Heat-induced denaturation and aggregation of ovalbumin at neutral pH described by irreversible first-order kinetics

The heat-induced denaturation kinetics of two different sources of ovalbumin at pH 7 was studied by chromatography and differential scanning calorimetry. The kinetics was found to be independent of protein concentration and salt concentration, but was strongly dependent on temperature. For highly pure ovalbumin, the decrease in nondenatured native protein showed first-order dependence. The activation energy obtained with different techniques varied between 430 and 490 kJ*mole(-1). First-order behavior was studied in detail using differential scanning calorimetry. The calorimetric traces were irreversible and highly scan rate-dependent. The shape of the thermograms as well as the scan rate dependence can be explained by assuming that the thermal denaturation takes place according to a simplified kinetic process where N is the native state, D is denatured (or another final state) and k a first-order kinetic constant that changes with temperature, according to the Arrhenius equation. A kinetic model for the temperature-induced denaturation and aggregation of ovalbumin is presented. Commercially obtained ovalbumin was found to contain an intermediate-stable fraction (IS) of about 20% that was unable to form aggregates. The denaturation of this fraction did not satisfy first-order kinetics.     

Freezing denaturation of ovalbumin at acid pH

The effects of rapid freezing and thawing at acid pH on the physiochemical properties of ovalbumin were examined. At low pH (around 2), UV difference spectra showed microenvironmental changes around the aromatic amino acid residues; elution curves by gel permeation chromatography showed decreasing numbers of monomers after neutralization. These changes depended on the incubation temperature (between -196 and -10 degrees C) and the protein concentration (0.5-10 mg/ml), and a low concentration of ovalbumin incubated at around -40 degrees C suffered the most damage to its conformation. With freezing and then incubation at -40 degrees C, three of the four sulfhydryl groups in the ovalbumin molecule reacted with 2,2'-dithiodipyridine. The CD spectra showed these changes in the secondary structure, but they were smaller than those when guanidine hydrochloride was used for denaturation. Supercooling at -15 degrees C or freezing at -196 degrees C had little or no effect on the conformation of the ovalbumin molecule. Thus, irreversible conformational changes of ovalbumin were caused under the critical freezing condition at an acid pH. These changes arose from partial denaturation and resembled those with thermal denaturation of ovalbumin at neutral pH.     

Conformational changes in ovalbumin at acid pH

Several physicochemical parameters of ovalbumin were examined at acid pH. The intrinsic viscosity and far UV-CD spectrum at pH 2 did not differ from those at pH 7. But the near UV-CD spectrum, difference absorption spectrum around 250-320 nm, and fluorescence spectrum showed micro-environmental changes around the aromatic amino acid residues in acid solution. The reactivity of one of the four sulfhydryl groups with 2,2'-dithiodipyridine increased at pH below 5. The rate of denaturation by urea and that of surface tension decay were high in the low pH range. We concluded that at low pH (around 2), ovalbumin molecules kept their native globular conformation, but that their chain flexibility increased and they were very susceptible to denaturation. This state might be equivalent to the molten-globule state observed with some globular proteins in acidic region.     

Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH

Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.     

Aging of tubulin at neutral pH

The aging of calf brain tubulin in neutral solution has been investigated using the techniques of sedimentation velocity and equilibrium, microtubule assembly, fluorescence spectroscopy, circular dichroism and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that tubulin incubated at 4 °C undergoes a slow association process leading to the generation of a 9 S component. The fraction of 9 S component increases progressively with incubation time and appears to follow mono-molecular kinetics. The generation of the 9 S species is paralleled closely by inhibition of microtubule assembly and loss of colchicine-binding ability. Fluorescence spectroscopy and circular dichroism spectra indicate that tryptophan moieties are perturbed during the aggregation process and that the tubulin dimer undergoes a conformational change. There is no protein degradation up to an incubation period of 50 hours. Sedimentation equilibrium experiments in 6 m-guanidine hydrochloride, both in the presence and absence of 2-mercaptoethanol, indicate that the aggregates are stabilized by disulfide bonds and hydrophobic interactions. At periods of incubation >50 hours, the protein starts to be degraded.     

Conformational state of disulfide-reduced ovalbumin at acidic pH.

Ovalbumin assumes a highly ordered molten-globule conformation at pH 2.2. To investigate whether or not such structural nature is related to the existence of an intrachain native disulfide bond, the structural characteristics of disulfide-reduced ovalbumin at the acidic pH were compared with those of the native disulfide-intact protein by a variety of analytical approaches. The disulfide-reduced protein was found to assume a partially denatured molten globule-like conformation similar to the disulfide-intact counterpart as analyzed by the CD and intrinsic tryptophan fluorescence spectra and by the binding of a hydrophobic probe of anilino-1-naphthalene-8-sulfonate. The results from size-exclusion chromatography also showed that the disulfide-reduced and disulfide-intact proteins have essentially the same compact, native-like hydrodynamic volume. The disulfide-reduced protein was, however, highly sensitive to proteolysis by pepsin at the acidic pH under the proteolytic conditions in which the disulfide-intact protein was almost completely resistant. Furthermore, on a differential scanning calorimeter analysis the disulfide-reduced protein had an endothermic transition at a much lower temperature (Tm = 48.5 degrees C) than the disulfide-intact protein (Tm = 57.2 degrees C). Taken together, we concluded that the intrachain disulfide bond should not be directly related to the highly ordered molten-globule conformation of ovalbumin, but that its conformational stability depends on the presence of the disulfide bond.     

