A 1H-NMR and MD study of intramolecular hydrogen bonds in methyl beta-cellobioside.

The existence of an HO-3...O-5' intramolecular hydrogen bond in methyl beta-cellobioside in solution in Me2SO-d6 and H2O-CD3OD (4:1 w/w) was studied by 500-MHz 1H-NMR spectroscopy and MD simulations. Temperature coefficients for the chemical shift of the hydroxyl resonances in these solvents were determined and the rates of proton exchange in the latter solvent were obtained from NOE data. With H2O-CD3OD as the solvent, the HO-3...O-5' hydrogen bond was insignificant, but its presence in Me2SO-d6 was confirmed.     

3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine: a novel antiretroviral analog of zidovudine. III. Solubility studies

The pH-solubility behavior and solubility of 3'-azido-3'-deoxy-5'-O-isonicotinoylthymidine (AZT-Iso), an antiretroviral derivative of zidovudine with important biological activity, was studied in water, ethanol, ethanol: water, and n-octanol. The N-pyridine pKa value was determined from its pH-solubility profile, which was in accordance with that of the experimental value of methyl isonicotinate. Also, the ethanol cosolvency in ethanol:water mixtures at 25 degrees C was studied, and log-linear and nonlinear solubilization models were applied to the experimental solubility AZT-Iso data, which allowed us to predict its solubility in those solvent mixtures at a determined content of cosolvent.     

Energetics of lectin-carbohydrate binding. A microcalorimetric investigation of concanavalin A-oligomannoside complexation.

Despite years of study, a comprehensive picture of the binding of the lectin from Canavalia ensiformis, concanavalin A, to carbohydrates remains elusive. We report here studies on the interaction of concanavalin A with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, the minimum carbohydrate epitope that completely fills the oligosaccharide binding site, and the two conceptual disaccharide "halves" of the trisaccharide, methyl 3-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside and methyl 6-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, using titration microcalorimetry. In all cases the interaction of protein and carbohydrate is enthalpically driven, with an unfavorable entropic contribution. The choice of concentration scales has an important impact on both the magnitude and, in some cases, the sign of the entropic component of the free energy of binding. The thermodynamic data suggest binding of the two disaccharides may take place in distinct sites, as opposed to binding in a single high affinity site. In contrast to carbohydrate-antibody binding, delta Cp values were small and negative, pointing to possible differences in the motifs used by the two groups of proteins to bind carbohydrates. The thermodynamic data are interpreted in terms of solvent reorganization. Cooperativity during lectin-carbohydrate binding was also investigated. Significant cooperativity was observed only for binding of the trisaccharide, and gave a Hill plot coefficient of 1.3 for dimeric protein.     

Fatty acid analysis of triacylglycerols: Preparation of fatty acid methyl esters for gas chromatography

A method to prepare fatty acid methyl esters was developed for fatty acid analysis of triacylglycerols by gas chromatography (GC). Triacylglycerols were mixed with methanolic CH₃ONa in hexane containing a mid-polar solvent for 10 s at room temperature. Under these conditions, trioleoylglycerol was converted to methyl oleate with an average yield of 99.3%. This procedure gave reliable and reproducible data on fatty acid compositions determined by GC.     

Dynamics of erabutoxin b as studied by nuclear magnetic resonance. Relaxation studies of methyl proton resonances

Longitudinal and transverse relaxation times were measured for well-resolved and assigned methyl proton resonances of erabutoxin b at 270 MHz, 300 MHz and 500 MHz. Both longitudinal and transverse magnetization decay curves are non-exponential due to cross-relaxation and cross-correlation effects. The longitudinal and transverse relaxation rates were obtained from the initial slope of both magnetization decay curves. The correlation times for the isotropic tumbling motion of the protein were determined to be 2.82 ns at 300 K and 1.62 ns at 330 K from the analysis of the relaxation data of some alpha protons. Using these values, the relaxation data of methyl protons were fitted to various theoretical models. Most of the methyl resonances could be fitted well to a model which allowed methyl rotation (in the range 0.01-0.05 ns) and an external contribution from protons assumed to be in positions derived from X-ray coordinates. The data for a few methyl groups, however, could not be fitted in this way. For these a smaller number of external protons than predicted by the X-ray coordinates was assumed. Additionally, a larger amplitude motion had to be introduced into the model for particular residues. This additional motion requires concerted protein motion close to these residues, since the X-ray structure suggests that steric hindrance would prevent local motion. These results are consistent with the idea of a flexible and dynamic structure for proteins.     

