The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin

A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.     

Rates of dissociation and recombination of the subunits of bovine thyrotropin

The rates of dissociation and recombination of the subunits of bovine thyrotropin have been measured under a variety of conditions using the fluorescence probe 1,8-anilinonaphthalenesulfonate. The method is based on the fact that the native hormone strongly enhances the fluorescence of 1,8-anilinonaphthalenesulfonate whereas the subunits have very little effect. The hormone can be easily dissociated into subunits, either in dilute acid (pH < 4) or in concentrated (8–10 m) urea solutions at pH 8.O. The rate of dissociation is first order with time and increases strongly with increasing temperature. The hormone is very stable in alkali, showing little tendency to dissociate below pH 12. After dissociation in acid, the subunits can be recombined between pH 7 and 9 at a rate which increases with increasing temperature and subunit concentration. The recombination is intermediate between first and second order suggesting a two-step mechanism: association of the subunits followed by a first-order refolding process in which the subunits acquire the tertiary structure characterisitc of the native hormone. Difference absorption measurements indicate that the dissociation is accompanied by the exposure of a substantial fraction of the 16 tyrosine residues to the more polar aqueous environment, suggesting major conformational changes in one or both subunits.     

Association/dissociation of gonadotropin subunits involves disulfide bridge disruption which is influenced by carbohydrate moiety

The association and dissociation rates of pituitary porcine luteinizing hormone (pLH) and equine LH (eLH) at oxidizing potential were slow and those of equine choriogonadotropin (eCG) were even much slower. At reducing potential mimicking endoplasmic reticulum condition, association of pLH subunits was observed in less than 5 min instead of 24 h at oxidizing potential. At neutral pH and 37 degrees C, DTNB and 2-nitro-5-thiocyanobenzoic acid (NTCB) were found to react with two cysteine residues (i.e., one S-S bridge) in pLH. The temperature dependence of the NTCB reaction on pLH was found to be similar to that of the dissociation of the hormone (Tm approximately 75 degrees C). The tight correlation between the reaction of two cysteines and dissociation of the subunits of pLH and eLH strongly suggests that transient opening of one fragile disulfide bridge is required for heterodimer assembly. Moreover, the absence of cysteine reaction with eCG indicates that its bulky carbohydrate chains exert a negative influence on the opening of this bridge leading to considerably diminished association-dissociation rates of its subunits.     

Dissociation of human follicle-stimulating hormone. Comparison with luteinizing hormone.

Rat testis tissue receptor assays were utilized to study the kinetics of dissociation of human follicle-stimulating hormone (hFSH) and luteinizing hormone (hLH) under varying conditions of urea concentration and pH. In these competitive protein binding assays, 125I-hFSH and 125I-hLH were the radioligands and hormone dissociation was followed by a decrease in the ability of the dissociating hormone to inhibit uptake of the radioligand by tissue receptors. Rate data for dissociation of the gonadotropins were analyzed for quality of fit to first or second order integrated rate equations by nonlinear regression analysis. Treatment of hFSH with 4 M urea at pH 8 and 25 degrees for 22 hours did not result in significant dissociation, whereas in 8 M urea, over 90% dissociation was observed. The rate of dissociation of hFSH in 8 M urea was increased approximately 4-fold by raising the temperature from 25 to 37 degrees. Similar results were obtained when dissociation of hFSH was followed through use of an accepted whole animal bioassay for FSH, thus confirming the reliability of the tissue receptor assay for such dissociation studies. Kinetic studies showed that hFSH was undissociated by incubation in 6 M urea of pH 8 after 4 hours at 25 degrees. In contrast, hLH was 90% dissociated under similar conditions. This differential rate of inactivation of hLH allowed preparation of hFSH having significant reduced levels of contaminating LH activity, as determined by tissue receptor assays and by whole animal bioassays. Marked differences were noted in the rate of dissociation of hFSH and hLH under acid conditions. hFSH completely dissociated after approximately 2 min of incubation of pH 2 (25 degrees), and over 90% dissociated after 15 min of incubation at pH 3. In contrast, hLH was dissociated 60% after 20 min of incubation at pH 2 (25 degrees) and 40% dissociated after 60 min at pH 3. Neither hormone was significantly dissociated at pH 4.4 after 60 min, but hFSH showed a slightly greater rate of dissociation than did LH in the period between 1 and 23 hours of incubation at that pH. hFSH and hLH were relatively resistant to dissociation after incubation at pH 12 for 1 hour, bu;t dissociated significantly after incubation for 22 hours at that pH. The time course for dissociation of hFSH or hLH under the various conditions described above did not conform clearly to either first or second order kinetics, indicating that the over-all dissociation process represents a mixed order reaction. It appears that urea or acid-induced denaturation of one or both subunits of hLH and hFSH may occur prior to their dissociation. The very rapid rate of dissociation at acid pH values, particularly of hFSH, indicate that ionic interactions contribute importantly to the subunit association phenomenon.     

