Effect of magnesium cations on the activity and stability of β-galactosidases

It was shown that the presence of magnesium cations in the reaction mixture increases, approximately twofold, the activity of bacterial Escherichia coli and yeast Kluyveromyces lactis β-galactosidases but does not affect the activity of bovine liver and fungous Penicillium canescens β-galactosidases. The catalytic constants for E. coli and yeast K. lactis β-galactosidases in the presence of 0.01 M and in the absence of Mg²⁺ cations were determined (490 and 220 s^?1 and 59.8 and 37.4 s^?1, respectively). It was shown that the Michaelis constants for these two enzymes are higher in the presence of Mg²⁺ cations, that the thermal stability of E. coli and K. Lactis β-galactosidases is higher in the presence of 0.01 M Mg²⁺, and that the effective rate constants of thermal inactivation of the enzymes are two-to eightfold lower, depending on conditions, in the presence of Mg²⁺ cations. The maximum stabilizing effect of magnesium cations was observed at weak alkaline pH values (7.5–8.5).     

Effect of urea on the structure and functional activity of proteins. Chymotrypsinogen A and α-chymotrypsin

The effect of urea on the structural stability and functional activity of globular proteins,viz., chymotrypsinogen A (ChtG) and -chymotrypsin (Cht), was studied over a wide range of concentrations (0.5–6 rnol L-1), and the existence of two different mechanisms of the action of urea on these proteins was demonstrated. No changes in the spatial structure of ChtG were observed in the concentration range from 0,5 to 3 mol L^–1 (region 1). Differential UV spectroscopy shows tile redistribution of aromatic groups between the inner volume and the outer surface of a protein molecule (protein denaturation) at concentrations >3 mol L^–1 (regionII), In regionI, urea changes the kinetic parameters of enzymatic reactions involving Cht, which is explained on the basis of millimeter spectroscopy data by its action on the structure and nucleophilic reactivity of water.Deceased.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 4, pp. 998–1002, April, 1996.     

The complex structures of ALKBH2 mutants cross-linked to dsDNA reveal the conformational swing of β-hairpin

Mammalian AlkB homologue 2(ALKBH2)is the primary housekeeping DNA demethylase,effectively repairing endogenously formed methylated lesions in double-stranded DNA.Our previous studies demonstrated that a hydrophobicβ-hairpin motif of ALKBH2 could play crucial roles in base-pair stability interrogation and damaged base flipping.Using chemical cross-linking strategy,we obtained two crystal structures of human ALKBH2 mutant bound to duplex DNA.The structural analysis suggests that theβ-hairpin motif is flexible in conformation and is likely to slide along the DNA duplex in local regions to search for damaged base.This study provides a new mechanistic insight into DNA damage detection by ALKBH2.     

Effect of urea on synchronous fluorescence spectra and electrochemical behaviour of cytochrome

The changes of the synchronous fluorescence spectra and the electrochemical behaviour of cytochrome c with the urea concentration are studied. It has been found that with the increase of urea concentration, there occur sequentially the deaggregation of cytochrome c molecules, the increase of exposure extent of the heme group to the solvent, the disruption of Fe-S bond of the heme group and the change in the electrochemical behaviour of cytochrome c. It is suggested that the reason why the electrochemical reaction of cytochrome c is irreversible is that cytochrome c molecules exist in the concentrated solution as oligomers which are electrochemically inactive.     

Effect of substituents on the solubility and the distribution of alkyl-substituted β-diketone iron chelates

The solubilities and the distributions of nine alkyl-substituted β-diketone iron chelates were measured in a number of common organic solvents, in order to establish the effect of substituents. In synthesis of iron chelates, acetylacetone, di-isobutyrylmethane and di-pivaloylmethane chelates were available with red-orange solid, while other chelates were red-orange oil. Using the solubilities and the respective molar volumes of these solid chelates, the solubility parameter values (δ_M) were calculated. The constancy of the calculated values was taken as evidence of the adequacy of the regular solution concept for explaining the observed solubility trends in the solvents studied. The molar volumes of iron chelates were nearly 1.5 times those of corresponding copper chelates and the solubility parameter values of the former were about the same as those of the latter. In the extraction of iron chelates, the extracted species were identified to be FeA₃ (A: β-diketone anion). The extraction constants (K_ex) of iron chelates does not depend on the nature of organic solvents. The distribution coefficients (P_Fe) of iron chelates increase regularly with the increase in the number of additional carbon atoms of the alkyl chain in the chelate molecule. The relationship between the solubilities or the distribution ratios of iron chelates and those of corresponding copper chelates were discussed.     

