Kinetics of the acid hydrolysis of heparin from its Cu (II) complex and its relation to biological activity

The acid-catalyzed hydrolysis of heparin from Cu(II) complex was studied as a function of time and temperature. Four independent calculations showed that the hydrolysis, during the 5-hr period examined, obeys the first-order kinetic law. Specific rate constants, calculated at 50°C, 57°C, 65°C, 71°C, and 80°C, were 3.3 × 10^?5 sec^?1, 6.5 × 10^?5 sec^?1, 10.4 × 10^?5 sec^?1, 15.1 × 10^?5 sec^?1, and 26.6 × 10^?5 sec^?1, respectively. Arrhenius plots of the data yielded 14.7 kcal as the energy of activation. An independent run of the self-hydrolysis of heparin at 57°C also obeyed first-order kinetics and its specific rate constant of 6.4 × 10^?5 sec^?1 is in excellent agreement with that of the hydrolysis of Cu(II)-heparin at 57°C. The anticoagulant activity of heparin and of the Cu(II)-heparin are not appreciably different. Further, the inactivation of heparin closely parallels Cu(II) release from the Cu(II) complex which in turn parallels desulfation.     

The stability of almond β-glucosidase during combined high pressure–thermal processing: a kinetic study

The thermal and the combined high pressure–thermal inactivation kinetics of almond β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) were investigated at pressures from 0.1 to 600 MPa and temperatures ranging from 30 to 80 °C. Thermal treatments at temperatures higher than 50 °C resulted in significant inactivation with complete inactivation after 2 min of treatment at 80 °C. Both the thermal and high pressure inactivation kinetics were described well by first-order model. Application of pressure increased the inactivation kinetics of the enzyme except at moderate temperatures (50 to 70 °C) and pressures between 0.1 and 100 MPa where slight pressure stabilisation of the enzyme against thermal denaturation was observed. The activation energy for the inactivation of the enzyme at atmospheric pressure was estimated to be 216.2?±?8.6 kJ/mol decreasing to 55.2?±?3.9 kJ/mol at 600 MPa. The activation volumes were negative at all temperature conditions excluding the temperature–pressure range where slight pressure stabilisation was observed. The values of the activation volumes were estimated to be ?29.6?±?0.6, ?29.8?±?1.7, ?20.6?±?3.2, ?41.2?±?4.8, ?36.5?±?1.8, ?39.6?±?4.3, ?31.0?±?4.5 and ?33.8?±?3.9 cm³/mol at 30, 35, 40, 45, 50, 60, 65 and 70 °C, respectively, with no clear trend with temperature. The pressure–temperature dependence of the inactivation rate constants was well described by an empirical third-order polynomial model.     

Effect of temperature on the survival and infectivity of <Emphasis Type="Italic">Pseudotheraptus devastans</Emphasis> vector

The aim of this study was to investigate under a controlled environment, the effect of temperature on the survival and infectivity of Pseudotheraptus devastans Distant, a cassava anthracnose disease vector. The insect P. devastans was collected from young cassava (Manihot esculenta Crantz) field plots, at the International Institute of Tropical Agriculture, (IITA), Ibadan, Nigeria. A mixture of the different developmental stages of eggs, first to fifth instar nymphs, and adults, were incubated in controlled environment chambers, under various constant temperatures of: 15, 17, 22, 25, 27, 30, and 35°C. Relative humidity at different temperature conditions were recorded and maintained at 90%, 85%, 80%, 75%, 70%, 65%, and 60%, respectively. A significant increase in insect survival was observed between 22 and 27°C temperature conditions while a significant decrease in survival was observed at 15°C and above 30°C. Lesion number, lesion diameter and infectivity among the insect stages varied as a function of temperature and relative humidity. Infectivity was highest at 22–25°C maintained at 75–80% RH and lowest at 15°C and above 30°C maintained respectively, at 65% RH and 90% RH. There was considerable low vector infectivity due to low survival of the insects at extreme temperatures.     

