Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

Kinetics of selenite uptake by mononuclear cells from peripheral human blood

The present study deals with the kinetics and thermodynamics of the uptake of⁷⁵Se-labeled SeO ₃ ^2? from incubation media to lymphocytes cultivated from eight normal individuals (14–55 years of age, two females). The uptake of SeO ₃ ^2? was evaluated on the assumption of pseudo-first-order kinetics with regard to a reacting cellular receptor pool. On the basis of the experimental observations, it was assumed that the suggested pool of receptor molecules-symbolically represented by “£H₄”—reacts with SeO ₃ ^2? in the hypothetical reaction: $$\pounds H_4 + SeO_3^{2 - } + 2H^ + \underset{{ - k_1 }}{\overset{{k_1 }}{\longleftrightarrow}}\pounds Se + 3H_2 O$$ The mean value of the change in standard free energy at 25°C was calculated to be ΔG ^o=?141.6±1.3 kJ/mol, while the corresponding mean value of the free energy of activation at 25°C was calculated to be ΔG ²⁺=?7.8±0.9 kJ/mol for the forward reaction. The calculated values of the corresponding individual changes in the respective standard enthalpies and entropies were mutually interdependent for all eight donors. ΔH ^o=?152+315ΔS ^o(kJ/mol) corresponding to the common value ΔG ^o??152 kJ/mol at 315°K. These mutual interdependencies are possibly the effect of variable conformational states (e.g., the macromolecular compactness) of the cellular receptor pools. This suggestion may furthermore be supported by the correlation traced between ΔH ^o vs the biological age in years of the donors: △H ^°?76.7?1.0 (age)kJ/mol (r = ?0.92) The calculated values of activation enthalpy ΔH ²⁺ kJ/mol and activation entropy ΔS ²⁺ (kJ/mol K) also mutually correlated linearly (r=0.998); the regression line was: △H ²⁺ = ?8.9 + 305△S²⁺ (kJ/mol) corresponding to the common value △H ²⁺ △ ?8.9 (kJ/mol) at 305°K Similarly the activation enthalpy ΔH ²⁺ vs the biological age in years correlated linearly: ΔH ²⁺=67.4?0.73(age) (kJ/mol) (r=?0.76) The range of ΔH ²⁺ studied was from 13.8 to 53.9 kJ/mol with a linearly corresponding range in ΔS ²⁺ from 73 to 205 J/mol K. The thermodynamic data reveal the selenite uptake during the hypothetical standard reaction to be exergonic and endothermic. Critical pH dependencies of the selenite uptake were explained.     

Thermodynamics of the B to Z transition in poly(dGdC)

The thermodynamics of the B to Z transition in poly(dGdC) was examined by differential scanning calorimetry, temperature-dependent absorbance spectroscopy, and CD spectroscopy. In a buffer containing 1 mM Na cacodylate, 1 mM MgCl₂, pH 6.3, the B to Z transition is centered at 76.4°C, and is characterized by ΔH_cal = 2.02 kcal (mol base pair)^?1 and a cooperative unit of 150 base pairs (bp). The t_m of this transition is independent of both polynucleotide and Mg²⁺ concentrations. A second transition, with ΔH_cal = 2.90 cal (mol bp)^?1, follows the B to Z conversion, the t_m of which is dependent upon both the polynucleotide and the Mg²⁺ concentrations. Turbidity changes are concomitant with the second transition, indicative of DNA aggregation. CD spectra recorded at a temperature above the second transition are similar to those reported for ψ(–)-DNA. Both the B to Z transition and the aggregation reaction are fully and rapidly reversible in calorimetric experiments. The helix to coil transition under these solution conditions is centered at 126°C, and is characterized by ΔH_cal = 12.4 kcal (mol bp)^?1 and a cooperative unit of 290 bp. In 5 mM MgCl₂, a single transition is seen centered at 75.5°C, characterized by ΔH_cal = 2.82 kcal (mol bp)^?1 and a cooperative unit of 430 bp. This transition is not readily reversible in calorimetric experiments. Changes in turbidity are coincident with the transition, and CD spectra at a temperature just above the transition are characteristic of ψ(–)-DNA. A transition at 124.9°C is seen under these solution conditions, with ΔH_cal = 10.0 kcal (mol bp)^?1 and which requires a complex three-step reaction mechanism to approximate the experimental excess heat capacity curve. Our results provide a direct measure of the thermodynamics of the B to Z transition, and indicate that Z-DNA is an intermediate in the formation of the ψ-(–) aggregate under these solution conditions.     