Studies on modification of tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone and its subunits.

Based on the regeneration of the hormonal activity following recombination, the alpha and beta subunits of human follicle-stimulating hormone have been designated as 'functional' or 'nonfunctional'. Chemical modifications of the tryptophan, methionine, tyrosine and arginine residues of human follicle-stimulating hormone, luteinizing hormone, and the 'functional' human follicle-stimulating hormone alpha and beta subunits have indicated that the tryptophan in human follicle-stimulating hormone-beta and human luteinizing hormone-beta is essential for the biological activity. The iodination of human follicle-stimulating hormone-alpha did not interfere with the hormonal activity. The modification of arginine abolishes the biological activity of the hormones. The accessibility of tyrosine and methionine in human follicle-stimulating hormone-alpha, of arginine in both native hormones and subunits, and the non-availability of the tryptophan residues to 2-hydroxy 5-nitrobenzyl bromide suggest that the alpha subunit lies on the surface of the native molecule.     

The effects of interstitial cell-stimulating hormone, its subunits, and recombinants on isolated rat Leydig cells.

The stimulation of cyclic AMP accumulation and testosterone synthesis in isolated rat Leydig cells by ovine and bovine ICSH, their subunits prepared by a new, mild procedure (dissociation of subunits at pH 3 and salt fractionation) and the recombined hormones have been studied. Whereas the isolated subunits exhibit less than 0.2% of the potency of the native hormones, recombination of the subunits results in full restoration of the biological activity. In contrast to this, recombination of the subunits prepared by a countercurrent distribution method resulted in only partial regeneration of the biological activity. The ovine hormone was found to be twice as active as the bovine ICSH. Both heterologous hybrids of the subunits of the ovine and bovine hormones were significantly more active than the bovine hormone. The utility of the isolated rat Leydig cell system as a rapid, sensitive bioassay for ICSH is also discussed.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.     

Kinetics of recombination of the subunits of human chorionic gonadotropin. Effect of subunit concentration.

The rate of formation of human chorionic gonadotropin from its alpha and beta subunits has been measured at neutral pH and 37 degrees C as a function of subunit concentration, using the fluorescence probe, 1,8-anilinoaphthalene-sulfonate (ANS), to monitor the reaction. The subunits were prepared by acid dissociation of the intact hormone (pH less than or equal to 2, 37 degrees C). Following neutralization, the rate of appearance of ANS fluorescence was identical with the rate of recovery of receptor binding activity and both of these properties were completely recovered. Kinetic data obtained over a 100-fold range of subunit concentrations (1.5 to 146 muM) were not compatible with a simple second-order reaction scheme, but required at least one additional step. The data were best fit by a model in which the subunits reversibly form an intermediate complex (alpha + beta in equilibrium alphabeta) which then undergoes a conformational rearrangement to form the native structure (alphabeta leads to H). Ultraviolet difference absorption measurements suggest that most of the change in the environment of the tyrosyl residues occurs during this second step.     

Intrinsic and extrinsic fluorescence probes of subunit interactions in ovine lutropin.

The fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) was enhanced in the presence of ovine lutropin (oLH). Fluorescence titration curves were sigmoidal with 50% saturation between 200 and 500 μm. Exclusion chromatography experiments indicated that the hormone self-associates to form dimers in the presence of excess ANS. By contrast, the isolated a and β subunits of oLH caused a much smaller enhancement of the fluorescence of ANS and did not self-associate in its presence. Dissociation of the intact hormone into its subunits was accompanied by 1) a loss in the ability to enhance ANS fluorescence, 2) the appearance of a negative differential absorption spectrum whose magnitude indicated the increased solvent-exposure of at least two tyrosines, and 3) a loss in conformational rigidity as evidenced by a decrease in polarization (P) of tyrosyl fluorescence from ~0.17 to ~0.13. Similar rates of dissociation were obtained by all three measurements and the first order rate constant at pH 3.6 and 37 °C under conditions of low ionic strength was k = 0.18 min^?1; at high ionic strength, e.g., 0.5 m KC1, dissociation was incomplete even after prolonged incubation. Acid-dissociated subunits recombined readily in 0.5 m acetate buffer, pH 5.3, and the recovery of the intrinsic absorption and fluorescence properties as well as the ability to enhance ANS fluorescence ranged between 70 and 90%. Titration of the isolated α and β subunits with acid or GdmCl had little or no effect on P, suggesting that residual secondary or tertiary structure is either absent, very stable, or its disruption does not alter the rigidity of the tyrosyl environment. The relatively high P for oLH-β (0.17) suggests a conformation which is rigid compared with oLH-α (0.13). P for both subunits decreased smoothly with increasing temperature between 20 and 70 °C. By contrast, oLH exhibited a thermal transition near 50 °C characterized by a drop in P from a value near that of β to a value near that of a as the subunits dissociated. Because α has more tyrosines with a higher average quantum yield, its fluorescence would be expected to dominate that of the hormone or of an equimolar mixture of subunits. Thus, most of the conformation changes which accompany dissociation and recombination appear to occur in the α subunit.     

