Ulrich Westphal

Steroid-protein interactions: XIII. Concentrations and binding affinities of corticosteroid-binding globulins in sera of man, monkey, rat, rabbit, and guinea pig☆

Abstract Endogenous corticosteroid hormones and other steroids were removed from sera by gel filtration over Sephadex at 45 °. At this temperature, the association constant of the corticosteroid-binding globulins (CBG)-corticosteroid complex is many times smaller, and the rate of dissociation considerably higher than at 4 °. The removal of exogenous hormone was practically complete; the CBG activity was not affected. Binding affinity of the filtered sera was determined by equilibrium dialysis in the presence of radiolabeled cortisol, corticosterone, and progesterone at different levels, at 4 ° and 37 °. Concentrations of CBG binding sites and apparent association constants for the various steroid hormone-CBG complexes were derived from the dialysis data. The concentration of binding sites was found to be approximately the same for the three steroid hormones studied, ranging from 3.4 × 10 −7 m for rabbit serum to 11.3 × 10 −7 m for rat serum, with an intermediate level of 7.2 × 10 −7 m for human serum. The association constant at 4 ° was between 10 and 40 times greater than at 37 °. As far as comparisons can be made, the values presented are in good agreement with those reported from other laboratories.

Steroid-protein interaction: from past to present.

In this review, the association between biologically active compounds and proteins is seen in the light of axioms expressed by Paracelsus and Paul Ehrlich long ago, and the physiological significance of the interactions is pointed out. Of the various types of proteins that form noncovalent complexes with steroid hormones, only the serum proteins will be discussed. Recent results, obtained in several laboratories, on the physicochemical properties of the human corticosteroid-binding globulin (CBG, transcortin) makes this glycoprotein perhaps the best known one among the steroid-binding serum proteins. Influence of pH on stability of the complexes, kinetics of the associations and their significance, as well as thermodynamic parameters of complex formation are being discussed. Characteristics of the binding sites are deduced from specificity studies. Influence of the entrance of hydrophilic or hydrophobic substituents into the steroid molecule illuminates the difference between typically hydrophobic binders such as the progesterone-binding globulin (PBG) of the pregnant guinea-pig and typically hydrophilic binders such as CBG. Complete elucidation of the steroid-binding proteins awaits the determination of the amino acid sequence and the X-ray crystallographic analysis of the steroid-protein complex.

Non-Esterified Higher Fatty Acids in Serum of CCl4-Treated and Normal Rats and Other Species

Summary 1. Available methods for the determination of the “free,” i.e. non-esterified, higher fatty acid fraction in serum were modified. These acids are firmly bound to albumin. Slightly less than one mole of them was found per mole albumin in normal rat, rabbit and bovine serum. 2. Serum of carbon tetra-chloride-treated rats contained more non-esterified higher fatty acids (1.30 moles per mole albumin) than that of normal rats (0.85 mole per mole albumin). The difference was statistically significant. 3. The results are considered indicative of the impaired liver function in the injured rats. They offer further evidence for the postulation that the lowering of the azorubin-binding capacity of serum albumin is caused by increased concentrations of firmly bound higher fatty acids.

Steroid hormone-binding proteins in blood plasma

Abstract Two types of proteins form dissociable complexes with the circulating steroid hormones in blood serum: albumin, the most abundant plasma protein, and the highly specific glycoproteins that occur in low concentrations: corticosteroid-binding globulin (CBG or transcortin), sex steroid-binding protein (SBP) and progesterone-binding globulin (PBG). Albumin interacts with low affinity, predominantly by hydrophobic bonding; the affinity constants of the steroid complexes with the specific globulins are higher by several orders of magnitude. CBG and SBP have molecular weights similar to that of albumin; PBG appears to be larger. A fairly general characteristic of these steroid-binding glycoproteins is their tendency to polymerize. Rat CBG forms dimers, tetramers and octamers upon removal of steroid from the complex; stoichiometric recombination with corticosterone completely reverses the polymerization. Estrogenic hormones increase the level of CBG and of SBP; androgenic hormones have the opposite effect. A CBG-depressing influence of corticosteroid hormones has been observed in the rat; adrenalectomized rats have increased CBG activity. The thyroid-stimulating hormone of the pituitary is required for the estrogen-induced rise of CBG synthesis in the rat; the effect is mediated by the thyroid gland. In accordance, thyroidectomy reduces the CBG activity whereas administration of thyroxin results in an increase. In contrast to all other species investigated, guinea pig serum contains separate proteins specific for binding of corticosteroid (CBG) and of progesterone (PBG). PBG levels increase about hundred-fold in pregnancy. PBG is a glycoprotein of χ 2 - or β -globulin mobility with an approximate molecular weight of 100,000 and an association constant for progesterone-binding higher than any of the other steroid-serum protein complexes. Association with serum proteins suppresses the biological activity of steroid hormones. Bound hormone in blood constitutes an inactive pool which protects the hormone from metabolic and chemical alterations and provides, by reversible dissociation, a buffer against sudden changes in active hormone concentrations.

