Local coupling of respiration processes and phosphorylation in rat liver mitochondria

One variant of the model of the local coupling of phosphorylation and respiration in intact mitochondria was experimentally verified. The model is based on the following postulates: (1). Upon the functioning of H+ pumps, hydrogen ions bound to the outer membrane surface do not enter the aqueous phase but are utilized for ATP synthesis in the membrane supercomplex respiratory H+ pump--ATP synthetase. (2). During the functioning of H+ pumps, an appreciable part of the energy of oxidation reactions can be stored in the form of the thermodynamic (solvation) potential of H+ ions bound to the outer membrane surface. According to the model, the hydration of hydrogen ions during the transition from the outer face of the inner membrane to the aqueous phase should lead to a decrease in the efficiency of the system of the coupling of respiration and phosphorylation. The model takes into account the ability of the nonpermeating buffer to catalyze the detachment of hydrogen ions from the membrane surface to the aqueous phase and provide their complete solvation. A preparation of phosphorylating mitochondria with the covalently bound pH probe was obtained. This made it possible to register for the first time the presence of a local H+ gradient on the outer side of the inner mitochondrial membrane during the stable functioning of the oxidative phosphorylation system. It was shown on these mitochondrial preparations that a decrease in the outer local H+ gradient by the action of increased concentrations of buffer is accompanied by a significant decrease in the ADP/O parameter and a partial dissociation of oxidative phosphorylation. Conditions were determined under which increased concentrations of buffer in the incubation medium cause a partial dissociation and a decrease in the ADP/O value from 20% to twofold (depending on the quality of mitochondrial preparations). The results obtained are in full agreement with the predictions of the model.     

Quantitative 2H NMR at natural abundance can distinguish the pathway used for glucose fermentation by lactic acid bacteria

For any given metabolic pathway, isotope redistribution coefficients (a(ij)) that characterize the specific derivation of each hydrogen atom can be defined. By using quantitative deuterium NMR, the redistribution of deuterium at natural abundance in lactic acid produced by the bacterial fermentation of glucose has been determined for each non-labile hydrogen atom of glucose or water and the hydrogen atoms of lactic acid. Distinct differences are observed in the lactic acid isolated from Lactococcus lactis and Leuconostoc mesenteroides that can be interpreted in terms of the different fermentative pathways used. Specifically, the affiliations observed between the H1, H3, and H4 positions of glucose with methyl and hydroxymethylene of lactic acid can give quantitative information on whether the glycolytic or the reductive pentose-phosphate pathway was involved in glucose catabolism.     

Optimization of the reaction medium for esterification catalyzed by A lipase from Pseudomonas fluorescens

The performance of a crude extract lipase from Pseudomonas fluorescensin esterification was evaluated in microaqueous, biphasic and surfactant-enriched biphasic systems containing various amounts of water (from almost no water to pure water). The results showed a strong negative influence of the water content on the thermodynamic equilibrium of the reaction in biphasic systems. From a kinetic point of view, the enzyme was more efficient in systems involving a water/organic solvent interface (4 times in the biphasic system, 12 times in the surfactant-enriched biphasic system).     

Inactivation of enterohemorrhagic Escherichia coli in rumen content- or feces-contaminated drinking water for cattle

Cattle drinking water is a source of on-farm Escherichia coli O157:H7 transmission. The antimicrobial activities of disinfectants to control E. coli O157:H7 in on-farm drinking water are frequently neutralized by the presence of rumen content and manure that generally contaminate the drinking water. Different chemical treatments, including lactic acid, acidic calcium sulfate, chlorine, chlorine dioxide, hydrogen peroxide, caprylic acid, ozone, butyric acid, sodium benzoate, and competing E. coli, were tested individually or in combination for inactivation of E. coli O157:H7 in the presence of rumen content. Chlorine (5 ppm), ozone (22 to 24 ppm at 5 degrees C), and competing E. coli treatment of water had minimal effects (<1 log CFU/ml reduction) on killing E. coli O157:H7 in the presence of rumen content at water-to-rumen content ratios of 50:1 (vol/wt) and lower. Four chemical-treatment combinations, including (i) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.05% caprylic acid (treatment A); (ii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.1% sodium benzoate (treatment B); (iii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.5% butyric acid (treatment C); and (iv) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 100 ppm chlorine dioxide (treatment D); were highly effective (>3 log CFU/ml reduction) at 21 degrees C in killing E. coli O157:H7, O26:H11, and O111:NM in water heavily contaminated with rumen content (10:1 water/rumen content ratio [vol/wt]) or feces (20:1 water/feces ratio [vol/wt]). Among them, treatments A, B, and C killed >5 log CFU E. coli O157:H7, O26:H11, and O111:NM/ml within 30 min in water containing rumen content or feces, whereas treatment D inactivated approximately 3 to 4 log CFU/ml under the same conditions. Cattle given water containing treatment A or C or untreated water (control) ad libitum for two 7-day periods drank 15.2, 13.8, and 30.3 liters/day, respectively, and cattle given water containing 0.1% lactic acid plus 0.9% acidic calcium sulfate (pH 2.1) drank 18.6 liters/day. The amounts of water consumed for all water treatments were significantly different from that for the control, but there were no significant differences among the water treatments. Such treatments may best be applied periodically to drinking water troughs and then flushed, rather than being added continuously, to avoid reduced water consumption by cattle.     

