DISSOCIATION,AGGREGATION AND DENATURATION OF SESAME α-GLOBULIN IN UREA AND GUANIDINE HYDROCHLORIDE SOLUTIONS*

The effect of urea and GuHCl on the major protein of sesame seed (Sesamum indicum L.), α-globulin, has been investigated by turbidity, sedimentation velocity, viscosity, difference spectra and fluorescence spectral measurements. The protein undergoes dissociation, aggregation and denaturation in the presence of the above denaturants. There is a critical concentration of the denaturant where aggregation is maximum. Both denaturation and aggregation are lower in buffers of high ionic strength. Dissociation and aggregation have been explained by considering two types of subunits present in the protein molecule, one leading to smaller sedimenting component and the other producing the aggregate. The amino acid analysis shows that the aggregated fraction is rich in aliphatic amino acid residues. The endothermic nature of the aggregation process has been considered to arise from hydrophobic interaction of aliphatic side chains of the relevant subunits. The protein exists in a more denatured state in GuHCl than in urea solution.     

Interaction of urea with the high molecular weight protein fraction,carmin, from safflower seed (Carthamus tinctorius)

The effect of urea on the high molecular weight protein fraction, carmin, from safflower seed Carthamus tinctorius L, was investigated by the techniques of sedimentation velocity, viscosity, fluorescence and difference spectral measurements, partial specific volume and circular dichroism. The protein underwent dissociation and denaturation simultaneously in the presence of increasing concentration of urea. The protein dissociated to a 2S component and in the process, the buried and exposed residues of tyrosines and tryptophans were perturbed. At 8 M urea concentration, the protein existed completely as a random coil; nearly 535 mol of urea were bound per mol of protein. The results indicated that the subunits of carmin appears to be held principally by noncovalent interactions and are dissociated/denaturated by increasing concentration of urea, leading to the destabilization of the native oligomer.     

DISSOCIATION AND DENATURATION BEHAVIOUR OF SESAME α-GLOBULIN IN SODIUM DODECYL SULPHATE SOLUTION*

The effect of anionic detergent, sodium dodecyl sulphate, on the major protein, α-globulin of sesame seed (Sesamum indicum L.) has been investigated by gel filtration, sedimentation velocity, viscosity, optical rotation, difference spectra and fluorescence measurements. The detergent causes dissociation of the protein first and then denaturation. In the detergent concentration range of 1.75–4.0 times 10^-3M four components are observed in the ultracentrifuge. The specific rotation of the protein increases with the detergent concentration above 2.5 times 10^-3M and shows a cooperative transition between 3–8 times 10^-3M detergent suggesting conformational change; above 8 times 10^-3M detergent the value of –[α] does not change. The reduced viscosity η_red however, increases above 2.5 times 10^-3M detergent and does not attain a plateau value. The difference spectrum of the protein indicates that both tryptophan and tyrosine groups have been affected by the detergent. The fluorescence intensity decreases and the maxima shifts towards red in the detergent solution resulting in an ‘isoemissive point’ at 355 nm. The double difference spectra in sucrose-detergent protein system show that below 5.0 times 10^-3M detergent, the difference absorption and fluorescence spectrum result from the binding of the detergent near the chromophoric groups and are not due to conformational change. Binding studies by equilibrium dialysis indicate the presence of 50 binding sites in the protein and a binding constant of 3.0 times 10³.     

CONFORMATIONAL STABILITY OF FRUCTOSE-1,6-BIPHOSPHATE ALDOLASE FROM CERATITIS CAPITATA

Fructose 1,6-biphosphate aldolase from Ceratitis capitata is a tetramer of identical subunits with 34% α-helix, 22% β structure and 44% of aperiodic order. Increase of urea concentration up to 4.0 M results in non-cooperative reversible dissociation of the enzyme. Sodium dodecylsulphate 0.06% (w/v) dissociates the tetramer cooperatively with retention of the helical content. Thermal denaturation was a non-reversible cooperative process with a midpoint for the transition at 55°. Cysteine residues are involved in this process and 2-mercapto-ethanol preserves partially the enzyme activity. The acidic dissociation of the enzyme is a non-reversible process in contrast to the reversible basic dissociation. Increase of ionic strength results in a more ordered secondary structure for the monomer after acidic dissociation.     