Enzymatically and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties.

A series of pectins with different levels and patterns of methyl esterification was produced by treatment of a very highly methylated pectin with acid, alkali, plant pectin methyl esterase and fungus pectin methyl esterase. The intrinsic pK values, as well as the free fractions of monovalent and calcium counterions, were determined on pectin salt-free solutions. The variations of pK(a) versus the ionisation degree were found to depend on the de-esterification process but a unique value of 2.90+/-0.15 was estimated for the intrinsic pK value. Calcium binding properties of chemically and enzymatically de-esterified pectins were investigated and experimental results were compared to Manning's theoretical values. A progressive dimerisation process for pectins with a blockwise distribution of carboxyl groups in the presence of calcium ions is hypothesised.     

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides

The 1H-NMR titration curves of chemical shifts versus pH were observed for imidazole, N1-methylimidazole, L-histidine, N1-methyl-L-histidine, N3-methyl-L-histidine, and other related compounds. With these results, the macroscopic pK values of these compounds were determined by a computer curve-fitting for a simple dissociation sequence. From the pK values of imidazole and N1-methylimidazole, the perturbation for the pK of the imidazole ring due to the substitution of a proton with a methyl group was estimated as -0.21 pH unit. The microscopic pK values of the individual tautomers of the imidazole ring were estimated with the pK values of N1-methyl-L-histidine, N3-methyl-L-histidine, and perturbation due to methyl substitution. The estimated pK values were 6.73 for the N1-H tautomer and 6.12 for the N3-H tautomer. These values were in good agreement with those obtained using carboxymethyl derivatives instead of methyl derivatives. Furthermore, the macroscopic pK value (6.02) calculated using the estimated microscopic pK values agreed with that (6.03) observed for the imidazole ring of L-histidine. Thus the method in this work was indicated to be self-consistent. The microscopic pK values of tautomers were also obtained for N alpha-acetyl-L-histidine and N alpha-acetyl-L-histidine methylamide. The molar ratios of tautomers were calculated on the basis of the microscopic pK values of tautomers. The intrinsic (or unperturbed) pK value of imidazole ring and perturbations due to the CO2- and NH3+ were obtained for each of the N1-H and N3-H tautomers.     

Chemical properties of bile acids. IV. Acidity constants of glycine-conjugated bile acids

The dissociation constants for the carboxyl group of a series of glycine (N-acyl)-conjugated and unconjugated bile acids were determined by potentiometric titration using dimethylsulfoxide-water and methanol-water mixtures of varying proportions. The pKa values in water were calculated by extrapolating the experimental values determined in different mole fractions of the organic solvent mixtures. The following values were obtained: 3.9 +/- 0.1 for glycine-conjugated bile acids and 5.0 +/- 0.1 for unconjugated bile acids, as general pKa values for the two classes of bile acids, respectively. The amidation of bile acids with glycine lowers the pKa value because of the proximity of the amide bond to the terminal carboxyl group. Bile acid dissociation constants are independent of the substituents in the steroid nucleus, since inductive effects of the hydroxyl groups on the steroid nucleus are too distant from the acidic group at the end of the side chain to influence its ionization.     

Behaviour of tyrosyl residues of calf-thymus histone F3. Difference-spectroscopy studies.