Effects of Temperature on Arachidonic Acid-Induced Cellular Edema and Membrane Perturbation in Rat Brain Cortical Slices

The effects of temperature on arachidonic acid-induced cellular edema in the first cortical brain slices of rats were studied. Incubation of the cortical slice in arachidonic acid at 37 degrees C induced cellular swelling, and increased intracellular Na+ and lactic acid contents concomitant with decreased intracellular K+. When the incubation temperature was reduced these changes were reduced in severity. The uptake of [3H]arachidonic acid in cortical slices was temperature-dependent. The incorporation of [3H]arachidonic acid into various lipid fractions was further studied by HPLC. The majority of [3H]arachidonic acid was incorporated into triacylglycerol and phosphatidylinositol (PI), but the incorporation of [3H]arachidonic acid into PI was temperature-dependent, unlike that into other phospholipids and neutrolipids. Further, cortical (Na+ + K+)-ATPase activity was inhibited whereas its subunit K+-activated p-nitrophenyl-phosphatase was activated by arachidonic acid at various incubation temperatures. The effects of arachidonic acid on these enzymes is similar to that of thimerosal, a lipid removal agent. These data suggest that both temperature and arachidonic acid play an important role in the development of cellular edema associated with membrane perturbation and inactivation of (Na+ + K+)-ATPase activity.     

Inactivation of Enterohemorrhagic Escherichia coli in Rumen Content- or Feces-Contaminated Drinking Water for Cattle

Cattle drinking water is a source of on-farm Escherichia coli O157:H7 transmission. The antimicrobial activities of disinfectants to control E. coli O157:H7 in on-farm drinking water are frequently neutralized by the presence of rumen content and manure that generally contaminate the drinking water. Different chemical treatments, including lactic acid, acidic calcium sulfate, chlorine, chlorine dioxide, hydrogen peroxide, caprylic acid, ozone, butyric acid, sodium benzoate, and competing E. coli, were tested individually or in combination for inactivation of E. coli O157:H7 in the presence of rumen content. Chlorine (5 ppm), ozone (22 to 24 ppm at 5°C), and competing E. coli treatment of water had minimal effects (<1 log CFU/ml reduction) on killing E. coli O157:H7 in the presence of rumen content at water-to-rumen content ratios of 50:1 (vol/wt) and lower. Four chemical-treatment combinations, including (i) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.05% caprylic acid (treatment A); (ii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.1% sodium benzoate (treatment B); (iii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.5% butyric acid (treatment C); and (iv) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 100 ppm chlorine dioxide (treatment D); were highly effective (>3 log CFU/ml reduction) at 21°C in killing E. coli O157:H7, O26:H11, and O111:NM in water heavily contaminated with rumen content (10:1 water/rumen content ratio [vol/wt]) or feces (20:1 water/feces ratio [vol/wt]). Among them, treatments A, B, and C killed >5 log CFU E. coli O157:H7, O26:H11, and O111:NM/ml within 30 min in water containing rumen content or feces, whereas treatment D inactivated approximately 3 to 4 log CFU/ml under the same conditions. Cattle given water containing treatment A or C or untreated water (control) ad libitum for two 7-day periods drank 15.2, 13.8, and 30.3 liters/day, respectively, and cattle given water containing 0.1% lactic acid plus 0.9% acidic calcium sulfate (pH 2.1) drank 18.6 liters/day. The amounts of water consumed for all water treatments were significantly different from that for the control, but there were no significant differences among the water treatments. Such treatments may best be applied periodically to drinking water troughs and then flushed, rather than being added continuously, to avoid reduced water consumption by cattle.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential Modulation by Divalent Cations of [3H]MK-801 Binding in Brain Synaptic Membranes

Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) to an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X-100 (Triton-treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone. In contrast, Zn2+ was invariably active as a potent inhibitor of the binding irrespective of the membrane preparations used. In untreated membranes, Ca2+ markedly accelerated the initial association rate of [3H]MK-801 binding without affecting the binding at equilibrium in a manner similar to that found with glycine, as well as with glutamate; Mg2+, however, facilitated the initial association rate with a concomitant reduction of the binding at equilibrium. Zn2+ was effective in accelerating the initial rapid phase of association, with the initial slow phase being delayed, and in markedly reducing the binding at equilibrium. Both Mg2+ and Ca2+ also facilitated dissociation of the bound [3H]MK-801 and Zn2+ slowed the dissociation in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pig heart lactate dehydrogenase. Binding of pyruvate and the interconversion of pyruvate-containing ternary complexes.