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

The nature of the steroid binding site in alpha1-acid glycoprotein (orosomucoid) was investigated by chemical modification of individual amino acids and subsequent examination of the binding affinity for progesterone. Equilibrium dialyses were performed under conditions that excluded contact with human skin. Reaction of the lysyl residues with trinitrobenzenesulfonic acid or arylisocyanates resulted in a reduction of active sites. In an alternate approach, one lysyl residue of alpha1-acid glycoprotein was protected from modification by trinitrobenzenesulfonic acid when progesterone was present to form the complex with alpha1-acid glycoprotein. We conclude that a lysyl residue is located in the binding site. Reaction of tetranitromethane with the tyrosine groups in alpha1-acid glycoprotein also reduced the number of active binding sites for progesterone. Again, a partial protection of this modification was seen in the presence of progesterone and other delta4-3-ketosteroids. The progesterone binding activity observed in the tyrosine-modified alpha1-acid glycoprotein by equilibrium dialysis and by fluorescence quenching titration can be interpreted best by the presence of one tyrosyl residue in the binding site, and involvement of a second tyrosine nearby. Modification of tryptophan in alpha1-acid glycoprotein by mild acid hydrolysis, N-bromosuccinimide, hydroxynitrobenzylbromide, and formic acid resulted in a decreased steroid binding; the formylation reaction was fully reversible. The approximate distance between progesterone and the tryptophan involved in the binding was calculated to be between 9.1 A and 14.1 A. When alpah1-acid glycoprotein was cleaved by the cyanogen bromide procedure according to Ikenaka et al. (1972, Biochemistry 11, 3817-3829), both the amino and the carboxyl fragment had a weak progesterone binding affinity which could be measured in 4 M NaCl. This result thus failed to specify the location of the steroid binding site in alpha1-acid glycoprotein. However, the closeness of tryptophan, lysine and tyrosine in the primary and presumably the tertiary structure of alpha1-acid glycoprotein is in agreement with the properties of the binding site suggested by our studies.

STEROID-PROTEIN INTERACTIONS. VII. INTERACTIONS OF PROGESTERONE AND CORTICOSTEROIDS WITH HUMAN PLASMA PROTEINS DETERMINED BY MULTIPLE EQUILIBRIUM DIALYSIS

Abstract Determination of binding of progesterone to human and rabbit serum by continuous paper electrophoresis shows high affinity to albumin and weak interaction with the globulins. The method of multiple equilibrium dialysis is described, including its application to the measurement of the binding of progesterone, corticosterone, and cortisol to electrophoretic serum components. The affinities of binding of progesterone and cortisol to prealbumin, α 1 -acid glycoprotein, fibrinogen, and globin have been determined. The interaction between α 1 -acid glycoprotein (orosomucoid) and progesterone is about 15 times as strong as that between albumin and progesterone.

Steroid-protein XIX. Complex formation between α1-acid steroid hormones☆

Interaction of cortexone, corticosterone, cortisol, testosterone and estradiol with α1-acid glycoprotein (orosomucoid) was investigated at 4° and 37°. The binding affinity of these steroids to α1-acid glycoprotein was found to follow the polarity rule in that the steroid with the least number of polar groups was bound most firmly. In analogy to previous results with progesterone, cortexone, corticosterone, cortisol and testosterone associated with α1-acid glycoprotein at a single primary binding site. The apparent association constants were determined. Estradiol interacted at several binding sites; as many as 7 and 3 were observed at 4° and 37°, respectively. The low solubility of the steroids studied did not permit a general analysis of secondary binding sites although their presence was suggested by the experimental data. In the case of the more soluble cortisol at 37°, approx. 30 secondary binding sites were indicated, with an apparent association constant several hundred times smaller than that of the primary binding site. The changes of free energy, ΔF°, and of enthalpy, ΔH°, for all complexes were found to be negative. The entropy changes, ΔS°, were positive in every case. Corticosterone and testosterone competed with progesterone for the single binding site, whereas estradiol did not show competition. This finding is in agreement with the assumption that the Δ4-3-keto grouping is involved in the interaction with α1-acid glycoprotein.

Steroid-Protein Interactions Revisited

There are two types of association complexes between steroid hormones and proteins. One group of proteins bind the steroids in such a way that their biological activity is suppressed; only the free, unbound form of the hormone is active. These binders are represented by the steroid-binding serum proteins as the best known examples. The second kind of protein is found in, or possibly on, the target cells for the hormones; they are called receptor proteins. They have the opposite effect on the biological potency: they mediate the hormonal action. The binding of the steroid hormone to the receptor protein is a necessary step for the manifestation of the biological effects.

Steroid-protein interactions XXVII. Progesterone binding to polymers of human serum albumin☆

Abstract Human serum albumin was separated by Sephadex gel filtration into monomeric, dimeric, and tr meric-polymeric fractions. Electrophoresis in dodecyl sulfate polyacrylamide gel showed that dimerization was followed by trimer and tetramer formation. Mild reduction with mercaptoethanol did not eliminate the polymerized human serum albumin species. In analytical gel filtration, the fractions were less clearly defined by molecular size, presumably due to protein-protein interaction. The absorbance values, A 1 cm 1% at 278 nm, for the unfractionated, monomeric, and dimeric fractions, after correction for light scattering, were 5.14, 5.17, and 5.38, respectively. Binding of progesterone to the delipidated human serum albumin fractions was determined by equilibrium dialysis at 4°C. Computer analysis of Scatchard plots for two independent sets of binding sites gave n 1 = 1; k 1 = 3.6 · 10 5 M −1 , and n 2 = 8; k 2 = 6 · 10 3 M −1 for the human serum albumin molecule in both the monomeric and dimeric species. The trimer-polymer fraction had a significantly lower binding affinity.

Steroid-protein interactions. III. Spectrophotometric demonstration of interaction between proteins and progesterone, deoxycorticosterone and cortisol

Abstract Use of water instead of alcohol as a solvent for progesterone, deoxycorticosterone, and cortisol resulted in a bathochromic shift of the ultraviolet absorption maximum of approximately 8 mμ. The molecular extinction in aqueous solution was inversely related to the number of polar groups. Interaction of the steroids with human and bovine serum albumin, β-lactoglobulin, casein, and some other proteins caused a depression of the molecular extinction coefficients and a slight displacement of the maximum to shorter wavelengths. These effects were found to decrease with increasing “polarity” of the steroids. The influence of Δ 4 -3-ketosteroid interactions on the ultraviolet absorptions is discussed.