DSC study of cold and heat denaturation processes of β-lactoglobulin A with guanidine hydrochloride

The cold and heat denaturations of bovine β-lactoglobuhn A (β-lg A) has been studied in solutions of guanidine hydrochloride (GuHCl) by differential scanning calorimelry (DSC) The experimental results are presented and discussed.It is shown that the number of protons bound by the monomeric molecules of β-lg A was unchanged before and after its heat denaturation below pH 3,and that the activation energy of the heat denaturation was depressed owing to the presence of GuHCl.In the solutions with 2.50 and 3.06 mol/L of GuHCl,both the cold and heat denat-urations of β-lg A were observed.In comparison with the heat denaturation,the activation energy of cold denaturation was far lower and the number of GuHCl molecules bound by the unfolded polypeptide chains after cold denaturation increased a lot.The absolute value of the enthalpy of cold denaturation was larger than that of heat denaturation It was found by the analysis that the contribution to the total denaturational enthalpy of conformational change i     

Calorimetric Study of Thermal Denaturation of Superoxide Dismutase

The thermal denaturation of superoxide dismutase (SOD) from bovine erythrocytes was studied at various pH values of different buffers and at various concentrations of solutions of two neutral salts by differential scanning calorimetry. The experiments performed indicate that the PIPES is a buffer non-coordinating with the SOD, and that the binding of the anions studied influences more or less the thermal denaturation of SOD, but the effect on the oxidation form of SOD is more apparent. A new conformer of SOD with lower thermostability was discovered by the experiments performed in different buffers at certain pH values higher than the isoelectric point of SOD, or at higher concentrations of neutral salt solutions. The new conformer may be converted irreversibly into the usual conformer with high thermostability during heating. Based on the thermodynamic parameters obtained in distilled water and by thermodynamic analysis using the Ooi's model, it is revealed that the large enthalpy △Hdc contributed by     

Denatured Thermodynamics of Proteins in Weak Cation－exchange Chromatography

The thermostability of some proteins in weak cation-exchange chromatography was investigated at 20—80 ℃. The results show that there is a fixed thermal denaturation transition temperature for each protein. The appearance of the thermal transition temperature indicates that the conformations of the proteins are de-stroyed seriously. The thermal behavior of the proteins in weak cation-exchange and hydrophobic interaction chromatographies were compared in a wide temperature range. It was found that the proteins have a higher thermostability in a weak cation-exchange chromatography system. The thermodynamic parameters (△H^0,△S^0) of those proteins were determined by means of Van′t t Hoff re|ationship(lnk′-1/T). According to stan-dard entropy change(△S^0) , the conformational change of the proteins was judged in the chromatographic pro-cess. The linear relationships between △H^0 and △S^0 can be used to evaluate “compensation temperature“ (β) at the protein denaturation and identify the identity of the protein retention mechanism in weak cation-ex-change chromatography.     

CHANGES IN CIRCULAR DICHROISM AND EXPOSURE OF BURIED THIOL GROUPS DURING DENATURATION OF RABBIT MUSCLE CREATINE KINASE

The denaturation of creatine kinase in guanidine solutions has been followed by both CD changes and the increase in rapid reacting SH groups. The rates of the exposure of SH groups axe in general agreement with the ultraviolet absorbance and fluorescence changes reported previously whereas changes in the ellipticity of the enzyme molecule can be detected at low guanidine concentrations before significant changes in the exposure of the aromatic residues could be observed. On the other hand, the rates of changes in the mean residue ellipticity at 220 nm are clearly slower than the changes in ultraviolet absorbance, fluorescence and exposure of SH groups. It is suggested that the secondaxy structure of the external regions of the peptide chains is affected at low guanidine concentrations followed by gross changes in the tertiary structure of the molecule resulting in the exposure of the buried aromatic residues. The destruction of ordered secondary structure of the peptide chain is a slower process than th     

EFFECT OF HYDRATION ON THE THERMAL STABILITY OF ENZYME

The thermal stability of New Zealand culture rabbit muscle aldolase wasinvestigated by differential scanning calorimetry in the water content range0.23-3.70 g water per g protein.The experimental results showed that at watercontents below 0.47g/g,an endothermic peak was observed and at water contentsabove 0.57g/g,an endothermic and an exothermic peak were both observed on DSCthermogram.Thermal denaturation result of the enzyme and the relationshipbetween two transition temperatures and water contents were first reported inthis paper.Up to now we have not seen any similar reports concerning theexothermic transition.     