Denaturation of glycinin by urea and guanidine hydrochloride

Denaturation of glycinin by urea and guanidine hydrochloride (GdnHC1) has been studied at 30° by viscosity and circular dichroism (CD) measurements. The kinetics of denaturation by 8M urea at 20, 30, 40 and 50°, and by 6M GdnHC1 at 20° have been measured by monitoring the increase in absorbance at 287 nm as a function of time. Viscosity increased with denaturant concentration and reached maximum value of 28 mL/g at 6M GdnHC1 and 7M urea. The (negative) molar ellipticity values in the region 250–200 nm decreased with increase in denaturant concentration. Analysis of viscosity and CD data indicated that both sets of data fitted the same curve of f_d (fraction of protein denatured) versus denaturant concentration. The kinetic data followed first order reaction kinetics, These suggest that denaturation of glycinin by urea/GdnHC1 is a two-state process and follows the same pattern as that of globular proteins.     

Characterization of forskolin binding sites in rat brain membranes using [14,15-3H]14,15-dihydroforskolin as a ligand

Summary [14, 15-³H]14, 15-Dihydroforskolin ([³H]DHF) has been used as a radioactive ligand to identify forskolin binding sites in rat brain membranes. The binding was saturable and reversible. The binding sites showed positive cooperative properties as evident from an upward convex Scatchard plot and a Hill coefficient of 1.6. The equilibrium dissociation constants (K _D) were in the range between 10 M and 10 nM as estimated from the limiting slopes of the curved Scatchard plot. Half-maximal saturation of the binding sites was observed at a ligand concentration of 225 nM. The binding kinetics were very rapid: Binding equilibrium was reached in less than 2 min and a large excess of cold forskolin displaced 80% of the radioligand within 2 min. The dissociation reaction was not first order, characterized by a decreasing dissociation rate constant. Bound [³H]DHF could be displaced with forskolin (IC₅₀ 0.3 M), 14,15-dihydroforskolin (IC₅₀ 0.8 M) and 7-desacetylforskolin (IC₅₀ 3 M). However, nucleotides (ATP, GTP) and other receptor ligands (adenosine, isoproterenol) had no effect on the binding. Although the density of the forskolin binding sites (3.2 pmole/mg protein) is similar to those of other adenylate cyclase linked receptors, discrepancies between the K _D and the ED₅₀ obtained in adenylate cyclase studies and the finding that activation of the enzyme by forskolin is negative cooperative makes it difficult to clearly relate the binding sites to adenylate cyclase.     

Denaturation of concanavalin A by urea at acid pH

The denaturation of dimeric concanavalin A induced by urea at pH 3 has been studied using optical activity and sedimentation velocity. Under the conditions employed Mn⁺² and Ca⁺² are dissociated from the protein, but the basic structural elements are little changed from those prevailing in the functional lectin at pH 5.5 [H.E. Auer and T. Schilz, preceding paper in this issue]. The protein passes through three stages as the urea concentration is varied from 0 to 10 M. Below 4M urea the only effect observed is the loss of optical activity of the aromatic amino acid residues. At 4 M, a conformational change occurs producing extensive aggregation, which persists to 7 M. At 8–10 M urea a disordered monomeric protein molecule prevails. The protein could be reactivated provided that dilution to native conditions was very rapid and the protein concentration remained very low. Kinetics of denaturation were monitored by optical activity at 218, 225 and 283 nm, Transients with one, two or three components were observed, which were resolved by nonlinear regression according to sequential first-order decay laws. First order character was confirmed by independence of the kinetic parameters from protein concentration over a two- to four-fold range. Enthalpies and entropies of activation for the various steps were also determined. The transients at the three wavelengths monitor changes in β structure, β turns and aromatic groups, respectively. The urea dependence of the rate constants is unique in most cases. It is concluded that different structural elements of the concanavalin A molecule unfold independently from one another.     

Kinetic control of guanine nucleotide binding to soluble Galpha(q).