The Gibbs Free Energy Threshold for the Invasion of a Microbial Population Under Kinetic Constraints

Possible chemotrophic metabolism at a site of interest is controlled not only by the catabolic energy expressed as the Gibbs energy of reaction (Δ_rG) but also by the kinetic constraints due to the availability of electron acceptors and donors. We introduced graphical and stochastic approaches for determining the Δ_rG threshold required to support a microbial population with a specific catabolic strategy under kinetic constraints. Invasibility as an indicator of the present reproductive ability of a microbial population was evaluated by simultaneously calculating Δ_rG for the catabolic reaction and the microbial catalytic rate. For example, the neutrophilic iron-oxidizing bacteria's invasibility was calculated by randomly choosing the Fe²⁺ and O₂ concentrations between 10^?8 and 10^?2 mol L^?1, and pH between 4 and 8, to determine the Δ_rG threshold for invasion. Parameters were estimated from batch experiments of neutrophilic iron-oxidizing bacteria reported in previous studies. Under the given conditions, the stochastic approach predicted that the neutrophilic iron-oxidizing bacteria can always invade a system in which the Δ_rG for Fe oxidation is below ?90 kJ mol Fe^?1, can occasionally invade if Δ_rG is between ?45 and ?90 kJ mol Fe^?1, and can never invade if Δ_rG is above ?45 kJ mol Fe^?1. The Δ_rG threshold for invasion is sifted by the growth yield coefficient, the loss rate of cells, the maximum cell-specific Fe oxidation rate constant, and the temperature. The Δ_rG threshold for invasion may be unable to rigorously predict the stable dominance of microbial metabolism, but can provide a rough indication for the possible microbial metabolism under current conditions.     

Interaction of iron nanoparticles with nervous system: an in vitro study

Nanoparticles (NPs) are one of the interesting and widely studying issues mainly because of their particular physico-chemical features and broad applications in the field of biomedical sciences, such as diagnosis and drug delivery. In this study, the interaction of iron nanoparticles (Fe–NPs) with Tau protein and PC12 cell, as potential nervous system models, was investigated with a range of techniques including dynamic light scattering, intrinsic fluorescence spectroscopy, circular dichroism, [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromid] assay, and acridine orange/ethidium bromide (AO/EB) dual staining method. An inverse correlation between Stern and Volmer constant (K_SV) and temperature indicated a probable static quenching mechanism occurred between Tau protein and Fe–NPs. The number of binding site (n = 0.86) showed that there is almost one binding site of Fe–NP per protein. The negative values of ?H (?53.21 kJ/mol) and T?S (?42.44 kJ/mol) revealed that Fe–NPs interacts with Tau protein with dominate role of hydrogen bonds and van der Waals interactions and this interaction was spontaneous (?G = ?10.77 kJ/mol). Also, Fe–NPs stabilized the random coil structure of Tau protein. Moreover, Fe–NPs reduced PC12 cells viability by fragmentation of DNA in an apoptotic manner. In conclusion, induced conformational changes of Tau protein and cytotoxicity of PC12 cells by Fe–NP were revealed to be in a concentration and time-dependent manner.     

Irradiance optimization of outdoor microalgal cultures using solar tracked photobioreactors

Photosynthetic activity and temperature regulation of microalgal cultures (Chlorella vulgaris and Scenedesmus obliquus) under different irradiances controlled by a solar tracker and different cell densities were studied in outdoor flat panel photobioreactors. An automated process control unit regulated light and temperature as well as pH value and nutrient concentration in the culture medium. CO₂ was supplied using flue gas from an attached combined block heat and power station. Photosynthetic activity was determined by pulse amplitude modulation fluorometry. Compared to the horizontal irradiance of 55 mol photons m^?2 d^?1 on a clear day, the solar tracked photobioreactors enabled a decrease and increase in the overall light absorption from 19 mol photons m^?2 d^?1 (by rotation out of direct irradiance) to 79 mol photons m^?2 d^?1 (following the position of the sun). At biomass concentrations below 1.1 g cell dry weight (CDW) L^?1, photoinhibition of about 35 % occurred at irradiances of ≥1,000 μmol photons m^?2 s^?1 photosynthetic active radiation (PAR). Using solar tracked photobioreactors, photoinhibition can be reduced and at optimum biomass concentration (≥2.3 g CDW L^?1), the culture was irradiated up to 2,000 μmol photons m^?2 s^?1 to overcome light limitation with biomass yields of 0.7 g CDW mol photons^?1 and high photosynthetic activities indicated by an effective quantum yield of 0.68 and a maximum quantum yield of 0.80 (F _v/F _m). Overheating due to high irradiance was avoided by turning the PBR out of the sun or using a cooling system, which maintained the temperature close to the species-specific temperature optima.     