A COMPARISON OF DENATURATION AND INACTIVATION RATES OF CREATINE KINASE IN GUANIDINE SOLUTIONS

Both the denaturation, as followed by UV absorbance and fluorescence changes, and inac-tivation of creatine kinase in guanidine solutions have been found to be first order reactions.In 3 M guanidine, at 30℃, the inactivation rate constant was found to be 5.9 sec~(-1) and thedenaturation rate constant 1.9 sec~(-1). At lower guanidine concentrations, the inactivation rateconstants were only little affected whereas the denaturation rate constants decreased markedly,being of the order of 0.04 in 1 M and 0.004 in 0.5 M guanidine. The kinetics of the inactiva-tion reaction in 0.5 M guanidine was found to be in agreement with a combination of two firstorder reactions. The enzyme lost activity first by a fast reaction with a rate constant onlyslightly lower than the rate constant in 3 M guanidine followed by a slower reaction with a rateconstant of 0.003 sec~(-1). In 0.3 M guanidine, very little change in either UV absorbance or influorescence was observed, but, in sharp contrast, the enzyme lost considerable activity by a fastreaction and this was followed by a slower reaction of inactivation. Even after prolongeddenaturation in 0.5 and 0.3 M guanidine, residual activities of 3.4% and 30% remained res-pectively. The above results suggest a very fragile active site although dissociation of thedimer and reversible guanidine inhibition may also contribute to the initial rapid inactiva-tion. It is also to be noted that the multiphasic courses of inactivation at lower guanidineconcentrations seem to suggest the presence of partly active intermediates during denaturation.     

STUDY ON THERMAL DENATURATION OF PROTEINS CONTAINING LOW WATER CONTENT BY DIFFERENTIAL SCANNING CALORIMETER——DEMONSTRATION OF PRE-THERMODENATURE NEW PEAK

Following the discovery that there is a low temperature endothermal peak for hydration bovine serum albumin, this work further measured DSC heating scanning curve for seven different kinds of hydration proteins with high purity from eleven sources. The measurements have shown that there is an obvious low temperature endothermal peak for all of the protein samples used. We term it "Pre-thermodenature New Peak". The appearance of pre-thermodenature new peak is the common property of proteins containing low water content. The studies of regularity and mechanism on new peak should help to understand the denaturation process of protein, protein folding, molecular association and/or molecular disassociation and solvent effect.     

EFFECT OF ANIONIC SURFACTANT UPON THE VISCOSITY OF POLYMER GUAR GUM SOLUTIONS

The reduced viscosity of polymer guar gum solutions containing a certain concentration of sodium dodecyl benzene sulfonate （SDBS） was measured. It has been found that the Huggins coefficient kH of polymer solutions is very sensitive to the concentration of the surfactant, CSDBS, in solutions. If CSDBS is lower than CMC, the critical micelle concentration of SDBS, kH increases rapidly with CSDBS. On the other hand, if CSDBS is larger than CMC, kH decreases rapidly with CSDBS. Comparatively, the intrinsic viscosity of polymer solution does not show a notable change with CSDBS. The experimental results indicate that the interchain association of polymer guar gum in solution is greatly associated with SDBS interacted with polymer chains through hydrogen bonds. However, the effect of SDBS upon the intrachain association of polymer guar gum solution is negligible, presumably due to the fact that guar gum is a slightly stiffened random-coil chain polymer.     

MECHANISTIC STUDY OF POLYNITROCOMPOUNDS BY XPS——MECHANISM OF THE THERMAL DECOMPOSITION OF POLYNITROPHENOLS

The subject-matter described in the paper reveals the importance of the hydroxyl groups within the molecules of polynitrophenols in the process of the thermal decomposition as studied by XPS (ESCA). It is evident that theintramolecular charge transfer with drastic change during the time of the thermal decomposition can be envisaged as an indicator capable of following the reaction process. The present work shows that combining with CNDO/2 SCF MO model XPS data allow information to be derived reasonably on the relative stability of nitrogen-containing species left in the surface of the energetic materials during decomposition.     