Binding of guanine nucleotides to heterotrimeric G proteins is controlled primarily by kinetic factors, such as the release of bound GDP, rather than by affinity alone. Detergent-solubilized Galpha(q) displays unusual guanine nucleotide binding properties in comparison with other G protein alpha subunits. Under conditions where most G proteins bind nearly stoichiometric GTPgammaS in 5-30 min at micromolar nucleotide concentrations, GTPgammaS binding to Galpha(q) is slow (>1 hr to completion), markedly substoichiometric, and dependent upon high concentrations of nucleotide (0.1 to 0.2 mM). Although the latter two properties suggest low affinity, GTPgammaS dissociation is immeasurably slow under commonly used conditions. We found that purified Galpha(q) can bind stoichiometric GTPgammaS, but that binding is controlled kinetically by a combination of factors. GDP (or IDP) dissociated slowly from Galpha(q), but the dissociation rate increased linearly with the concentration of (NH4)2SO4 up to 0.75 M (approximately 20-fold acceleration). The resulting GDP-free Galpha(q) was labile to rapid and irreversible denaturation, however (rate constant > or = 1 min(-1) at 20 degrees). Denaturation competed kinetically with relatively slow GTPgammaS association, such that stoichiometric binding was only attained at 100 microM GTPgammaS. These findings reconcile the slowly reversible binding of GTPgammaS to Galpha(q) with the other behaviors that suggested lower affinity, and point out that events subsequent to GDP dissociation can markedly influence the rates and extents of guanine nucleotide binding to G protein alpha subunits. Understanding these interactions allowed the direct, accurate quantitation of active Galpha(q) by a simple GTPgammaS binding assay in the presence of (NH4)2SO4, and similarly can prevent underestimation of the concentrations of other G proteins.     

DENATURATION OF THERMOPHILIC AND MESOPHILIC 6-PHOSPHOGLUCONATE DEHYDROGENASE BY 8M UREA

The denaturation reaction of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate: NADP oxidoreductase (decarboxylating EC 1.1.1.44) from B. Stearothermophilus and E. coli in 8 M urea has been compared. The rates of denaturation were evaluated from the fluorescence quenching that occurs at 334 nm. The thermophilic enzyme has been found about ten times more resistant to the action of the denaturating agent, and showed a biphasic denaturation process, whereas the E. coli enzyme followed a single first-order kinetics.     

AGGREGATION,DISSOCIATION AND DENATURATION OF SESAME (Sesamum indicum L.) α-GLOBULIN IN CETYL TRIMETHYL AMMONIUM BROMIDE SOLUTION*

The behaviour of the major protein of sesame seed (Sesamum indicum L.) α-globulin has been studied in a cationic detergent, cetyl trimethyl ammonium bromide solution. Up to a critical detergent concentration the protein is precipitated from solution, above which redissolution of the protein is observed. Sedimentation velocity patterns indicate the presence of higher aggregates in the detergent concentration range 5 times 10^-5– 1 times 10^-3 M. These are considered to be the soluble precursors of the insoluble aggregates. Fluorescence measurements show that tryptophanyl groups of the protein which are in contact with the aqueous phase are perturbed by the detergent. The difference spectra of the protein in higher concentration of detergent indicate considerable red shift in the spectrum. Spectrophotometric titration of phenolic groups in 1 times 10^-2 M CTAB indicate that a conformational change in the protein has taken place.     

Some physico-chemical properties of the rigid form of the Sendai virus nucleocapsid

The effect of some dissociation agents (SDS, beta-mercaptoethanol, urea, EDTA) on the rigid form of the Sendai virus nucleocapsid was studied. Polyacrylamide gel electrophoresis in the presence of lytic mixture (1% SDS, 2% beta-mercaptoethanol, 5 M urea, for 2 min at 100 degrees C) revealed two types of polypeptide subunits (mol. wts. 46,000 and 14,000), as well as the dissociation in the presence of 0.1% SDS only. The EDTA treatment leads to a disorganization of the protein part (10(-2) M) or of the nucleocapsid structure (5 x 10(-2) M).     

微管蛋白的分离鉴定及其在抗癌药物筛选中的应用

用两个循环聚合——解聚的方法,从猪脑分离到部分纯化的微管蛋白,在聚丙烯酰胺凝胶电泳图谱上显示两个主峰带,即。微管蛋白(M:W.58,000)和β微管蛋白(M.W.54,000)。在ATP或GTP存在的MES缓冲液中37℃保温b微管蛋白迅速聚合为微管,A 350呈典型S形聚合曲线。在电子显微镜下显示网状微管结构。抗癌药物筛选实验表明:秋水仙碱明显抑制微管聚合;长春新碱低浓度(0.75-6μM)抑制微管蛋白聚合,高浓度(>6μM)时导致微管蛋白聚集(Aggregation),且在低温下不解聚。三尖杉酯类生物碱对微管蛋白的聚合及解聚未显示明显的影响。     

ASSOCIATION-DISSOCIATION AND DENATURATION BEHAVIOUR OF AN OLIGOMERIC SEED PROTEIN α-GLOBULIN OF SESAMUM INDICUM L. IN ACID AND ALKALINE SOLUTIONS