Molecular dynamics simulation study of probe diffusion in liquid n-alkanes

We performed molecular dynamics simulations for the probe diffusion and friction dynamics of Lennard-Jones (LJ) particles modelled for methyl yellow (MY) in liquid n-alkanes of C₁₂–C₂₀₀ at temperatures of 318, 418, 518 and 618 K. Two LJ particles are chosen: MY1 with a mass of 114 g/mol, LJ parameters of σ = 4.0 Å and ? = 0.4 kJ/mol, and MY2 with a mass of 225 g/mol, σ = 6.0 Å and ? = 0.6 kJ/mol. We observed a clear transition in the power law dependence of MY2 diffusion on the molecular weight of n-alkanes at lower temperatures of 318 and 418 K. The sharp transitions occur near n-dotriacontane (C₃₂). However, no such transition is found for MY1 at all the temperatures and for MY2 at higher temperatures of 518 and 618 K. We also calculated the friction constants of both MY probe molecules in liquid n-alkanes. For the larger probe molecule (MY2), at lower temperatures, a large deviation of slope from the linear dependence of the friction of MY2 on the chain length of n-alkane is observed, which indicates a large reduction of friction in longer chains when compared with the shorter chains, enhancing the diffusion of the probe molecules (MY2).     

Two-Probe Microfluorometry Estimation of Transmembrane Potential (ΔΨ p) Slight Changes in Individual Hepatocytes Under Short-Term Hormone Action

Early events in individual hepatocytes of rat activated by adrenaline (10^?6M) and phenylephrine (10^?5M) have been investigated by quantitative image microfluorometry and microspectrofluorometry. Cationic DiOC₂ and anionic SqSC₄ probes have been used for image analysis and transmembrane potential (ΔΨ _p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. Based on the mean fluorescence intensity, the magnitude of ΔΨ _p was calculated by Nernst equation adapted for lipophilic cationic probes. DiOC₂ has revealed that both hormones induce biphasic hyperpolarization of hepatocytes membrane with α-agonist phenylephrine causing ΔΨ _p changes at higher amplitude. The first increase of ΔΨ _p within 2 and 5 min (ΔΔΨ _p = ?8.6 ± 4.2 mV) apparently related to Na⁺/K⁺-ATPase activation by the Ca²⁺-mobilizing hormone. The second peak of hyperpolarization (ΔΔΨ _p = ?13.2 ± 3.2 mV) between 25 and 30 min, after a transient decrease of ΔΨ _p (ΔΔΨ _p = 10.9 ± 4.3 mV) over 15 min experiment, probably is mediated by phenylephrine stimulating action on K⁺-channels. K⁺ channel blocker (Ba²⁺ or 4-aminopyridine) as well as elevating of extracellular K⁺ prevented the hyperpolarization. Modulation of PLD-dependent signal transduction pathway by 0.4 % butanol had a weak influence on the first increase of ΔΨ _p but it abolished the second phase of hyperpolarization. That points to PLD involvement in the ΔΨ _p fluctuations mediated by K⁺-channels in response to phenylephrine. Based on SqSC₄, fluorescent parameters estimation of relative changes of ΔΨ _p revealed similar character of time dependence with two phases of hyperpolarization. Synchronic fluctuation of ΔΨ _p determined by oppositely charged probes demonstrate that the quantitative microfluorometry allows to evaluate slight ΔΨ _p changes separately from ΔΨ _m in non-excitable individual cells at the short-term hormone action.     