We have studied the behaviour of microenvironments containing tyrosine of calf thymus histone F3 (or histone H3) by using the difference spectroscopy techniques of thermal and solvent perturbation. By comparison of the parameters found for the models L-tyrosine methyl ester and N-acetyl-L-tyrosine ethyl ester with those for the protein at various conditions, several aspects of the tertiary structure of histone F3 become apparent. The raising of ionic strength produces a general burial of tyrosyl residues of the histone, whereas low pH or urea treatment causes a complete exposure of tyrosyl groups with respect to the solvent. Anomalously high values can also be observed of accessibility of the perturbants sucrose and ethylene glycol at low concentrations of phosphate buffer. The relevance of these findings towards a better understanding of the tertiary structure of histone F3 and of its interactions with DNA is discussed.     

Optico-hydrodynamic properties of high molecular weight DNA. II. Effect of aqueous glycerol solvents

The optical and hydrodynamic properties of T2 bacteriophage DNA have been determined by steady-state flow birefringence and viscosity in glycerol–aqueous buffer solvents at 25°C. Flow birefringence and extinction angle data were obtained over a velocity gradient range of 0.1 to 5 sec^?1 and at concentrations from 3 to 55 μg/ml in solvents containing approximately 30, 42, and 48 vol-% glycerol. Large optical backgrounds were observed in the mixed solvent flow birefringence studies which presented special experimental difficulties; these are described and their effect upon the flow birefringence data are discussed. The data on extinction angle provide no evidence for an internal viscosity effect on the stationary-state hydrodynamic properties of high molecular weight DNA over a range of solvent viscosity from 0.9 to 4.6 cP. Both the optical and hydrodynamic properties under present conditions of measurement appear to be self-consistent in terms of the values for these quantities in neutral aqueous buffer solution. Interpretation of the birefringence is complicated by uncertainties inherent in calculating the form anisotropy of DNA in non-aqueous solvents, but the data imply no large changes in helical structure with increasing glycerol concentration. Both intact and slightly degraded DNA samples were investigated, and no significant polydispersity effects were observed under the experimental conditions described.     

Electrostatic forces in two lysozymes: calculations and measurements of histidine pKa values.

In order to examine the electrostatic forces in globular proteins, pKa values and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human lysozyme (HUML) were measured, and they were compared with those calculated numerically. pKa values of His residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by 1H-nmr measurements. The associated changes in pKa values in HEWL and HUML were calculated by solving the Poisson-Boltzmann equations numerically for macroscopic dielectric models. The calculated pKa changes and their ionic strength dependence agreed fairly well with the observed ones. The contribution from each residue of each alpha-helix dipole to the pKa values and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKa of His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKa values is determined by the overall charge distributions and their accessibilities to solvent; and (3) alpha-helix dipoles make a significant contribution to the pKa, when the His residue is close to the helix terminus and not fully exposed to the solvent.     

Solution properties of synthetic polypeptides. 18. Helix-coil transition of poly-N5-(2-hydroxyethyl)L-glutamine

The helix–coil transition of poly-N⁵-(2-hydroxyethyl)L -glutamine (PHEG) in aqueous isopropanol was examined by means of optical rotatory dispersion (ORD) and intrinsic viscosity [η] measurements. The Zimm–Bragg parameters σ and s for the transition were determined from the ORD data as a function of molecular weight. It was found that the transition was characterized by a relatively low cooperativity; the values of \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt \sigma $\end{document} were in the range from 0.039 to 0.066, depending on the solvent composition. These σ values are much larger than those reported for other polypeptide–solvent systems. The transition enthalpy was negative and its magnitude varied with the solvent composition, with a maximum of 620 cal/mol at 40 wt% isopropanol. The curve of [η] versus helical content for a high-molecular-weight sample exhibited a very broad minimum, and this behavior was attributed to the low cooperativity of the transition.     

Molecular dynamics simulations of an enzyme surrounded by vacuum, water, or a hydrophobic solvent.