Effect of constant temperatures on development of the walnut husk fly,Rhagoletis completa

The developmental rates of various life stages ofRhagoletis completa Cresson (Diptera: Tephritidae) were determined in the laboratory at seven different constant temperatures: 8, 12, 16, 20, 24, 28, and 32±1°C, RH 80±10%, photoperiod L 16∶D8. Preoviposition developmental rate was fastest at 28°C (10±1 days, mean±SD) and slowest at 12°C (26±1 days). About 83% of the females deposited eggs at 20 and 24°C and only 25% oviposited at 32°C. Females laid the highest number of eggs at 24°C and the lowest at 8°C. Egg development increased with increasing temperatures up to 28°C, then declined. The fastest egg development was noticed at 28°C (55±1 h) and slowest at 8°C (389±2 h). Over 90% egg hatch was observed at temperatures between 12 and 32°C, but decreased to 73% at 8°C. Larval development was fastest also at 28°C (20±0.2 days). Over 65% pupation was recorded at 20 and 24°C, but decreased to 15% at 32°C and 12% at 8°C. Pupal development was most rapid at 24°C (53±1 days) and slowest at 8°C (162±2 days). More than 70% of adult emergence was noticed in treatments between 16 and 24°C but decreased to 20% at 8°C. Based on a linear regression model of temperature-development rate relationship, the lower developmental thresholds were determined to be 6.6, 5.3, 2.9, and 5°C for preoviposition, egg, larval, and pupal stages, respectively. Based on a non-linear developmental rate model, the upper developmental thresholds were 34°C for preoviposition, egg, and larval stages and 30°C for pupal stage.     

Proline peptide isomerization and the reactivation of denatured enzymes

The kinetics of slow phase reactivation of 11 single chain denatured enzymes containing between 6 and 28 proline residues were each found to be first-order having half-times ranging from 0.15 to 12.1 minutes, respectively, at 25 °C. The reactivation kinetics of selected enzymes are independent of solvent viscosity and give an activation energy of 19 kcal/mol. These results are consistent with the proposal that cis/trans proline isomerization in the denatured state is responsible for the slow phase of enzyme refolding/reactivation and with biosynthetic rates for enzyme production.     

Biochemical characterization and kinetic/thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase with transfructosylating activity

This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45–65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol⁻¹, and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3–290.4 kJ mol⁻¹, 568.7–571.0 J mol⁻¹ K⁻¹, and 97.9–108.8 kJ mol⁻¹, respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.     

The response of bacterial spores to vacuum treatments. II. Germination and viability studies.

The viability of Bacillus megaterium spores has been determined after exposure to vacuum dehydration at temperatures between 0 and 65 °C, for periods up to 24 hr. A curvilinear relationship has been demonstrated between viability and drying temperature, with minimum viability occuring around 15 °C and increases in viability being shown above 35 °C. In contrast to vegetative bacteria, reequilibration of the dried spores to 2 × 10^?3 or 10 Torr aqueous vapor pressure, and/or subsequent exposure to oxygen had no effect on viability. Dehydration, rehydration and oxygen treatments had no effect on the time for outgrowth of the spores or on the growth rate of the resultant vegetative cells. Physical loss of spores from samples was not demonstrated during any of these treatments. Evidence has been presented for a novel type of spore activation, which occurs during vacuum dehydration at high temperatures, to an extent that is dependent upon drying time. The mechanism of this activation is unlike that of conventional heat or chemical activation but is oxygen independent and unaffected by reequilibration to 2 × 10^?3 or 10 Torr.     

Saccharification of cellolulose by the cellulolytic enzyme system of Thermonospora sp. I. Stability of cellulolytic activities with respect to time,temperature, and pH

The stabilities and optima with respect to temperature and pH of the β-glucosidase, Avicelase, and carboxymethylcellulase (CMCase) activity of Thermomonospora sp., in the culture filtrate, culture whole broth, and filtrate after sonication of culture solids, are reported. The β-glucosidase is cell associated and has an optimal activity at about pH 6.5 and 55°C. In the whole culture broth, it has a half-life of about 8 hr at 55°C and less than 1 hr at 60°C, while the half-life of the activity in the sonicated, cell-free filtrate is less than 1 hr at 55°C. The Avicelase and CMCase activities occur in the extracellular culture fluid and have optima at about pH 7.0 and 5.9, and 65 and 70°C, respectively. The CMCase activity is stable over 24 hr at 60°C, but declines by 50% in the same period at 65°C. The Avicelase activity declines by 15% over 24 hr at 55°C, and by 50% at 60°C. The highest pH studied (pH 7.3) was the most destabilizing for all three activities. The thermostable characteristics of the cellulases from Themomonospora appear to make them suitable for commercial saccharification processes operating at elevated temperatures.     