Effects of saline root environment (NaCl) on nitrate and potassium uptake kinetics for rose plants: a Michaelis–Menten modelling approach

Greenhouse-grown cut flower roses are often irrigated with moderately saline irrigation water. The salt/ballast ions are either present initially in poor quality raw water or reclaimed municipal water, or accumulated in greenhouse irrigation water that is captured and reused. Such ions can inhibit root absorption of essential nutrients. The objective of this work was to quantify the influence of NaCl concentration on the uptake of nitrate and potassium by roses and develop a predictive model of uptake inhibition based on NaCl, NO₃ ^?, and K⁺ concentration. One year-old rose plants (Rosa spp. ‘Kardinal’ on ‘Natal Briar’ rootstock) were moved into growth chambers where nitrogen and potassium depletion were monitored during 6 days. Eight different initial NaCl treatments varying from zero to 65 mol m^?3 were used and within these there were two initial NO₃ ^? and K⁺ concentrations: high concentration (HC, 7.0 mol m^?3 and 2.6 mol m^?3 NO₃ ^? and K⁺ respectively) or low concentration (LC, 3.5 mol m^?3 and 1.3 mol m^?3 NO₃ ^? and K⁺ respectively). Plant NO₃ ^? uptake was negatively affected by NaCl concentration. NO₃ ^? maximum influx (I_max) declined from 5.1 µmol to 2.5 µmol per gram of plant dry weight per hour as NaCl concentration increased from zero to 65 mol m^?3. A modified Michaelis–Menten (M–M) equation taking into account inhibition by NaCl provided the best fit for NO₃ ^? uptake in response to varying NaCl concentration. K⁺ uptake was unaffected by NaCl concentration. A M–M equation that did not include inhibition was suitable for describing K⁺ uptake at varying NaCl concentration. The resulting empirical models could assist with decision making, such as: adjustment of NO₃ ^? fertilization based on NaCl concentration, necessity of water desalinization, or determination of the desired leaching fraction.     

Energetics of Acidianus ambivalens growth in response to oxygen availability

All life requires energy to drive metabolic reactions such as growth and cell maintenance; therefore, fluctuations in energy availability can alter microbial activity. There is a gap in our knowledge concerning how energy availability affects the growth of extreme chemolithoautotrophs. Toward this end, we investigated the growth of thermoacidophile Acidianus ambivalens during sulfur oxidation under aerobic to microaerophilic conditions. Calorimetry was used to measure enthalpy (ΔH_inc) of microbial activity, and chemical changes in growth media were measured to calculate Gibbs energy change (ΔG_inc) during incubation. In all experiments, Gibbs energy was primarily dissipated through the release of heat, which suggests enthalpy‐driven growth. In microaerophilic conditions, growth was significantly more efficient in terms of biomass yield (defined as C‐mol biomass per mole sulfur consumed) and resulted in lower ΔG_inc and ΔH_inc. ΔG_inc in oxygen‐limited (OL) and oxygen‐ and CO₂‐limited (OCL) microaerophilic growth conditions resulted in averages of ?1.44 × 10³ kJ/C‐mol and ?7.56 × 10² kJ/C‐mol, respectively, and average ΔH_inc values of ?1.11 × 10⁵ kJ/C‐mol and ?4.43 × 10⁴ kJ/C‐mol, respectively. High‐oxygen experiments resulted in lower biomass yield values, an increase in ΔG_inc to ?1.71 × 10⁴ kJ/C‐mol, and more exothermic ΔH_inc values of ?4.71 × 10⁵ kJ/C‐mol. The observed inefficiency in high‐oxygen conditions may suggest larger maintenance energy demands due to oxidative stresses and a preference for growth in microaerophilic environments.     

Triple helix–coil transition of covalently bridged collagenlike peptides

The thermal triple helix–coil transition of covalently bridged collagenlike peptides with repeating sequences of (Ala-Gly-Pro)_n, n = 5–15, was studied optically. The peptides were soluble in water/acetic acid (99:1) and were found to form triple-helical structures in this solvent system beginning with n = 8. The thermodynamic analysis of the transition equilibrium curves for n = 9–13 yielded the parameters ΔH°_s = ?7.0 kJ per tripeptide unit, ΔS°_s = ?23.1 J deg^?1 mol^?1 per tripeptide unit for the coil-to-helix transition, and the apparent nucleation parameter σ ? 5 × 10^?2. It was suggested through double-jump temperature experiments that the rate-limiting step during refolding is not only influenced by the difficulties of nucleation, but also by cis–trans isomerization of the Gly-Pro peptide bond.     

