactivation. A comparison of the affinity constants of PBG complexes with steroids of different structures leads to a conceptual image of the binding site and to localization of the various forces of interaction over the binding site area.

Kinetic analysis of calcium binding to concanavalin A

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a kobs of 0.012 sec-1. Kinetic measurements were used to calculate the association constant, Ka, for calcium binding to concanavalin A of 2.7 x 10(4) M-1, in reasonable agreement with values obtained by equilibrium methods.