Production of non-defective and defective oligomers of viral DNA in mouse 3T3 cells transformed by a thermosensitive mutant of polyoma virus

Cultures of three lines of mouse 3T3 cells transformed independently by the thermosensitive ts-a mutant of polyoma virus yield virus upon lowering their incubation temperature to 31°C. At 31°C, the internal pools of DNA of all three lines contain not only superhelical viral monomers, but also a small proportion of viral oligomers.From one of these three cell lines, several sublines of different clonal morphology were isolated at 38.5°C. The viral DNA synthesized at 31°C by each different subline displayed a unique oligomer pattern which has been stable through many cell passages and further reclonings. In contrast to the parental line, the monomer in most of these sublines is a minor component of the viral DNA pool. In one subline, more than 80% of the viral DNA consists of superhelical molecules about 1.6-times the size of a monomer. The specific infectivity of these molecules is only about one-tenth that of monomers, whereas the efficiency in transforming hamster (BHK21) cells is about twice that of monomers.     

Reaction of peroxynitrite with L-tryptophan

Summary

The reaction between peroxynitrous acid (hydrogen oxoperoxonitrate) and L-tryptophan is 130 M^?1s^?1 at 25°C. The pH dependence of the second-order rate constant shows a maximum at pH 5.1. The enthalpy and entropy of activation at pH 7.1 are 10.6 ± 0.4 kcal.mol^?1 and -16 ± 2 cal.mol^?1K^?1 respectively. High-performance liquid chromatography analysis revealed a number of reaction products, two of which were identified as 5- and 6- nitrotryptophan. Hydroxytryptophans were not observed, even at low peroxynitrite concentrations where most of the peroxynitrite decays to nitrate via a first-order process. These results support the hypothesis that isomerization of protonated peroxynitrite to nitrate does not involve formation of the hydroxyl radical.     

Temperature dependence of ultrasound-induced cell killing: The role of membrane fluidity

Chinese hamster cells in suspension were exposed to 20 kHz ultrasound (US) at 54 W/cm² and various temperatures between 2 and 44 °C. Activation energies were 2.6 and 24 kcal/mole below and above 35 °C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 °C, increasing the activation energy to 62 kcal/mole. The inactivation of the bacterium Salmonella typhimurium by US was also dependent on the exposure temperature, with an activation energy of 2.9 kcal/mole between 2 and 44 °C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.     

The temperature dependence of the exchange transport of glucose in human erythrocytes

The temperature dependence of the uptake of glucose by exchange transport is investigated at two pH values (7.5 and 5). The Arrhenius's energy of activation, the heat of activation, the free energy of activation and the entropy of activation are calculated. The parameters are different at pH 7.5 and 5. For both pH levels the heat of activation and the entropy of activation change abruptly at 20°C. This finding leads us to assume that at this temperature a change in the structure of the membrane takes place.     

Cross-mating experiments with geographically different populations of <Emphasis Type="Italic">Amblyomma cajennense</Emphasis> (Acari: Ixodidae)

The effect of temperature on the development and fecundity of Sancassania polyphyllae fed on tissues of Polyphylla fullo larvae was studied at 15, 20, 25, 30, and 35 ± 1°C and 65 ± 10% RH in a dark incubator. Mean developmental period of immature stages decreased significantly with increasing temperatures from 15 to 30°C. Developmental periods at 30–35°C were not significantly different. The estimated lower developmental thresholds of the various immature stages ranged between 10.1 and 11.5°C. The thermal constant for the egg-to-female adult was 93.5 degree-days. The pre-oviposition, oviposition, and post-oviposition periods and female longevity were significantly longer at 15°C than at higher temperatures. Mean total and daily fecundity were the highest at 25°C, which were significantly different from those obtained at 15, 20 and 30°C. The net reproductive rate (R ₀) was the highest at 25°C (588.3 ♀/♀). The longest mean generation time (T ₀) occurred at 15°C (36 days) and the shortest occurred at 30°C (9.2 days). The highest intrinsic rate of increase (r _m) for S. polyphyllae was observed at 25 (0.61 ♀/♀/day) and 30°C (0.62 ♀/♀/day).     