Partial Purification and Characterization of Chromate Reductase of a Novel Ochrobactrum sp. Strain Cr-B4

Hexavalent chromium contamination is a serious problem due to its high toxicity and carcinogenic effects on the biological systems. The enzymatic reduction of toxic Cr(VI) to the less toxic Cr(III) is an efficient technology for detoxification of Cr(VI)-contaminated industrial effluents. In this regard, a chromate reductase enzyme from a novel Ochrobactrum sp. strain Cr-B4, having the ability to detoxify Cr(VI) contaminated sites, has been partially purified and characterized. The molecular mass of this chromate reductase was found to be 31.53 kD, with a specific activity 14.26 U/mg without any addition of electron donors. The temperature and pH optima for chromate reductase activity were 40°C and 8.0, respectively. The activation energy (E_a) for the chromate reductase was found to be 34.7 kJ/mol up to 40°C and the activation energy for its deactivation (E_d) was found to be 79.6 kJ/mol over a temperature range of 50–80°C. The frequency factor for activation of chromate reductase was found to be 566.79 s^?1, and for deactivation of chromate reductase it was found to be 265.66 × 10³ s^?1. The reductase activity of this enzyme was affected by the presence of various heavy metals and complexing agents, some of which (ethylenediamine tetraacetic acid [EDTA], mercaptoethanol, NaN₃, Pb²⁺, Ni²⁺, Zn²⁺, and Cd²⁺) inhibited the enzyme activity, while metals like Cu²⁺ and Fe³⁺ significantly enhanced the reductase activity. The enzyme followed Michaelis–Menten kinetics with K_m of 104.29 µM and a V_max of 4.64 µM/min/mg.     

Physicochemical Aspects of Chitosan Dispersibility in Acidic Aqueous Media: Effects of the Food Acid Counter-Anion

Differences in formation of colloidal dispersions of chitosan in aqueous solutions of citric acid or lactic acid (25, 50 or 100 mM) were quantitatively studied. Protonation enthalpies, electrical conductivity and ζ-potential measurements were additionally undertaken, aiming at better understanding these differences at a molecular level. In dispersion kinetics assays, experimental data were well fitted (R²?>?0.9; MAPE?<?4 %) by a first-order kinetics model with two terms - one accounting for the fast, direct dispersion of biopolymers chains and another accounting for the slow dispersion of chains from lumps. In all cases, maximal dispersibility was reached after about 20?30 min of stirring. For both acids, the higher the acid concentration in the medium, the higher was the chitosan dispersibility. At a given acid concentration, chitosan showed higher dispersibility in lactic acid than in citric acid solutions. Protonation of chitosan -NH₂ groups was strongly exothermic, with ΔH values three times higher for citric acid (triprotic) than lactic acid (monoprotic) (ΔH?=??120 kJ?mol^{- 1} and ΔH?=??40 kJ?mol^{- 1}, respectively), indicating that chitosan -NH₂ protonation itself was not dependent on the type of acid. However, the electrical conductivity of suspensions of powdered chitosan in water evolved differently as these systems were titrated with citric or acid lactic. With citric acid, electrical conductivity remained virtually constant for acid concentration?<?of 15 mM, and then increased linearly as the acid concentration increased until 75 mM. Instead, with lactic acid, electrical conductivity progressively increased with increasing of acid concentration from 0 to 75 mM. The ζ-potential of chitosan dispersed particles was +28.5 mV and +52.1 mV in dispersions containing 10 mM of citric and lactic acids, respectively. The conjoint analysis of data from physicochemical analyses suggested that, contrarily to lactate anions, citrate anions bind more strongly on the electrical double layer of protonated, positively charged chains of chitosan, diminishing the inter-chains electrostatic repulsion, thus leading to a lower dispersibility of this polysaccharide in aqueous solutions of citric acid, compared to equimolar solutions of lactic acid.     