Ultraviolet absorption spectral changes accompanying the association of the subunits of human luteinizing hormone

Mixtures of the alpha and beta subunits of human luteinizing hormone, at 40°C and pH 7.50, demonstrate time dependence in their ultraviolet absorption spectra. Spectra of the native hormone or the separated subunits are stable with respect to time. The spectral changes are qualitatively consistent with a transfer of aromatic chromophores from an aqueous environment to less polar, possibly hydrophobic, surroundings during the process of subunit combination.     

Structure-activity relationships in tryptophanyl transfer ribonucleic acid synthetase from beef pancreas. Influence of the alkylation of the sulfhydryl groups on the dimer-monomer equilibrium.

Upon reaction with N-ethylmaleimide, tryptophanyl-tRNA synthetase from beef pancreas dissociates into subunits. At pH7, the rate of the dissociation is close to both the reaction rate of the buried--SH groups and the rate of inactivation (Iborra, F., Mourgeon, G., Labouesse B., and Labouesse, J. (1973) Eur. J. Biochem. 39, 547-556). The pH and enzyme concnetration dependences of the reaction rate of the 16 cysteinyl residues of the enzyme as well as that of its inactivation support the idea that inactivation by alkylation of the--SH groups is due essentially to the dissociation of the protein into inactive subunits and not to the chemical blocking of a catalytic residue. This is confirmed by the independence on N-ethylmaleimide concentration of the reaction of the buried--SH groups and of the inactivation of the enzyme at high N-ethylmaleimide concentration. The dissociation becomes in this case the rate-limiting step of the chemical reaction. The monomeric structure is stabilized by the blocking of the--SH groups exposed during the dissociation. The dissociation constant of the dimeric enzyme is progressively increased during the alkylation. The tightness of the associated structure depends on the protonation of groups titrating between pH 7 and pH 9.     

The carboxylic acid groups of bovine luteinizing hormone. The effects of their modification on receptor site binding and subunit-subunit interaction.

The modification of the carboxyl groups of the subunits of bovine luteinizing hormone to neutral derivatives by carbodiimide-mediated coupling with glycine methyl ester has been studied. The modified alpha subunit, which has 8 residues of glycine methyl ester incorporated, will no longer recombine with native beta (hormone-specific) subunit, but the modified beta subunit, with 6 to 7 glycine methyl esters incorporated, will recombine with native alpha to yield a partially active hormone. Derivatization of the intact hormone results in dissociation to subunits together with formation of a major side product which is covalently cross-linked. Significant cross-linked product was not obtained during modification of individual subunits, thus indicating an orientation between an activated carboxyl group(s) and a nucleophile(s) in the intact hormone which favors coupling. Separation of subunits from the derivatized, noncross-linked fraction by countercurrent distribution reveals a heterogeneous preparation of the modified alpha subunit which also will not recombine with either a native or modified beta subunit. The beta subunit from the modified intact hormone was indistinguishable from the modified isolated beta subunit in amino acid composition and in ability to recombine with native alpha subunit. The results are consonant with data from this and other laboratories in which various modifications of the alpha chain, the subunit common to the glycoproteins, more seriously affect recombination than similar modifications of the beta subunits. The number of carboxyl groups modified in each subunit is compatible with but not in total agreement with assignments of amides reported from sequence studies.     

Direct demonstration of intrinsic follicle-stimulating hormone receptor-binding activity in acid-treated equine luteinizing hormone

After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20–30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of acid-treated equine LH and FSH and intact equine LH and FSH were compared in five types of radioligand receptor assays. LH and FSH receptor-binding activities of equine LH were unaffected by pH 3. Equine LH showed 19- and 32-times more activity in the rat testis FSH assay than it did in chicken or horse FSH assays, respectively, directly demonstrating the intrinsic FSH receptor-binding activity of equine LH and the relative lack of specificity for these hormone preparations of the rat FSH receptor. Acid-treated equine FSH lost 95% of its biologic activity in FSH assays. In LH assays, the slight (0.2%) activity of equine FSH was relatively unaffected by acid treatment, suggesting that contamination by equine LH accounts for this activity.     