1. Lactate oxidation catalysed by pig heart lactate dehydrogenase was studied in the presence of inhibitory concentrations of pyruvate. Experimental results show the presence of an intermediate which occurs immediately after the hydride transfer step, but before the dissociation of pyruvate and the H+ produced by the reaction. The rate constant for pyruvate dissociation and the dissociation constant for pyruvate from the ternary complex differ from those obtained in pyruvate reduction experiments. 2.In single-turnover pyruvate reduction by pig heart lactate dehydrogenase at pH8.0 pyruvate can bind to the enzyme before a H+ is taken up, and the subsequent uptake of a H+ is governed by a step that is also rate-limiting for single-turnover and steady-state NADH oxidation. 3. Observation of various intermediates in the single-turnover pyruvate reduction experiments has made it possible to determine separately the dissociation constant and Km value for pyruvate at pH8.0, and also the catalytic turnover rate and Km for pyruvate under first-order conditions at different pH values. 4. Further studies on single-turnover pyruvate reduction carried out in 2H2O, or in water at low temperature, show another step which, under these conditions, is slower than that controlling H+ uptake and rate-limiting for NADH oxidation. A scheme is presented which explains these results.     

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.     

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nature of the nerve receptor for the acid taste as indicated by the absorption of organic acids by fats and proteins

Summary Distribution coefficients of thirteen organic acids between olive oil and water have been measured. The concentration in the water phase was about.02N. The acids were chosen so as to bring out the influence of certain polar groups, OH, CO, SO₃H, NH₂ and C=C. The effects of these groups are clearly marked, and are in general in the same direction and of the same order of magnitude as their effects on the concentration gradients of the same undissociated acids between water and the nerve receptor in the taste experiments ofTaylor, Farthing andBerman. Specific effects are noted for the NH₂ and the phenyl groups.Measurements have been made of the hydrogen ion concentration of gelatine to which these acids were added. A family of curves was obtained in which the arrangement is determined by the dissociation constant of the acid. The various polar groups affect the position of the curves only so far as they affect the dissociation constants. The results do not parallel the results of the experiments on taste. It is concluded that the nerve ending which the acid penetrates and stimulates to give the sensation of sourness is essentially like a fat (olive oil) rather than like a protein (gelatine) in character.Submitted for publication December 13, 1929.It is a pleasure to acknowledge the aid of MrLindsay Helmholz, a senior student in chemistry of the Johns Hopkins University. He has ably assisted in the experimental part of this investigation.     

Isolation and properties of a bacteriocin-producing Carnobacterium piscicola isolated from fish.

A facultative psychrotrophic lactic acid bacterium isolated from fresh fish was identified as Carnobacterium piscicola on the basis of carbohydrate utilization, G + C content and 16S RNA analysis. Its bacteriocin, designated carnocin UI49, is produced during the mid-exponential phase of growth at temperatures between 15 degrees C and 34 degrees C. Carnocin UI49 is active against a large number of closely-related lactic acid bacteria including carnobacteria, lactobacilli, pediococci and lactococci. Furthermore, the bacteriocin has a bactericidic mode of action which results in lysis of sensitive cells. Maximum bactericidal activity is observed at 34 degrees C with a decrease in activity down to 15 degrees C where it is completely abolished.     

Solid model compounds and the thermodynamics of protein unfolding.

Analysis of thermodynamic data on the dissolution of solid cyclic dipeptides into water in terms of group additivity provides a rationale for the enthalpy and entropy convergence temperatures observed for small globular protein denaturation and the dissolution of model compounds into water. Convergence temperatures are temperatures at which the extrapolated enthalpy or entropy changes for a series of related compounds take on a common value. At these temperatures (TH* and TS*) the apolar contributions to the corresponding thermodynamic values (delta H degrees and delta S degrees) are shown to be zero. Other contributions such as hydrogen bonding and configurational effects can then be evaluated and their quantitative effects on the stability of globular proteins assessed. It is shown that the denaturational heat capacity is composed of a large positive contribution from the exposure of apolar groups and a significant negative contribution from the exposure of polar groups in agreement with previous results. The large apolar contribution suggests that a liquid hydrocarbon model of the hydrophobic effect does not accurately represent the apolar contribution to delta H degrees of denaturation. Rather, significant enthalpic stabilizing contributions are found to arise from peptide groups (hydrogen bonding). Combining the average structural features of globular proteins (i.e. number of residues, fraction of buried apolar groups and fraction of hydrogen bonds) with their specific group contributions permits a first-order prediction of the thermodynamic properties of proteins. The predicted values compare well with literature values for cytochrome c, myoglobin, ribonuclease A and lysozyme. The major thermodynamic features are described by the number of peptide and apolar groups in a given protein.     