Stable isotope fractionation of strontium in coccolithophore calcite: Influence of temperature and carbonate chemistry

Marine calcifying eukaryotic phytoplankton (coccolithophores) is a major contributor to the pelagic production of CaCO₃ and plays an important role in the biogeochemical cycles of C, Ca and other divalent cations present in the crystal structure of calcite. The geochemical signature of coccolithophore calcite is used as palaeoproxy to reconstruct past environmental conditions and to understand the underlying physiological mechanisms (vital effects) and precipitation kinetics. Here, we present the stable Sr isotope fractionation between seawater and calcite (Δ^88/86Sr) of laboratory cultured coccolithophores in individual dependence of temperature and seawater carbonate chemistry. Coccolithophores were cultured within a temperature and a pCO₂ range from 10 to 25°C and from 175 to 1,240 μatm, respectively. Both environmental drivers induced a significant linear increase in coccolith stable Sr isotope fractionation. The temperature correlation at constant pCO₂ for Emiliania huxleyi and Coccolithus braarudii is expressed as Δ^88/86Sr = ?7.611 × 10^?3 T + 0.0061. The relation of Δ^88/86Sr to pCO₂ was tested in Emiliania huxleyi at 10 and 20°C and resulted in Δ^88/86Sr = ?5.394 × 10^?5 pCO₂ – 0.0920 and Δ^88/86Sr = ?5.742 × 10^?5 pCO₂ – 0.1351, respectively. No consistent relationship was found between coccolith Δ^88/86Sr and cellular physiology impeding a direct application of fossil coccolith Δ^88/86Sr as coccolithophore productivity proxy. An overall significant correlation was detected between the elemental distribution coefficient (D_Sr) and Δ^88/86Sr similar to inorganic calcite with a physiologically induced offset. Our observations indicate (i) that temperature and pCO₂ induce specific effects on coccolith Δ^88/86Sr values and (ii) that strontium elemental ratios and stable isotope fractionation are mainly controlled by precipitation kinetics when embedded into the crystal lattice and subject to vital effects during the transmembrane transport from seawater to the site of calcification. These results provide an important step to develop a coccolith Δ^88/86Sr palaeoproxy complementing the existing toolbox of palaeoceanography.     

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10^?7 cm² s^?1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (?5.78 kcal mol^?1) and positive ΔS (11.01 cal mol^?1 deg^?1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (?9.19 kcal mol^?1) value implies a spontaneous CBC-papain interaction.     

Purification,kinetic and thermodynamic characterization of soluble acid invertase from sugarcane (Saccharum officinarum L.)

We report for the first time kinetic and thermodynamic properties of soluble acid invertase (SAI) of sugarcane (Saccharum officinarum L.) salt sensitive local cultivar CP 77-400 (CP-77). The SAI was purified to apparent homogeneity on FPLC system. The crude enzyme was about 13 fold purified and recovery of SAI was 35%. The invertase was monomeric in nature and its native molecular mass on gel filtration and subunit mass on SDS-PAGE was 28 kDa. SAI was highly acidic having an optimum pH lower than 2. The acidic limb was missing. Proton transfer (donation and receiving) during catalysis was controlled by the basic limb having a pKa of 2.4. Carboxyl groups were involved in proton transfer during catalysis. The kinetic constants for sucrose hydrolysis by SAI were determined to be: k_m = 55 mg ml^?1, k_cat = 21 s^?1, k_cat/k_m = 0.38, while the thermodynamic parameters were: ΔH* = 52.6 kJ mol^?1, ΔG* = 71.2 kJ mol^?1, ΔS* = ?57 J mol^?1 K^?1, ΔG*_E–S = 10.8 kJ mol^?1 and ΔG*_E–T = 2.6 kJ mol^?1. The kinetics and thermodynamics of irreversible thermal denaturation at various temperatures 53–63 °C were also determined. The half -life of SAI at 53 and 63 °C was 112 and 10 min, respectively. At 55 °C, surprisingly the half -life increased to twice that at 53 °C. ΔG*, ΔH* and ΔS* of irreversible thermal stability of SAI at 55 °C were 107.7 kJ mol^?1, 276.04 kJ mol^?1 and 513 J mol^?1K^?1, respectively.     