中性体系中超高压－Nisin协同作用下的细菌致死机理

摘要：【目的】为保证超高压中性食品的杀菌强度,可以??????????通过添加Nisin等细菌素协同杀菌以达到商业无菌要求。本文从分子水平和超微结构揭示二者协同作用下的细胞致死机理,为超高压杀菌在中性食品中的应用奠定理论基础。【方法】采用pH7.0的环境体系,100－500 MPa的超高压处理,Nisin浓度为200 IU/mL。通过荧光染色法和紫外吸收法检测细胞膜通透性,傅里叶转换红外光谱法检测细菌细胞壁、蛋白以及核酸的变化,透射电镜观察细菌在协同作用下的形态变化。【结果】结果发现：中性条件下,超高压与     

A ratiometric fluorescent chemosensor for the detection of cysteine in aqueous solution at neutral pH

A ratiometric fluorescent chemosensor 2‐(2′‐hydroxy‐3′‐formyl‐5′‐methoxyphenyl)benzothiazole ( 1 ) was developed for the detection of cysteine (Cys). In aqueous solution at neutral pH, 1 exhibited a ratiometric fluorescent response to Cys with a remarkable red‐to‐green shift in the emission wavelength. This fluorescence change was attributed to the cyclization reaction between the formyl group in 1 and the amino and sulfhydryl group in Cys in a stoichiometry of 1: 1 according to the proposed mechanism. At neutral pH, 1 displayed a significant fluorescence ratio signal enhancement with the addition of Cys. Furthermore, 1 showed good selectivity toward Cys. The detection limit and linear range were 5.6 and 0–100 μmol/L, respectively, which demonstrated that 1 could recognize relatively low concentrations of Cys and is a good candidate for applications in detecting Cys.     

Thermothrix thioparus gen. et sp. nov. a facultatively anaerobic facultative chemolithotroph living at neutral pH and high temperature.

Thermothrix thioparus gen. et ep. nov. occurs naturally in a New Mexico hot spring at a temperature of 74 degrees C, a pH of 7.0, and a HS- concentration of 1 mg/litre. The organism is gram-negative, non-motile, 0.5-1.0 X 3-20 mum, and forms cell chains up to 1 cm in length. The resulting filaments do not possess a sheath. Sulfur is deposited extracellularly. The organism was isolated using an autotrophic medium with HS- as the energy source and NO3- as the terminal electron acceptor. Anaerobically either NO2- or NO3- is required, NO2- is formed from NO3-, and no observable gas is evolved. Oxygen can also be used as the terminal electron acceptor, but growth is poor because of the decreased solubility of O2 at temperatures required for growth. Alternate energy sources used aerobically and anaerobically include hexose, HS-, SO3-, and S2O3=. The temperature optimum is 70-73 degrees C and growth occurs from 62 to 77 degrees C. The organism's thermal and physiological characteristics are compared to those of Bacillus stearothermophilus, Methanobacterium thermoautotrophicum, Sulfolobus acidocalderius, Thermus aquaticus, Thermus flavus, as well as Thiobacillus denitrificans, the latter being the only other facultatively anaerobic chemolithotroph which has been isolated and described.     

Viscosity analysis of the temperature dependence of the solution conformation of ovalbumin

The viscosity of ovalbumin aqueous solutions was studied as a function of temperature and of protein concentration. Viscosity-temperature dependence was discussed on the basis of the modified Arrhenius formula at temperatures ranging from 5 to 55 degrees C. The activation energy of viscous flow for hydrated and unhydrated ovalbumin was calculated. Viscosity-concentration dependence, in turn, was discussed on the basis of Mooney equation. It has been shown that the shape parameter S decreases with increasing temperature, and self-crowding factor K does not depend on temperature. At low concentration limit the numerical values of the intrinsic viscosity and of Huggins coefficient were calculated. A master curve relating the specific viscosity etasp to the reduced concentration c[eta], over the whole range of temperature, was obtained and the three ranges of concentrations: diluted, semi-diluted and concentrated, are discussed. It has been proved that the Mark-Houvink-Kuhn-Sakurada (MHKS) exponent for ovalbumin does not depend on temperature.     

Butanol formation byClostridium thermosaccharolyticum at neutral pH

Summary Clostridium thermosaccharolyticum can produce up to 40 mM butanol. The formation of ethanol and butanol from starch and glucose by strain DSM 571 and by the new isolate 021 was compared. The ratios for ethanol/acetate and butanol/butyrate were higher during growth at neutral pH than at acidic pH. Butanol formation was greatly stimulated by the addition of butyrate.     

