Progesterone Binding Globulin Research Articles

Bifunctional Gonadotropin-Releasing Hormone Antagonist-Progesterone Analogs with Increased Efficacy and Duration of Action

GnRH peptide analogs are widely used to treat diverse clinical conditions. However, they have poor oral activity and exhibit rapid metabolic clearance, thus requiring injection and depot formulation. Because steroid hormones are bound to plasma proteins, we explored the possibility of conjugating hydroxylated progesterones to GnRH analogs to reduce metabolic clearance of the peptides. Conjugation of [D-Lys6]GnRH agonist to the alpha11-hydroxyl of alpha11-hydroxyl progesterone via a hemi-succinate bridge increased the plasma half-life after iv injection in rabbits by 3.6-fold while retaining high binding affinity, thus providing proof of concept. Five GnRH antagonists were then synthesized with 21-hydroxyprogesterone conjugated via C21-hydroxyl to positions six (conjugates A and B) and position seven (conjugates C and D) of GnRH antagonists. In the fifth compound the NH2 terminus of a GnRH antagonist lacking the first two amino acids was conjugated via the C21-hydroxyl to 21-hydroxyprogesterone (conjugate E). All five analogs bound to guinea pig progesterone binding globulin with relatively high affinities (264-1020 nM). Moreover, all five conjugates retained high progestogenic activity in stimulating a progesterone-response-element-driven chloramphenicol acetyltransferase reporter gene in the T47D breast cancer cell line. Conjugation via the epsilon-amino function of D-Lys6 (conjugates A and B) produced compounds with high binding affinity for the human GnRH receptor (15 and 7 nM) comparable to that of the unconjugated GnRH antagonists (4 and 26 nM). Conjugation via the epsilon-amino function of Lys7 (conjugates C and D) or the NH2 terminus of an N-terminally truncated antagonist (conjugate E) produced compounds of low binding affinity. Conjugates A and B also exhibited high functional antagonism of GnRH stimulation of inositol phosphate production in COS-7 cells expressing the human GnRH receptor (2.6 and 16 nM) compared with the unconjugated antagonists (1.3 and 122 nM). In accordance with their poor receptor binding affinity, conjugates C, D, and E had poor functional antagonism. Preliminary dose-finding studies in female marmosets showed transitory progesterone inhibition by 0.25 mg and prolonged suppression of 12 and 17 d by 0.5- and 1.0-mg doses. Injection of conjugate A in adult male marmosets (0.5 mg sc) rapidly suppressed plasma testosterone levels, which remained suppressed for at least 3 d. In contrast, the unconjugated parent antagonist alone or with progesterone suppressed testosterone for only 8 h to 1 d. The findings demonstrate that conjugation of progesterone to GnRH antagonists conveys plasma binding and progestogenic properties and increases their efficacy and duration of action in vivo. These new GnRH antagonists show promise as therapeutic agents for hormone-dependent diseases and as contraceptives.

Purification and characterization of progesterone-binding globulin in the pregnant Cape porcupine, Hystrix africaeaustralis

Progesterone-binding globulin (PBG) was purified from pooled pregnant Cape porcupine ( Hystrix africaeaustralis) plasma by SP-Sephadex Chromatography, followed by Sephacryl S-200 gel filtration. The final product was homogeneous according to high performance capillary electrophoresis, SDS-polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. A molecular mass of 65,300 Da was calculated from sedimentation equilibrium centrifugation data. Scatchard analysis indicated a dissociation constant of 0.82 × 10 −9M, and PBG concentration of 18.4 nmole/mg protein.

Evidence for the direct transfer of corticosteroid-binding globulin from plasma to whey in the guinea pig.

Two steroid-binding proteins, corticosteroid-binding globulin (CBG) and progesterone-binding globulin (PBG), are known to be present in the milk whey of lactating guinea pigs. After injection of radioiodinated CBG into the maternal circulation, labeled CBG was found in the milk whey. The labeled whey CBG was identical to its plasma counterpart on the basis of size (sucrose gradients, Sephacryl S-200 gel filtration, and sodium dodecyl sulfate-gel electrophoresis), charge (DEAE-chromatography and polyacrylamide gel electrophoresis), and immunoprecipitability. In contrast, radioiodinated ovalbumin was not transferred to the milk. These results demonstrate that the CBG present in guinea pig whey results from the direct transfer of CBG from plasma to milk.