The association-dissociation and denaturation behaviour of the major protein fraction,α-globulin of sesame seed (Sesamum indicum L.), in acid and alkaline solutions in the ranges of pH 4.2–1.5 and pH 7–12 have been studied. The results of gel filtration, fluorescence and viscosity measurements indicate dissociation and denaturation of the protein up to pH ? 3. The difference spectrum in this region arises from a combination of dissociation, denaturation and charge effect on the chromophore. In still stronger acid solution, reassociation of the dissociated fraction takes place by hydrophobic interaction. In alkaline solution dissociation takes place around pH 8, and above pH 10 dissociation and denaturation proceed simultaneously as has been evidenced by sedimentation, fluorescence, spectral change, optical rotation and viscosity measurements. The phenolic group (pK_Int = 10.6) in the protein is abnormal and denaturation in alkaline solution is irreversible. Above pH 11.5 further dissociation of the protein takes place. Characteristic pH values of transition from 10.6–10.8 indicate that the transition of the protein involves a single step in alkaline solution.     

DISSOCIATION,AGGREGATION OF SESAME α-GLOBULIN IN NONIONIC DETERGENT SOLUTION

Nonionic detergents Triton X-100 and Brij 36T induce dissociation and aggregation of the protein sesame α-globulin above the critical micelle concentrations (cmc) of the detergents. Spectrophotometric titration in Triton shows no change in the pK_Int value of the tyrosyl groups at 1 × 10^-3M detergent where both dissociation and aggregation of the protein are observed. Fluorescence measurement does not indicate any change in the environment of the tryptophan groups of the protein in Brij. Viscosity measurements show no major conformational change of the protein in the detergent solution. Binding measurements suggest that perhaps micelles of the detergent predominantly bind to the protein. The detergent micelles preferentially bind to the exposed hydrophobic surfaces of the protein subunits. The association of the protein detergent complex through electrostatic interaction is probably responsible for the formation of the aggregates.     

ON THE 3α-HYDROXYSTEROID DEHYDROGENASE FROM PSEUDOMONAS TESTOSTERONI. The effect of denaturing agents

Highly purified preparations of the 3α-hydroxysteroid:NAD- oxidoreductase (E.C.1.1.1.50) from Pseudomonas testosteroni (ATCC 11996) which consist of two major isoenzymes, with traces of a third, have been split into two enzymatically inactive polypeptides A and B by the use of sodium dodecylsulphate, urea and guanidinium hydrochloride. Both polypeptides have a molecular weight of 25,000 ± 2,500 as shown by thin-layer gel chromatography and ultracentrifugations. They differ, however, in charge as shown by electrophoresis on cellulose acetate strips in the presence of 8 M urea. Each of the isoenzymes, have molecular weight of about 50,000 and thus consist of two subunits. The presence of the three isoenzymes may be explained by the following combinations of the subunits AA, AB and BB. Close to 100% of the original activity towards the three substrates, androsterone, tetrahydrocortisone and desoxycholate could be restored within 24 h when the inactivated enzyme was diluted in order to remove the effect of the denaturant.     

Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying

Purpose The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying.Methods The cold denaturation of both phosphoglycerate kinase (PGK) and β-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (T_zero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution.Results Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at −10°C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with β-lactoglobulin. The half-lives of PGK and β-lactoglobulin unfolding are estimated to be 5.5 × 10¹¹ and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20°C above T_g′. Thus, freeze-drying below T_g′ should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60°C above the system T_g′ in a thermodynamically unstable system during freeze-drying.Conclusions Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above T_g′. Thus, it is not always necessary to freeze-dry protein formulations at temperature below T_g′ to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the T_g′, thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.     

FURTHER CHARACTERIZATION OF THE OVINE LUTROPIN α AND β SUBUNITS PREPARED BY THE SALT PRECIPITATION METHOD

The subunits of ovine lutropin prepared by acid dissociation and salt precipitation were characterized by end group analysis, tryptic peptide mapping, SDS gel electrophoresis and biological activity. No evidence of internal peptide cleavage was found in the α subunit. The subunits possessed low activity. The α and β subunits recombined effectively to generate a complex that had full receptor binding activity and in vitro biological activity. The recombinants of subunits prepared by countercurrent distribution showed only 50% activity in both assays. The salt precipitation method α subunit could be completely reduced and reoxidized in the absence of denaturants. The reoxidized α subunit combines with the native β subunit generating full activity. However, this recombined hormone tends to lose activity with time, suggesting that the reoxidation may not fully restore the native structure of the reduced α subunit. The native lutropin α subunit effectively combined with follitropin β subunit generating complete follitropin activity.     