Comparative Study on the Effect of NaBr on the Interaction Between Alkyltrimethylammonium Bromide and β-Cyclodextrin

The effect of NaBr on the interaction between alkyltrimethylammonium bromide (CnTAB (n = 14 for TTAB and 16 for CTAB)) and ß-CD was studied by surface tension method. The first decrease in the γ_cmc of TTAB/ß-CD in the presence of NaBr mainly due to the elongated effective chain length of the hydrophobic part of the TTAB/ß-CD complexes; while the increase in the γ_cmc of both of the TTAB/ß-CD and CTAB/ß-CD in the presence of NaBr could be attributed to the close pack of CnTAB at the air/water interface.     

Synthesis and structural investigations of functionalizable hybrid β-hairpin

The solution and solid state conformations of a designed β-hairpin containing functionalizable α,β-unsaturated γ-amino acids at the antiparallel β-strands and a single step transformation to its saturated γ-peptide analogue are studied.     

Comparison of the results of thermal denaturation of β-lactoglobulin obtained by DSC and UV-spectroscopy

The thermal denaturation of β-lactoglobulin in the presence of urea and alkylurea solutions were measured. In the presence of a high concentration of urea this protein shows not only heat but also cold denaturation. For studying the effect of temperature two methods were used, differential scanning calorimetry (DSC) and UV-spectroscopy. DSC provides direct model-independent determination of the transition enthalpy in comparison with UV-spectroscopy, which gives only apparent or van't Hoff enthalpy of transition. The UV-melting curves were analyzed on the basis of a two-state approximation. The apparent standard enthalpies of thermal denaturation, ΔH _app. ^o , were compared with calorimetric ones.     

Enhancement of sortase A-mediated protein ligation by inducing a β-hairpin structure around the ligation site

A Staphylococcus aureus transpeptidase, sortase A (SrtA), catalyzes selective peptide/protein ligations that have been applied to cell imaging and protein engineering, while the ligations do not proceed to completion due to their reversibility. We successfully enhanced SrtA-mediated protein ligation through the formation of a β-hairpin around the ligation site.     

Thermodynamic denaturation of β-lactoglobulin in the presence of cetylpyridinium chloride

In this work, we determined the stability parameters of bovine β-lactoglobulin, variant A, (BLG-A), in relation to their transition curves induced by cetylpyridinium chloride (CPC) as a cationic surfactant. The experiments took place over the temperature range of 298 K to 358 K. For each transition curve at any specific temperature, the conventional method of analysis, which assumes a linear concentration dependence of the pre- and post-transition base lines, gave the most realistic values for ΔG_D(H₂O). Results show that the minimum value of ΔG_D(H₂O) occurs at T = 328 K. Using the Gibbs–Helmholtz equation, the values of enthalpy, ΔH_D, and entropy, ΔS_D, of denaturation have been calculated considering temperature dependence of ΔG_D at any specified concentration of CPC. The values of 12.05 kJ · mol⁻¹, 18.54 kJ · mol⁻¹, and 18.32 J · mol⁻¹ · K⁻¹, were obtained for ΔG_D(H₂O), ΔH_D(H₂O), and ΔS_D(H₂O), respectively. The results show that the enthalpy term dominates the entropy term.     

Anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol and lupeol isolated from Diospyros lotus L.

In this study, the anti-tumour-promoting and thermal-induced protein denaturation inhibitory activities of β-sitosterol (1) and lupeol (2), isolated from Diospyros lotus L., were explored. Compound 1 showed a marked concentration-dependent inhibition against 12-O-tetradecanoylphorbol-13-acetate (20 ng/32 pmol)-induced Epstein–Barr virus early antigen activation in Raji cells with IC₅₀ of 270 μg/ml, without significant toxicity (70% viability). Compound 2 showed significant anti-tumour-promoting effect with IC₅₀ of 412 μg/ml, without significant toxicity (60% viability). In heat-induced protein denaturation assay, compound 1 exhibited a concentration-dependent attenuation with a maximum effect of 73.5% at 500 μg/ml with EC₅₀ of 117 μg/ml, while compound 2 exhibited a maximum effect of 59.2% at 500 μg/ml with EC₅₀ of 355 μg/ml. Moreover, in silico docking studies against the phosphoinositide 3-kinase enzyme also show the inhibitory potency of these compounds. In short, both the compounds exhibited a marked anti-tumour-promoting and potent inhibitory effect on thermal-induced protein denaturation.     