We report on molecular dynamics simulations of a medium-sized protein, a lipase from Rhizomucor miehei, in vacuum, in water, and in a nonpolar solvent, methyl hexanoate. Depending on force field and solvent, the molecular dynamics structures obtained as averages over 150 ps had root-mean-square deviations in the range of 1.9 to 3.6 A from the crystal structure. The largest differences between the structures were in hydrogen bonding and exposed surface areas of the protein. The surface area increased in both solvents and became smaller in vacuum. The change of surface exposure varied greatly between different residues and occurred in accordance with the hydrophobicity of the residue and the nature of the solvent. The fluctuations of the atoms were largest in the external loops and agreed well with crystallographic temperature factors. Root-mean-square fluctuations were significantly smaller in the nonpolar solvents than they were in water, which is in accordance with the notion that proteins become more rigid in nonpolar solvents. In methyl hexanoate a partial opening of the lid covering the active site occurred, letting a methyl hexanoate molecule approach the active site.     

Dependence of the catalytic activity of papain on the ionization of two acidic groups.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.     

Quantum mechanical and NMR spectroscopy studies on the conformations of the hydroxymethyl and methoxymethyl groups in aldohexosides

The potential energy surfaces of the hydroxymethyl and methoxymethyl groups in methyl hexopyranosides have been extensively studied, employing quantum mechanical calculations and high resolution NMR data. The structure and energy of the C-5-C-6 rotamers were calculated at the B3LYP level of the density functional theory (DFT). For all, geometry optimizations were carried out for 264 conformers of 16 methyl D-gluco- and methyl D-galactopyranoside derivatives 1-16 at the B3LYP/6-31G** level. For all calculated minima, single-point calculations were performed at the B3LYP/6-311++G** level. Solvent effects were considered using a self-consistent reaction field method. Values of the vicinal coupling constants 3J(H-5-H-6R), 3J(H-5-H-6S), 3J(C-4-H-6R), and 3J(C-4-H-6S) for methyl D-glucopyranosides, methyl D-galactopyranosides and their 6-O-methyl derivatives 9-16 were measured in two solvents, methanol and water. The calculated gg, gt, and tg rotamer populations of the hydroxymethyl and methoxymethyl groups in 9-16 agreed well with experimental data. The results clearly showed that the population of gg, gt, and tg rotamers is sensitive to solvent effects. It was concluded that the preference of rotamers in 1-16 is due to the hydrogen bonding and solvent effects.     

Structural studies by proton magnetic relaxation of stereochemical probes. II. Allosteric effects in human methaemoglobin.

The haem-iron accessibility to solvent molecules in human aquomet- and fluoromethaemoglobin was studied by the magnetic relaxation of protons from a stereochemical probe (methanol in deuterated solutions) in its dependence on allosteric effects induced by inositol hexaphosphate and pH between 5.5 and 8.5. The exchange of methanol with bulk solvent was observed only when inositol hexaphosphate was bound to aquomethaemoglobin, which is consistent with a widening of the haemcrevice compared to the conformation in the absence of inositol hexaphosphate. An increase in alkalinity in the physiological range of the Bohr effect results in a gradual impedence of the solvent dynamics inside the haem-pocket. The fast-relaxation phase of methyl protons indicates that a large number of methanol molecules are under the strong influence of the protein; this effect is considerably smaller with inositol hexaphosphate bound to aquomethaemoglobin. The hypothesis which implies a proton from the coordinated water molecule is responsible for the observed relaxation rates has been critically discussed. The model with a water molecule exchanging between a position next to the sixth-ligand site of the haem-iron and the bulk solvent is further substantiated experimentally. This model has been found to be the simplest and most self consistent in the interpretation of all these proton magnetic relaxation data.     

HIV-1 protease: characterization of a catalytically competent enzyme-substrate intermediate.