Progesterone-binding globulin interaction with its steroid ligands; study of the protein binding site topography using spin labeled steroids and electron spin resonance spectroscopy

The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice.

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.

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.

Changes in the metabolic clearance rate and plasma production rate of testosterone in the pregnant guinea-pig

The metabolic clearance rate of testosterone (MCR T) in the pregnant and non-pregnant guinea-pig was established by a constant infusion method to steady state levels of 3H testosterone (T). Simultaneously, the concentration of endogenous T was determined by radioimmunoassay and the production rate of T (PR T) was calculated from the product of MCR T and endogenous T concentration. Results indicate important alterations in the three parameters between non-pregnant ( MCR T = 64.0 ± 2.6 1 24 h ; T concentration = 51 ± 8 pg/ml; PR T = 3.24 ± 0.13 μg/24 h) and the pregnant guinea-pig. In these animals mean values from day 43 were about 33 1 24 h for MCR T, 378 Pg/ml for T concentrations and 12.5 μg/24 h for PR T. The significance of these results is discussed in relation to the specific binding of testosterone with the progesterone binding globulin during pregnancy in this species.

Progesterone-binding globulin and progesterone in guinea-pigs after ovariectomy, abortion and parturition

The level of progesterone in the serum of pregnant guinea-pigs which continued their pregnancies after ovariectomy on Day 30 fell and then rose again. In contrast, progesterone-binding globulin (PBG) was apparently not affected. PBG did however fall in guinea-pigs which aborted and also after parturition. It is apparent therefore that high PBG levels were associated with continued pregnancy. The half life of PBG was found to be approximately 2 days.

Progesterone-binding proteins in plasma of guinea-pigs and other hystricomorph rodents.

Progesterone-binding plasma proteins (PBPP, progesterone-binding globulin, PBG, and corticosteroid binding globulin, CBG) have been measured in plasma of guinea-pigs, casiragua, cuis, degu and plains viscacha. During pregnancy PBPP increased to reach peak values between Days 20-25 and Days 50-term in guinea-pigs, immediately before parturition in casiragua, and in mid-gestation in cuis, degu and viscacha. The pattern of PBPP concentration during pregnancy was similar to that of plasma progesterone concentration, and the ratio of molar concentration of PBPP and progesterone was greater than 1 in all species. Plasma proteins were separated by chromatography on SP-Sephadex C-50, and from its chromatographic behaviour and progesterone-binding property evidence was obtained for an anionic, high-affinity PBG in the plasma of all the animals studied during gestation: Electrophoretic mobility of SP-Sephadex-purified PBG applied to 7% polyacrylamide gels differed between species, being highest for casiragua PBG and lowest for guinea-pig PBG. Binding capacities ranged from 0.5 to 4.8 X 10(-6) M, and association constants from 7.6 to 17.4 X 10(8) M-1. Ligand specificities differed between species and marked contrasts were found for certain steroid isomers. The average molecular weights measured by gel electrophoresis of PBG under denaturing conditions were similar in cuis, degu and viscacha and close to PBG I of guinea-pigs, while coypu PBG had a molecular weight of about one half, approximately 75000. It is concluded that PBG has been adopted in pregnancy as a progesterone-conserving mechanism in this suborder of rodents with long gestation periods relative to maternal body weight. Restricted homologies in the physico-chemical characteristics of PBG from various hystricomorph rodents suggest that interspecific differences in structure are considerable.

Concentration and relationship to progesterone of progesterone-binding globulin in pregnant guinea-pigs: measurement by progesterone tracer binding assay.

A single-point progesterone tracer binding assay (TBA) was developed to measure the concentration of progesterone-binding globulin (PBG) in the serum of pregnant guinea-pigs. Tritiated progesterone was added to dilutions of a reference serum, the percentage bound calculated and a standard curve constructed. The amount of progesterone tracer bound to serum samples was determined, and the concentration of binding protein in the sample relative to the reference was calculated. The molar concentration in the reference serum was determined by Scatchard analysis. The TBA could readily process 30 samples per day. There was a rapid rise in PBG concentrations between days 15 and 20 of gestation, in parallel with progesterone concentrations. The molar ratio of PBG: progesterone was approximately 10:1 after this time, until about day 45 when there was an increase in ratio.