Mutual effects of proton and sodium chloride on oxygenation of liganded human hemoglobin

The different effects of pH and NaCl on individual O2-binding properties of alpha and beta subunits within liganded tetramer and dimer of human hemoglobin (HbA) were examined in a number of laser time-resolved spectroscopic measurements. A previously proposed approach [Dzhagarov BM & Lepeshkevich SV (2004) Chem Phys Lett390, 59-64] was used to determine the extent of subunit dissociation rate constant difference and subunit affinity difference from a single flash photolysis experiment. To investigate the effect of NaCl concentration on the association and dissociation rate constants we carried out a series of experiments at four different concentrations (0.1, 0.5, 1.0 and 2.0 m NaCl) over the pH range of the alkaline Bohr effect. As the data suggest, the individual properties of the alpha and beta subunits within the completely liganded tetrameric hemoglobin did not depend on pH under salt-free conditions. However, different effects NaCl on the individual kinetic properties of the alpha and beta subunits were revealed. Regulation of the O2-binding properties of the alpha and beta subunits within the liganded tetramer is proposed to be attained in two quite different ways.     

Effects of buffer salt on the geminate recombination of photodissociated carboxyhemoglobin and its isolated subunits

The geminate recombination of photodissociated carboxyhemoglobin (human adult) and its isolated subunits was studied by nanosecond laser photolysis. The rebinding kinetics were found not to depend upon the kind of buffer used or on pH but changed appreciably with the buffer concentration. The results are compared to oxygenation properties under the same conditions.     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

Site-site interactions among insulin receptors. Characterization of the negative cooperativity.

By studying the dissociation of 125I-instulin from its receptors in the absence and phe negatively cooperative type for the insulin receptors. In the present study we extend oy purified mouse and rat liver membranes as well as in human circulating monocytes and human cultured lymphocytes demonstrated negative cooperativity that was extraordinarily simn membranes more slowly than it does from its receptors on whole cells. The dissociaty a small percentage of the receptor sites (1 to 5%), are sufficient to accelerate dissociation of hormone from receptor. At these insulin concentrations insulin is entirely monomeric, and in fact at higher concentrations of insulin (greater than 10(-7) M) where insulin dimers predominate, the cooperativity effect is progressively lost. The dissociation rate of 125I-insulin alone (that is at very low fractional saturation of receptors) was markedly accelerated by dripping the pH from 8.0 to 5.0, whereas the dissociation of 125I-insulin at high receptor occupancy was only slightly accelerated by the fall in pH. The dissociation rate was directly related to temperature, but the dissociation rate of 125I-insulin at low receptor occupancy was much more affected by reduction in temperature and showed a sharp transition at 21 degrees. Urea at concentrations as low as 1 M produced a marked acceleration of 125I-insulin dissociation. Divalent cations (calcium and magnesium) appear to stabilize the insulin-receptor interaction, since higher degrees of receptor occupancy were required to achieve a given rate of dissociation of 125I-insulin. These data make it likely that the insulin receptors exist as oligomeric structures or clusters in the plasma membrane. Insulin receptor sites appear to switch from a "slow dissociating" state to a "fast dissociating" state when their occupancy increases; the proportion of sites in each state is a function of occupancy of the receptor sites by the insulin monomer as well as of the physiochemical environment. Other models which could explain apparent negative cooperativity besides site-site interactions, i.e. polymerization of the hormone, steric or electrostatic hindrance due to ligand-ligand interactions, or unstirred (Noyes-Whitney) layers are considered unlikely in the case of insulin receptors on both experimental and theoretical grounds.     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

Physico-chemical properties of pregnant mare serum gonadotropin

Pregnant mare serum gonadotropin exhibits a dissociation at acid pH as shown by the drop of s20,w values from 3.52 S at pH 8.1 to 2.52 S at pH 2.0. The dissociation is accompanied by an absorbance change with a maximum at 287 nm and a parallel loss of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activities as followed by radioreceptor assays. The apparent pKa of the acid transition is 3.45 with an extremely slow and temperature-dependent rate at pH 2.0 (1.8 . 10(-4) s-1 at 37 degrees C). By gel filtration the molecular weight of the active hormone is estimated to be 45 000 (rather than the previously reported 53 000-64 000). The active conformation of the hormone includes beta sheet structure (34%) as for other gonadotropin hormones with a minor but significative amount of alpha-helix. Four tyrosine residues were titrated, two of pKa = 10.3 and two of pKa = 11 out of a total of seven tyrosines. The parallel changes in FSH and LH activities during the preparation and the acid transition suggest that the two biological activities are intrinsic properties of the same molecular entity.