Dynamics of molecular organization in agarose sulphate

Nongelling solutions of structurally regular chain segments of agarose sulphate show disorder–order and order–disorder transitions (as monitored by the temperature dependence of optical rotation) that are closely similar to the conformational changes that accompany the sol–gel and gel–sol transitions of the unsegmented polymer. The transition midpoint temperature (T_m) for formation of the ordered structure on cooling is ～25 K lower than T_m for melting. Salt-induced conformational ordering, monitored by polarimetric stopped-flow, occurs on a millisecond time scale, and follows the dynamics expected for the process 2 coil ? helix. The equilibrium constant for helix growth (s) was calculated as a function of temperature from the calorimetric enthalpy change for helix formation (ΔH_cal = ?3.0 ± 0.3 kJ per mole of disaccharide pairs in the ordered state), measured by differential scanning calorimetry. The temperature dependence of the nucleation rate constant (k_nuc), calculated from the observed second-order rate constant (k_obs) by the relationship k_obs = k_nuc(1 ? 1/s) gave the following activation parameters for nucleation of the ordered structure of agarose sulphate (1 mg mL^?1; 0.5M Me₄NCl or KCl): ΔH* = 112 ± 5 kJ mol^?1; ΔS* = 262 ± 20 J mol^?1 K^?1; ΔG*₂₉₈ = 34 ± 6 kJ mol^?1; (k_nuc)₂₉₈ = (7.5 ± 0.5) × 10⁶ dm³ mol^?1 s^?1. The endpoint of the fast relaxation process corresponds to the metastable optical rotation values observed on cooling from the fully disordered form. Subsequent slow relaxation to the true equilibrium values (i.e., coincident with those observed on heating from the fully ordered state) was monitored by conventional optical rotation measurements over several weeks and follows second-order kinetics, with rate constants of (2.25 ± 0.07) × 10^?4 and (3.10 ± 0.10) × 10^?4 dm³ mol^?1 s^?1 at 293.7 and 296.2 K, respectively. This relaxation is attributed to the sequential aggregation processes helix + helix → dimer, helix + dimer → trimer, etc., with depletion of isolated helix driving the much faster coil–helix equilibrium to completion. Light-scattering measurements above and below the temperature range of the conformational transitions indicate an average aggregate size of 2–3 helices.     

Helix-coil dynamics of a Z-helix hairpin

The helix–coil transition of a Z-helix hairpin formed from d(C-G)₅T₄(C-G)₅ has been characterized by equilibrium melting and temperature jump experiments in 5M NaClO₄ and 10 mM Na₂HPO₄, pH 7.0. The melting curve can be represented by a simple all-or-none transition with a midpoint at 81.6 ± 0.4°C and an enthalpy change of 287 ± 15 kJ/mole. The temperature jump relaxation can be described by single exponentials at a reasonable accuracy. Amplitudes measured as a function of temperature provide equilibrium parameters consistent with those derived from equilibrium melting curves. The rate constants of Z-helix formation are found in the range from 1800 s^?1 at 70°C to 800 s^?1 at 90°C and are associated with an activation enthalpy of ?(50 ± 10) kJ/mole, whereas the rate constants of helix dissociation are found in the range from 200 s^?1 at 70°C to 4500 s^?1 at 90°C with an activation enthalpy +235 kJ/mole. These parameters are consistent with a requirement of 3–4 base pairs for helix nucleation. Apparently nucleation occurs in the Z-helix conformation, because a separate slow step corresponding to a B to Z transition has not been observed. In summary, the dynamics of the Z-helix–coil transition is very similar to that of previously investigated right-handed double helices.     

Reactions of ruthenium(II) para-substituted tetraphenylporphyrin complexes with carbon monoxide oxygen: A kinetic investigation

The reaction of Ru(XTPP)(DMF)₂, where XTPP is the dianion of para substituted tetraphenylporphyrins and X is MeO, Me, H, Cl, Br, I, F, with O₂ and CO were studied in DMF. The process was found to be first-order in metalloporphyrin, first-order in molecular oxygen and carbon monoxide, and second-order overall. Second-order rate constants for the CO reaction ranged from 0.170 to 0.665 M^?1 s^?1 at 25°C, those for the O₂ reaction from 0.132 to 0.840 M^?1 s^?1 at 25°C. Similar activation parameters (ΔH_CO^± = 87 ± 1 kJ mol^?1, ΔS_CO^± = 22 ± 4 JK^?1 mol^?1; ΔH_O2^± = 81 ± 1 kJ mol^?1, and ΔS_O2^± = 11 ± 5 JK^?1 mol^?1) were found within each series. Reactivities of X substituted metalloporphyrins were found to follow different Hammett σ functions. The CO reactions correlated with σ_? following normal behavior; the O₂ reactions correlated with σ₈° indicating O₂ is π-bonded in the transition states. A dissociative mechanism is postulated for the process.