Characterization,purification, and temperature/pressure stability of polyphenol oxidase extracted from plums (Prunus domestica)

In this study, polyphenol oxidase (PPO) was extracted from Prunus domestica and partially purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and ion exchange chromatography. The final purification step revealed a 32.81-fold purification, and the molecular mass was estimated to be 65 kDa by SDS-PAGE. The purified PPO showed enzymatic activity mainly toward five substrates, namely catechol, catechin, 4-methyl catechol, chlorogenic acid, and L-3,4-dihydroxyphenylalanine, whereas it showed no activity toward caffeic acid, ferulic acid, p-coumaric acid, p-cresol, and l-tyrosine. The optimum pH and temperature values were 6.0 and 25 °C, respectively. The enzyme showed high stability in the pH range of 5.0–7.0 and in the temperature range of 25–65 °C. The most effective inhibitors of this enzyme were found to be ascorbic acid and l-cysteine. The thermal inactivation followed a first-order kinetic model, with activation energy of E_a 150.46 ± 1.29 kJ/mol. PPO extracted from plum showed stability at high pressure, with enzyme activation at 500 MPa.     

Mathematical modeling of maize starch liquefaction catalyzed by α-amylases from Bacillus licheniformis: effect of calcium, pH and temperature

The first step of starch hydrolysis, i.e. liquefaction has been studied in this work. Two commercial α-amylases from Bacilllus licheniformis, known as Termamyl and Liquozyme have been used for this purpose. Using starch as the substrate, kinetics of both enzymes has been determined at optimal pH and temperature (pH 7, T = 80 °C) and at 65 °C and pH 5.5. Michaelis–Menten model with uncompetitive product inhibition was used to describe enzyme kinetics. Mathematical models were developed and validated in the repetitive batch and fed-batch reactor. Enzyme inactivation was described by the two-step inactivation model. All experiments were performed with and without calcium ions. The activities of both tested amylases are approximately one hundred times higher at 80 °C than at 65 °C. Lower inactivation rates of enzymes were noticed in the experiments performed at 65 °C without the addition of calcium than in the experiments at 80 °C. Calcium ions in the reaction medium significantly enhance amylase stability at 80 °C and pH 7. At other process conditions (65 °C and pH 5.5) a weaker calcium stabilizing effect was detected.     

Acid-Catalyzed nucleophilic substitution and racemization of 3-methoxy-N-desmethyldiazepam enantiomers in methanol

pK_a1 values of 3-methoxy-N-desmethyldiazepam in acetonitrile and methanol containing various acid concentrations were determined by spectrophotometry to be 3.5 and 1.3, respectively. Temperature-dependent racemization of enantiomeric 3-methoxy-N-desmethyldiazepam in methanol containing 0.5 M H₂SO₄ was studied by circular dichroism spectropolorimetry and the racemization reactions were found to follow apparent first-order kinetics. Thermodynamic parameters of the racemization reaction were found to be: E_act = 18.8 kcal/mol, and at 25°C: ΔH^? = 18.3 kcal/mol, ΔS^? = ?14.8 entropy unit, and ΔG^? = 22.7 kcal/mol, respectively. The racemization had an isotope effect (k_H/k_D) of 1.6 at 42°C. Based on the results of this report and those of earlier reports by other investigators, a nucleophilically solvated C3 carbocation intermediate resulting from either a P (plus) or an M (minus) conformation is proposed to be an intermediate and responsible for the stereoselective nucleophilic substitution and the subsequent racemization of 3-methoxy-N-desmethyldiazepam enantiomers. © 1993 Wiley-Liss, Inc.     

Interactions of bovine fibrinogen and thrombin. Early events in the development of clot structure.

Interactions which determine the rate of conversion of fibrinogen into monomer fibrin and the retention of monomer fibrin in a noncompactible form through interaction with residual fibrinogen (solution stabilization) were examined through the kinetics of formation of equilibrium compactible network at pH 7 and ionic strength 0.15. For studies of conversion, reactions with thrombin were at 29 or 2 °C, hirudin was added at successive times to inhibit thrombin, and compactible network was equilibrated at 2 °C, where solution stabilization is negligible. A substrate dependency of initial rate is interpreted on the basis of inactive complex formation between thrombin and both fibrinogen and monomer fibrin. At 29 or 2 °C specific rate constants are 32 or 2.9 × 10⁶ liter/mol, and association constants for inactive complex formation are 5.2 or 2.0 × 10⁵ liter/mol. The second peptide-A is removed from fibrinogen ~ 40-fold as rapidly as the first.With equilibration at 29 °C, compactible network does not appear until the solution stabilization ratio of residual fibrinogen/monomer fibrin is four. Thereafter, increasing amounts of compactible network appear. However, the stabilization ratio progressively decreases to approximately two, a situation which indicates the complexity of the stabilization mechanism.The thrombin-hirudin association constant is estimated to be 4.9 or 17 × 10¹¹ liter/mol at 29 or 2 °C.     