3-Methoxycarbonyl-5-nitrophenyl boronic acid: high affinity diol recognition at neutral pH

Several boronic acids were screened for their ability to bind to diols. 3-methoxycarbonyl-5-nitrophenyl boronic acid bound to both a catechol dye as well as fructose with a comparable affinity to that of an ortho-methylamino substituted boronic acid. This work suggests a greater role for appropriately functionalized electron deficient boronic acids in diol and carbohydrate recognition.     

4-aminophenylalanine as a biocompatible nucleophilic catalyst for hydrazone ligations at low temperature and neutral pH

Hydrazone formation and similar reactions are highly versatile and specific, but their application to biological systems has been limited by their characteristically slow reaction kinetics at neutral pH. Catalysis of these reactions through imine formation with aromatic amines such as aniline has broadened the applicability of these reactions to biomolecular labeling. High concentrations of the catalyst are necessary, which may be incompatible with the native structure of certain proteins. In this study, we investigated the utility of 4-aminophenylalanine (4a-Phe) as a catalyst for these reactions. We find that 4a-Phe is nearly as effective as aniline in catalyzing hydrazone formation between the reactive amino acid 3-formyltyrosine (3f-Tyr) and hydrazine-containing fluorophores, both free in solution and incorporated into the protein tubulin. The catalyst 4a-Phe maintains ～70% of the catalytic efficacy of aniline and is less detrimental to the native structure of tubulin. Examination of the temperature dependence of imine formation between 3f-Tyr and 4a-Phe shows an increase in imine concentration accompanying a decrease in temperature, confirming the exothermic nature of the equilibrium reaction. Interestingly, decreasing the temperature of the 4a-Phe-catalyzed hydrazone reaction between 3f-Tyr and the fluorophore 7-hydrazinyl-4-methylcoumarin increases the overall rate of the reaction. This result indicates that the temperature dependence of the catalyst-aldehyde equilibrium is greater than the temperature dependence of the rate constant for hydrazone formation from this intermediate, and that the rate of hydrazone formation a direct function of the concentration of the intermediate imine. These results provide a platform for conducting nucleophilic catalysis under conditions that are more compatible with biomolecular targets than previously demonstrated, thereby expanding the utility of hydrazone ligations in biological systems.     

An acid-cleavable linker stable at neutral pH that releases doxorubicin at lysosomal pH

A Kemp's triacid amide of doxorubicin (DOX), esterified at a second carboxyl, releases DOX at lysosomal pH at a reasonable rate but is extremely stable at neutral pH.     

The conformation of polyriboadenylic acid at low temperature and neutral pH. A single-stranded rodlike structure.

The conformation of single-stranded polyrA in aqueous solution has been measured at temperatures down to ?12°C. The radius of gyration of low-molecular-weight polyrA varies very little with temperature in this range. By studying the dependence of radius of gyration on temperature for several polyrA fractions, we show that the dependence of the radius upon chain length is consistent with formation of a single-stranded rodlike structure at low temperature. The structure has an approximate length of 3.2 Å/nucleotide.     

Human hemi-myeloperoxidase. Initial chlorinating activity at neutral pH, compound II and III formation, and stability towards hypochlorous acid and high temperature.

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two promoters, 'hemi-myeloperoxidase'. We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of H2O2 for complete conversion. Hemi-MPO was less stable at a high temperature (80 degrees C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (IC50 = 32 microM HOCl) compared to dimeric MPO (IC50 = 50 microM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.     

Polyamines favor DNA triplex formation at neutral pH

The stability of triplex DNA was investigated in the presence of the polyamines spermine and spermidine by four different techniques. First, thermal-denaturation analysis of poly[d(TC)].poly[d(GA)] showed that at low ionic strength and pH 7, 3 microM spermine was sufficient to cause dismutation of all of the duplex to the triplex conformation. A 10-fold higher concentration of spermidine produced a similar effect. Second, the kinetics of the dismutation were measured at pH 5 in 0.2 M NaCl. The addition of 500 microM spermine increased the rate by at least 2-fold. Third, in 0.2 M NaCl, the mid-point of the duplex-to-triplex dismutation occurred at a pH of 5.8, but this was increased by nearly one pH unit in the presence of 500 microM spermine. Fourth, intermolecular triplexes can also form in plasmids that contain purine.pyrimidine inserts by the addition of a single-stranded pyrimidine. This was readily demonstrated at pH 7.2 and 25 mM ionic strength in the presence of 100 microM spermine or spermidine. In 0.2 M NaCl, however, 1 mM polyamine is required. Since, in the eucaryotic nucleus, the polyamine concentration is in the millimolar range, then appropriate purine-pyrimidine DNA sequences may favor the triplex conformation in vivo.     

Proteolytic activity at neutral pH in bovine spleen

Proteolytic activity at neutral pH can be demonstrated in extracts from beef spleen. This activity is completely due to an aminopeptidase - or a number of aminopeptides - which is able to hydrolyse proteins completely to amino acids. No evidence was found for the presence of endopeptidases active at neutral pH. The enzyme resembles to some extent swine kidney aminopeptides.