Influence of protein hydrolysis on the growth kinetics of β-lg fibrils

Recently it was found that protein hydrolysis is an important step in the formation of β-lactoglobulin fibrils at pH 2 and elevated temperatures. The objective of the present study was to further investigate the influence of hydrolysis on the kinetics of fibril formation. Both the hydrolysis of β-lactoglobulin and the growth of the fibrils were followed as a function of time and temperature, using SDS polyacrylamide gel electrophoresis and a Thioflavin T fluorescence assay. As an essential extension to existing models, the quantification of the effect of the hydrolysis on the fibrillar growth was established by a simple polymerization model including a hydrolysis step.     

Effect of β- and γ-cyclodextrins and their methylated derivatives on the degradation rate of benzylpenicillin

Benzylpenicillin is characterized by instability in aqueous media, low oral bioavailability, and short biological half-life. Since their degradation products can cause allergic reactions, β-lactam antibiotics are supplied as a powder for injection and require special precaution when stored and re-constituted in water. An efficient method for β-lactam stabilization in the aqueous environment could reduce allergic side effects and facilitate their handling. Previously we proposed that complexation with cyclodextrins (CDs) is a way to achieve this goal. The purpose of the present study was to investigate the effect of methylation of CD on the chemical stability of benzylpenicillin. Kinetic studies revealed that degree of methylation of the CD molecule determines whether the CD has destabilizing or stabilizing effect on the β-lactam. The fully methylated βCD derivative Trimeb stabilizes benzylpenicillin while partial methylation of βCD only decreases to some extent the catalytic effect of native βCD. The complexes of all investigated CDs were also studied by DSC, FT-IR and NMR spectroscopy.     

Effect of functionalized multiwall carbon nanotubes on the curing kinetics and reaction mechanism of bismaleimide–triazine

Functionalized multiwall carbon nanotubes (f-MWCNTs) with varying functionalization degrees were prepared by chemical methods. The effect of f-MWCNTs on the cure kinetics of bismaleimide–triazine (BT) resin was studied through nonisothermal differential scanning calorimetry (DSC) methods. The reaction activation energy (E _α ) was determined by Flynn–Wall–Ozawa method. The results show that f-MWCNTs have more acceleration ability than pristine MWCNTs, due to more groups on the surface of f-MWCNTs than that of pristine MWCNTs. The activation energy was decreased from a value of 91.3 kJ mol^?1 for the neat BT resin to 74.2 kJ mol^?1 at the small mass loading (1.0 %) of f7-MWCNTs. The effect of f-MWCNTs on the reaction mechanism has been investigated. It shows that the f-MWCNTs accelerate the cure reaction of BT resin by providing the Lewis acids (H⁺) to make the “Diels–Alder” reaction and “ENE” reaction of BT resins more efficient. These findings offer useful insights into the cure technology of thermosetting resin filled with f-MWCNTs, without negative effect on the cure reaction.     

Effect of substituents on the solubilities of alkyl-substituted β-diketone copper chelates

The solubilities of alkyl-substituted β-diketone copper chelates were measured in a number of common organic solvents and an aqueous solution, in order to establish the effect of substituents. Using these data and the respective molar volumes of the chelate, the solubility parameters were calculated. The molar volumes for the chelates were in direct proportionality to the additional carbon atoms in the molecule and the solubility parameters were proportional to the reciprocal of them. The constancy of the calculated values was taken as evidence of the adequacy of the regular solution concept for explaining the observed solubility trends in the solvents studied. The distribution coefficients which can be calculated on the basis of the solubility parameter and the molar volume of copper chelates were in fair agreement with those obtained by an extraction method. The higher the solubilities of the chelates were in the organic solvent, the higher the distribution coefficients of the chelates were also. The relationship between the distribution coefficients and the solubilities of the chelates is discussed.     