The capacity for increase at a low temperature of some Australian populations of the granary weevil,Sitophilus granarius (L.)

The capacity for increase (r_c) of one laboratory and seven field populations of young adult S. granarius from different sites in Australia was determined over thirty-two weeks at 15°C in wheat of 14% moisture content. The mean value of r_c was 0.0704 ±0.0016 and the populations differed significantly with respect to this parameter. Variation in the net reproductive rate (R₀), which averaged 25.4± 1.29, had a greater effect on the value of I_c than did variation in the cohort generation time (T_c), which averaged 45.7±0.37 weeks. The populations did not differ significantly in terms of adult survivorship and 94% of females were still alive at thirty-two weeks. The maximum rate of oviposition, about ten eggs per female per week, was achieved in the eighteen-twenty week age-interval. About 41% of the immature stages survived to adulthood. Estimates of r_c over a twenty-four week period were only slightly lower than those over thirty-two weeks. The capacity for increase at 15°C of a given population was shown to be correlated with its fertility at 29°C, an optimal temperature, and with its body weight rather than with its cold tolerance, as indicated by its chill-coma temperature, or its previous temperature-history. The temperature experienced by the immature stages had a marked influence on r_c in that weevils reared at 15°C and 27°C had respective values of 0.0654 and 0.0786 when subsequently held at 15°C. The differences in the survivorship and fertility of S. granarius and S. oryzae (L.) when both species were reared at 15°C are considered.     

Effect of temperature and relative humidity on the rate of development,fecundity and life table parameters of the red spider mite Oligonychus mangiferus (Rahman and Sapra) (Acari: Tetranychidae)

Studies on biology of Oligonychus mangiferus (Rahman and Sapra) at combination of eight constant temperatures and relative humidities (RHs) viz., 7.0°C with 85% RH, 10°C with 80% RH, 15.0°C with 75% RH, 23.0°C with 70% RH, 31.0°C with 65% RH, 34.0°C with 65% RH, 36.0°C with 60% RH and 40.0°C with 55% RH revealed that the optimal condition for the development of these mites are 15.0–31.0°C and 65–75% RH. The highest temperature and the lowest RH accelerated the rate of development and induced more reproduction of O. mangiferus. Its population also multiplied 30.81 times in a generation time of 27.36 days at 31.0°C and 65% RH, while the same population only increased 7.46 times in a generation time of 48.07 days at 15.0°C and 75% RH. Fecundity was highest at 31.0°C and 65% RH with 46.43 eggs per female. The highest intrinsic rate of natural increase was observed at 31.0°C as 0.125 per day.     

Acclimation of photosynthesis, H2O2 content and antioxidants in maize (Zea mays) grown at sub-optimal temperatures

Maize plants were grown at 14, 18 and 20 °C until the fourth leaf had emerged. Leaves from plants grown at 14 and 18 °C had less chlorophyll than those grown at 20 °C. Maximal extractable ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity was decreased at 14 °C compared with 20 °C, but the activation state was highest at 14 °C. Growth at 14 °C increased the abundance (but not the number) of Rubisco breakdown products. Phosphoenolpyruvate carboxylase (PEPC) activity was decreased at 14 °C compared with 20 °C but no chilling-dependent effects on the abundance of the PEPC protein were observed. Maximal extractable NADP-malate dehydrogenase activity increased at 14 °C compared with 20 °C whereas the glutathione pool was similar in leaves from plants grown at both temperatures. Foliar ascorbate and hydrogen peroxide were increased at 14 °C compared with 20 °C. The foliar hydrogen peroxide content was independent of irradiance at both growth temperatures. Plants grown at 14 °C had decreased rates of CO₂ fixation together with decreased quantum efficiencies of photosystem (PS) II in the light, although there was no photo-inhibition. Growth at 14 °C decreased the abundance of the D1 protein of PSII and the PSI psaB gene product but the psaA gene product was largely unaffected by growth at low temperatures. The relationships between the photosystems and the co-ordinate regulation of electron transport and CO₂ assimilation were maintained in plants grown at 14 °C.