CHANGES IN BLOOD AND RED CELL VOLUME IN THE NEONATAL LAMB AND THE EFFECT OF INSULIN-LIKE GROWTH FACTOR I

1. Blood volume was measured weekly using [⁵¹Cr]-labelled red cells in 10 lambs from 3 to 10 weeks of age. Red cell and plasma volumes were calculated using the measured blood volume and haematocrit. Other parameters, including plasma erythropoietin, urea, creatinine and glucose, were measured twice weekly. The results were compared to a group of five lambs that received an infusion of insulin-like growth factor I (IGF-I). 2. In control lambs, plasma volume increased linearly by 47 ± 7 mL/week over the experimental period. Red cell volume only increased by 10 ± 2 mL/week during weeks 3–7, but then increased by 25 ± 2 mL/week over weeks 7–10. Haematocrit declined from 28.0 ± 1.6 to 24.7 ± 1.7% over weeks 3–7 and then increased to 30.7 ± 1.1% by week 10. 3. In 10 control lambs infused for 8 days (starting at 22–26 days of age) with 10 mmol/L HCl, there was a decrease in plasma IGF-I concentrations, 3 days after the start of infusion. In five lambs infused for 8 days with IGF-I (6 μg/kg per h) plasma IGF-I concentration was maintained significantly (P < 0.01) higher than that of the controls. 4. There was no significant difference in haematocrit, red cell or plasma volumes between the treatment groups and no reticulocytosis was observed. Plasma erythropoietin concentrations did not change over the infusion period in either group. 5. Serum urea decreased significantly in the IGF-I infused group but serum creatinine did not change in either group during the infusion period. In both the groups, there was a significant decrease in glucose, urea and creatinine over weeks 3–10 after birth. There was no difference in growth rates between the two groups. 6. Thus, it appears that the observed changes in haematocrit are due to a constant increase in plasma volume with varying rates of red cell volume increases. 7. IGF-I infused at a dose that maintains physiological concentrations and alters protein metabolism does not result in increased erythropoietin or erythropoiesis during the neonatal period of the lamb.     

EFFECT OF SODIUM DODECYL SULPHATE ON THE 12S FRACTION OF MUSTARD PROTEINS (B. JUNCEA)

The effect of SDS* on the 12S protein fraction of mustard seed (B. juncea) has been followed by the techniques of ultracentrifugation, gel filtration, gel electrophoresis, viscosity, ultraviolet difference spectra and fluorescence spectra. At low concentrations of SDS, up to 0.1%, both aggregation and dissociation of the protein occurs. Only dissociation occurs at higher SDS concentrations and is complete at 0.5% SDS. Viscosity increases sharply up to 0.15% SDS, remains constant between 0.15 and 0.30% and then increases markedly again, SDS induces also difference spectra with minima at 280, 288 and 295 nm. Fluorescence emission intensity increases at SDS concentrations < 0.05% and quenching occurs at higher SDS concentrations. The results suggest that SDS causes association, dissociation and denaturation of the protein molecule.     

REDUCTION AND REOXIDATION OF BOVINE PROINSULIN. EFFECT OF pH,ZINC IONS,TEMPERATURE AND CONCENTRATION

The influence of pH, temperature and the concentrations of zinc ions and reduced proinsulin upon the yields of reoxidized proinsulin were studied using the insulin radioimmunoassay. The optimal conditions were found to be pH 8.5, absence of zinc ions and a low protein concentration. However, even at a concentration of reduced proinsulin of 1.47 μM, the reoxidized proinsulin did not compete with ¹²⁵I-insulin for the insulin antibodies as strongly as did genuine proinsulin. At high concentrations of reduced proinsulin (14.7 μM), the yield increased with decreasing temperature; at a 10-times lower concentration the temperature had no influence upon the yields. Adsorption of reduced prosinulin to glassware was either irreversible or had a denaturing effect rendering the products immunologically inactive. Spontaneous reoxidation of reduced proinsulin is a rather delicate process, and the optimal conditions are different from those present in vivo. When the reduction of proinsulin was carried out at a concentration of 31.5 μM, zinc ions were found to stabilize the reduced protein against denaturation and precipitation for more than 24 hours. However, at this concentration, reduced proinsulin is in a thermodynamic ally unstable state, both with and without zinc.