Effect of end groups on the raman spectra of lycopene and β-carotene under high pressure

The Raman spectra of all-trans-lycopene in n-hexane were measured under high pressure, and the results compared with those of β-carotene. The different pressure effects on Raman spectra are analyzed taking into account the different structures of lycopene and β-carotene molecules. It is concluded that: (a) the vibronic coupling between the S? and S? states of β-carotene is stronger than that of lycopene, (b) the diabatic frequency increment of the ν? mode is more susceptible to pressure than that of the ν? mode for lycopene, and (c) β-rings rotation can relieve the pressure effect on the C=C bond length in β-carotene. This work provides some insights for elucidating the structural and environmental effects on Raman spectra of carotenoids.     

On the Effect of the Aglycon Structure of Three Aryl β-D-Galactosides on the Yield and the Regioselectivity of the Transglycolytic Synthesis of N-Acetyllactosamine

ABSTRACT

We examined the effect as donors of three aryl β-D-galactosides (i.e. p-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-galactopyranoside and phenyl β-D-galacto-pyranoside) on the regioselectivity and the yield of the synthesis of N-acetyllactosamine obtained from the transglycosylation reaction catalyzed by a crude preparation of β-D-galactosidase from Bacillus circulans at 25 °C, 37 °C and 55 °C, respectively. Using p-nitrophenyl β-D-galactopyranoside the reaction results were fully regiospecific at all the temperatures considered: the maximum molar yield (74%) was obtained at an incubation temperature of 55 °C. Using o-nitrophenyl β-D-galactopyranoside as the donor the reaction was still highly regioselective and the maximum molar yield (50%) was achieved at an incubation temperature also of 55 °C. Using phenyl β-D-galactopyranoside transglycolytic products appear only at an incubation temperature of 55 °C but at very low molar yield (about 14%) and lower regioselectivity.     

Effect of the Formation of Inclusion Complexes With β-Cyclodextrin on the Photoisomerization and Fluorescence Properties of Aromatic Norbornadiene Derivatives

The valence photoisomerization of four aromatic norbornadiene (NBD)derivatives has been studied in ethanol and in 0.01 M -cyclodextrin(-CD) water–ethanol (v/v, 99/1) solution (WECD). Observedfirst-order rate constants are found to be of the same order of magnitude inethanol and WECD, ranging between 0.1 and 0.28 s^-1, accordingto the compound. These photoisomerization kinetic properties are attributedto the formation of inclusion complexes between NBDs and -CD. Thestoichiometry is 1 : 1, and association constants ranging between 310 and390 M^-1 have been determined fluorimetrically, usingBenesi–Hildebrand plots and a nonlinear regression method. Thestructure of the inclusion complexes is discussed on the basis of AMIsemiempirical dimension calculations and photophysical properties.     

Crowding alters the folding kinetics of a β-hairpin by modulating the stability of intermediates

Crowded environments inside cells exert significant effects on protein structure, stability, and function, but their effects on (pre)folding dynamics and kinetics, especially at molecular levels, remain ill-understood. Here, we examine the latter for, as an initial candidate, a small de novo β-hairpin using extensive all-atom molecular dynamics simulations for crowder volume fractions φ up to 40%. We find that crowding does not introduce new folding intermediates or misfolded structures, although, as expected, it promotes compact structures and reduces the accessible conformational space. Furthermore, while hydrophobic-collapse-mediated folding is slightly enhanced, the turn-directed zipper mechanism (dominant in crowder-free situations) increases many-fold, becoming even more dominant. Interestingly, φ influences the stability of the folding intermediates (FI(1) and FI(2)) in an apparently counterintuitive manner, which can be understood only by considering specific intrachain interactions and intermediate (and hierarchical) structural transitions. For φ values <20%, native-turn formation is enhanced, and FI(1), characterized by a hairpin structure but slightly mismatched hydrophobic contacts, increases in frequency, thus enhancing eventual folding. However, higher φ values impede native-turn formation, and FI(2), which lacks native turns, re-emerges and increasingly acts as a kinetic trap. The change in the stability of these intermediates with φ strongly correlates with the hierarchical folding stages and their kinetics. The results show that crowding assists intermediate structural changes more by impeding backward transitions than by promoting forward transitions and that a delicate competition between reduction in configuration space and introduction of kinetic traps along the folding route is key to understanding folding kinetics under crowded conditions.