Free and bound androgens in the female guinea pig during gestation and after parturition and in the offsprings during the perinatal period

Abstract. Total amounts of testosterone (T) and dihydrotestosterone (DHT) and their distribution between non-protein-bound (free), albumin-bound and PBG-(progesterone binding globulin)-bound fractions were determined by radioimmunoassay and equilibrium dialysis from plasma samples. The samples were taken from male guinea pigs during the perinatal period and from pregnant females during gestation and after parturition. Plasma proteins of the foetus and newborn animals appeared to have no high binding affinity for androgens. On the other hand albumin having a binding capacity of 56% for testosterone and 75% for DTH irrespective of the total androgen concentration may be considered to be an important low affinity binding protein. Maternal plasma developed a specific binding activity with the appearance of PBG in early pregnancy. Alterations in the binding capacity of PBG for T or DHT paralleled changes in plasma concentrations of both androgens, the highest values being observed on day 48 of pregnancy, with a prompt return to normal after parturition. Irrespective of the total androgen concentration, it was evident that PBG was capable of maintaining the free T and DHT at low concentrations (about 0.20 and 0.17 ng/10 ml, respectively). Besides the specific binding due to PBG a non-specific binding due to albumin was observed. The competition which exists between these two binding systems for T and DHT was evident when the quantity of bound androgens was expressed as a percentage. Neither the sex, nor the number of the foetuses, nor the interaction between the two, was found to have any significant effect in the maternal androgens, whether total or free hormone was considered.

Steroid-binding proteins in guinea pig milk and plasma.

The levels of two steroid-binding proteins, progesterone-binding globulin (PBG) (1) and corticosteroid-binding globulin (CBG), present in the plasma of pregnant guinea pigs were determined just before and after parturition. Both PBG (3.37 +/- 2.1 microM) and CBG (10.1 +/- 1.7 microM) were present in high levels just before parturition and were found to decrease with a half-life of 2 days after delivery. PBG and CBG were also found in the milk whey of lactating guinea pigs but at much lower levels (26.5 +/- 12 and 375 +/- 18 nM, respectively, on day 1 post partum). The whey content of each protein declined with a half-life of 3 days. The whey steroid-binding proteins were indistinguishable from their plasma counterparts on the basis of ion exchange and gel filtration chromatographies, sucrose gradient centrifugation, susceptibility to heat denaturation, and hormone binding specificity.

Interaction of steroid-binding serum proteins with concanavalin A-Sepharose 4B.

The interaction between steroid-finding serum proteins and concanavalin A was studied using affinity chromatography on concanavalin A-Sepharose 4B. The sex hormone-binding globulins of man and rabbit together with the corticosteroid-binding globulins of man, rabbit and guinea pig were all bound quantitatively by the affinity medium. Only two thirds of rat corticosteriod-binding globulin showed an affinity for concanavalin A indicating this protein to be heterogeneous with respect to terminal mannose or glucose residues. The progesterone-binding globulin in pregnant guinea pig serum, possessing the highest carbohydrate content of the proteins investigated, did not bind to concanavalin A-Sepharose 4B. Affinity chromatography on concanavalin A-Sepharose 4B is documented to be a valuable supplement to existing methods for analysing steroid-protein interactions.

Alterations in the ultraviolet absorption spectra of steroids upon binding to serum proteins.

Difference spectra of progesterone-binding globulin (PBG) complexes with progesterone and testosterone were measured. The contributions of steroid and protein to the difference spectra were resolved by use of 5alpha-pregane-3,20-dione and dihydrotestosterone to compensate for the perturbation of PBG. The absorption spectra of seven bound steroids all showed increased extinction coefficients, sharpened absorption bands, a small blue shift, and an increased area implying an enhanced transition moment. This is in contrast to the steroid complexes with the low affinity binders, human serum albumin, and alpha 1-acid glycoprotein, which exhibit decreased extinction coefficients and reduced transition moments.

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.

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

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.

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.

Steroid interactions with progesterone-binding globulin

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.

PH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.

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

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