The steady-state and pre-steady-state kinetic parameters for the interaction of E with the fluorogenic substrate 2-aminobenzoyl-Thr-Ile-Nle-Phe(p-NO(2))-Gln-Arg-NH(2) were determined in 1.25 M NaCl, 0.1 M MES-TRIS at pH 6.0 at 25 degrees C. At low concentrations of enzyme, the values of the K(m) and k(cat) calculated from steady-state data were 2.1 microM and 7.4 s(-1), respectively. At high concentrations of enzyme, the time-courses of fluorescence enhancement associated with catalysis were very dependent on the excitation wavelength used to monitor the reaction. Because the absorbance spectrum of the substrate overlapped the fluorescence emission spectrum of the enzyme, these abnormalities were attributed to fluorescence energy transfer between the enzyme and the substrate in an enzyme-substrate intermediate. The kinetic data collected with lambda(ex) = 280 nm and lambda(em) > 435 nm were analyzed according to the following mechanism in which EX was the species with enhanced fluorescence relative to substrate or products: [formula see text]. The values of the kinetic parameters with (1)H(2)O as the solvent were K = 13 microM, k(2) = 150 s(-1), k(-2) = 25 s(-1), and k(3) = 11 s(-1). The values of the kinetic parameters with (2)H(2)O as the solvent were K = 13 microM, k(2) = 210 s(-1), k(-2) = 12 s(-1), and k(3) = 4.4 s(-1). These values yielded solvent isotope effects of 2 on k(cat) and 0.9 on k(cat)/K(m). From analysis of the complete time-course of the fluorescence change (lambda(ex) = 280 nm and lambda(em) > 435 nm) during the course of substrate hydrolysis, the intermediate EX was determined to be 6.3-fold more fluorescent than the product, which, in turn, was 4.5-fold more fluorescent than ES or S. Rapid quench experiments with 2 N HCl as the quenching reagent confirmed that EX was a complex between enzyme and substrate. Consequently, the small burst in fluorescence observed when monitoring with lambda(ex) = 340 nm (0.3 product equiv per enzyme equivalent) was attributed to the fluorescence change upon transfer of substrate from an aqueous environment to a nonaqueous environment in the enzyme. These results were consistent with carbon-nitrogen bond cleavage being the major contributor to k(cat).     

On the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the reaction catalyzed by choline oxidase

Recent kinetic studies established that the positive charge on the trimethylammonium group of choline plays an important role in substrate binding and specificity in the reaction catalyzed by choline oxidase. In the present study, pH and solvent viscosity effects with the isosteric analogue of choline 3,3-dimethyl-butan-1-ol have been used to further dissect the contribution of the substrate positive charge to substrate binding and catalysis in the reaction catalyzed by choline oxidase. Both the kcat and kcat/Km values with 3,3-dimethyl-butan-1-ol increased to limiting values that were approximately 3- and approximately 400-times lower than those observed with choline, defining pKa values that were similar to the thermodynamic pKa value of approximately 7.5 previously determined. No effects of increased solvent viscosity were observed on the kcat and kcat/Km values with the substrate analogue at pH 8, suggesting that the chemical step of substrate oxidation is fully rate-limiting for the overall turnover and the reductive half-reaction in which the alcohol substrate is oxidized to the aldehyde. The kcat/Km value for oxygen determined with the substrate analogue was pH-independent in the pH range from 6 to 10, with an average value that was approximately 75-times lower than that previously determined with choline as substrate. These data are consistent with the positive charge headgroup of choline playing important roles for substrate binding and flavin oxidation, with minimal contribution to substrate oxidation.     

Chromatographic mobilities and partition coefficients of synthetic corticosteroid glucosiduronates

Analytical data are presented on the free acids, the methyl esters, the methyl ester triacetates and the methyl ester triacetate semicarbazones of C-21 glucosiduronic acid conjugates of six adrenal hormones. Chromatographie mobilities of all of these compounds in three or more solvent systems are given. The stability of the steroidal glucosiduronic acids in alkali, their hydrolysis by β-glucuronidase and their partition coefficients in several solvent systems are also given.