Comparison Between Catalytic Activity and Activity Changes During Denaturation by Guanidine Hydrochloride of Enzymes in Crystalline State and in Solution

The catalytic activity and activity changes during denaturation by guanidine hydrochloride of glyceraldehyde-3-phosphate dehydrogenase,lactate dehydrogenase and α-chymotrypsin in crystalline state and in solution have been compared.The catalytic activities are lower in crystalline state than in solution. Enzymes in crystalline state are more stable than in solution during denaturation by guanidine hydrochloride.Ammonium sulfate has different effects on catalytic activities of different enzymes and shows protection on all enzymes studied during denaturation by guanidine hydrochloride.The protection is more obvious at high concentrations of guanidine hydrochloride than at low concentrations.It is suggested that the flexibility or mobility of enzyme is required for the catalytic activity and related to the stability of enzymes. Enzymes with less flexibility or mobility are more stable.     

PROPERTIES OF DILUTE SOLUTIONS OF HYDROPHOBICALLY MODIFIED POLY (4-VINYL PYRIDINE) (POLYSOAPS)

Hydrophobically modified poly(4-vinyl pyridines) by alkyl bromides arekinds of polysoap similar to the surfactant. Properties of dilute solutions were studiedthrough the viscosity measurements in pure water and NaCl solutions. In aqueous solu-tions of polysoaps hydrophobic interaction can be attributed to aggregation of hydrophobicgroups of the polysoap main chains. The hydrophobic groups of polysoap can aggregate toform hydrophobic microdomains (micelles) in aqueous solution. This is a compact confor-mation. The formation of such microdomains is a process of dynamic equilibrium.     

Dynamic High Pressure Micro-fluidization Effects on Structure and Physico-chemical Properties of Egg White Protein

The authors dealt with the effects of dynamic high pressure micro-fluidization(untreated and 20―160 MPa treated) on the structure and physico-chemical properties of egg white solutions[6%(mass fraction) or 61.2 mg protein/mL]. Micro-fluidization treatment resulted in a decrease in mean particle size,and little change in solubility. It was found that the residual denaturation enthalpy was decreased with the increase of pressure from 20 to 100 MPa and from 120 to 160 MPa,reaching into the maximum at 160 MPa. ...     

STUDIES ON THE MECHANISM OF INDIRUBIN ACTION IN THE TREATMENT OF CHRONIC GRANULOCYTIC LEUKEMIA——V.BINDING BETWEEN INDIRUBIN AND DNA AND IDENTIFICATION OF THE TYPE OF BINDING

The binding between indirubin and calf thymus DNA in vitro has been verified by meansof the isotope labelling method, spectrophotometric method and thermal denaturation meas-urements. The λ_max 207 nm of indirubin shifted toward longer wave length with decrease ofabsorbance after the incubation of indirubin with DNA. The escalation of Tm value of DNAinduced by indirubin was about 2.4°C and it was reproducible. The binding force between themwas rather weak, as indirubin molecules were easily released during the precipitation withalcohol or the gel filtration. The binding was not affected by sodium chloride even at high con-centration but greatly decreased (to 20-30% of the control) in the presence of 8 M urea.These results showed that the binding between indirubin and DNA might be of hydrogen bondrather than ionic. The amount of bound ~3H-indirubin was directly proportional to the con-centration of indirubin. However, it increased abruptly when the concentration of indirubinreached 1.5×10~(-4) M. This     

INFLUENCE OF THERMAL AND NON—THERMAL EFFECTS OF ULTRASOUND ON ISOTHERMS OF PHENOL ON NKAⅡ RESIN

Adsorption equilibrium experiments of phenol on the NKA II resin are separately conducted in the presence and absence of ultrasound at ambient temperature.The isotherm of phenol on the polymer adsorbent in the presence of ultrasonic field is firstly reported.Results indicated that the isotherm of phenol determined in the presence of ultrasound is lower than that in the absence of ultrasound.In addition,experiments also show that the use of ultrasound to the adsorption system of the phenol aqueous solution and NKA Ⅱ resin could cause the rising of the temperature of the system in the order of 6^-C.The effect of ultrasound on the isotherm of the phenol on the NKA Ⅱ resin mostly ascribes to the thermal effect and the non-thermal effect of ultrasonic field.and the role of the later is greater than that of the former.     

A DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF TRICHOSANTHIN AT LOW RESOLUTION

Trichosanthin, extracted from a Chinese herb medicine, is a plant protein having a molec-ular weight of ca. 24,000 and containing 219 amino acid residues. The crystal is mono-clinic, belonging to the space group C2, with the following cell dimensions: a=75.64A, b=75.52A, c=88.85A β= 99.51°. Each asymmetric unit contains two such protein molecules. The UO_2Ac_2 and K_2Pt(NO_2)_4 derivatives of trichosanthin have been prepared by soakingin the respective solutions of high dilution. The joint probability of isomorphous replacementand anomalous scattering has been used to calculate the native phases. In the electron densitymaps, the boundary between the two protein molecules can be recognized, leading to thefollowing approximate molecular dimensions: 45×40×35A~3. Each molecule contains fourrod-shaped segments easily known as α-helices.     

STUDIES ON ELECTROANALYTICAL CHEMISTRY OF RARE EARTH ELEMENTS——POLAROGRAPHIC INVESTIGATION OF CARBOXYNITRAZO (CNA) AS A LIGAND OF RE-CNA COMPLEXES

The polarographic behaviour of carboxynitrazo (CNA) has been investigated in detail by means of single-sweep, DC, AC polarography and cyclic voltammetry. It has been shown that CNA has different reduction mechanisms in acidic and basic solutions. In acidic solution, the first wave is reversible and the second involves the disproportionation mechanism with acid catalysis, while in basic solution the first wave splits into two and the second wave has no disproportionation characteristics. The third wave is the reduction of nitro-group in the molecule.     

THERMAL BEHAVIOR OF THERMOTROPIC HYDROXYETHYL CELLULOSE ACETATE/POLYETHYLENE BLENDS

The thermal behavior of thermotropic hydroxythyl cellulose acetate (HECA)/polyethy-lene (PE) blends has been studied by DSC. It is found that the blends of HECA and PEare immiscible but the crystallization of PE is affected by HECA chains in the blends withmore than 50% HECA, which results in the subordinate crystallization of PE and the for-mation of imperfect structures in the PE crystals. The imperfection of PE crystals in theblends can be eliminated after annealing at 393K.     

EFFECT OF ULTRASONIC WAVE ON THE ELECTROPOLYMERIZATION OF PYRROLE

Electropolymerization of pyrrole under ultrasonic field at 20kHz was performed ina series of aqueous and propylene carbonate (PC) solutions. The ultrasonic wave withmoderate intensity at the power of 44W, which is the power threshold of the ultrasonicgenerator used in this work to produce cavitation effect, enhance the conductivity andtensile strength of the polypyrrole films as prepared. However, too high intensity of theultrasonic wave is harmful to the polymerization.     

RAPID DETERMINATION OF PROTEIN IN MILLET BY FOURIER TRANSFORM NEAR-INFRARED(FTNIR)DIFFUSE REFLECTANCE SPECTROSCOPY

In this paper,the Fourier transform near-infrared(FTNIR)diffuse reflectance spectroscopy is applied for therapid determination of protein in millet.The partial least-squares(PLS)regression is successfully used as aneffective multivariate calibration technique.The calibration set is composed of 20 standard millet samples thatthe protein contents were determined by the traditional Kjeldahl method.The optimal model dimension is foundto be 5 by cross-validation.22 millet samples were determined by the proposed FTNIR-PLS method.Thecorrelation coefficient between the concentration values obtained by the FTNIR-PLS method and the traditionalKjeldahl method is 0.9805.The standard error of prediction(SEP)is 0.28% and the mean recovery is 100.2%.Theproposed method has been successfully applied for the routine analysis of protein in about 10,000 grain samples.