Stimulation of Lactic Acid Bacteria by a Micrococcus Isolate: Evidence for Multiple Effects

Growth of, and rate of acid production by, six cultures of lactic acid bacteria were increased in the presence of Micrococcus isolate F4 or a preparation of its capsular material. Concentrations of hydrogen peroxide found in pure cultures of the lactic acid bacteria were not detectable, or were greatly reduced, in mixed culture with Micrococcus isolate F4. The capsular material was not as effective as whole cells in preventing accumulation of H(2)O(2). Catalase stimulated growth of, and the rate of acid production by, the lactic acid bacteria, but not to the same extent as Micrococcus isolate F4 in some cultures. The existence of two mechanisms for micrococcal stimulation of the lactic acid bacteria is postulated. One mechanism involves removal of H(2)O(2); the other has not been characterized.     

Deuterons cannot replace protons in active transport processes in yeast

Replacement of ordinary water with heavy water causes a sharp reduction of the rates of both primary hydrogen ion transport (at the plasma membrane ATPase) and secondary symports (H(+)-associated transports of sugars and amino acids) in several species of yeast. At the same time, the hydrolytic activity of the ATPase is affected only very little. Likewise, the membrane potential, the delta pH and, correspondingly, the accumulation ratios of the various symported solutes are altered much less. This serves as evidence that H+ or H3O+ ions are direct participants in the various active transports of nutrients in yeast.     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

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.     

Interaction of aromatic donor molecules with manganese(III) reconstituted horseradish peroxidase: proton nuclear magnetic resonance and optical difference spectroscopic studies

The interaction of aromatic donor molecules with manganese(III) protoporphyrin-apohorseradish peroxidase complex [Mn(III)HRP] was investigated by optical difference spectroscopy and relaxation rate measurements of 1H resonances of aromatic donor molecules (at 500 MHz). pH dependence of substrate proton resonance line-widths indicated that the binding was facilitated by protonation of an amino acid residue (with a pKa of 6.1), which is presumably distal histidine. Dissociation constants were evaluated from both optical difference spectroscopy and 1H-NMR relaxation measurements (pH 6.1). The dissociation constants of aromatic donor molecules were not affected by the presence of excess of I-, CN- and SCN-. From competitive binding studies it was shown that all these aromatic donor molecules bind to Mn(III)HRP at the same site, which is different from the binding site of I-, CN- and SCN-. Comparison of the dissociation constants between the different substrates suggests that hydrogen bonding of the donors with distal histidyl amino acid and hydrophobic interaction between the donors and active site contribute significantly towards the associating forces. Free energy, entropy and enthalpy changes associated with the Mn(III)HRP-substrate equilibrium have been evaluated. These thermodynamic parameters were found to be all negative. Distances of the substrate protons from the paramagnetic manganese ion of Mn(III)HRP were found to be in the range of 7.7 to 9.4 A. The Kd values, the thermodynamic parameters and the distances of the bound aromatic donor protons from metal center in the case of Mn(III)HRP were found to be very similar as in the case of native Fe(III)HRP.     

A three-dimensional solubility parameter approach to nonaqueous enzymology

Widespread commercial application of enzymes as catalysts for specialty or commodity chemical synthesis will require their use in nonaqueous systems. While a number of non-aqueous enzyme applications have been demonstrated, the lack of useful rules for predicting enzyme-solvent interactions has hindered the development of this technology. Both Hildebrand and solvent hydrophobicity (octanol-water partition coefficient) parameters have been used previously to correlate and predict enzyme activity in nonaqueous systems, with some success, but any single-parameter approach is inherently limited in its ability to reflect the spectrum of possible enzyme-solvent interactions. Therefore, this study evaluates the three-dimensional solubility parameter space, as proposed by Hansen, to correlate and predict enzyme activity in microaqueous, miscible, and biphasic nonaqueous systems. Preliminary results suggest that Hansen parameters may be useful for correlating nonaqueous enzyme activity, and that the dispersive and polar parameters may be disproportionately important in single-phase microaqueous systems. The Hansen hydrogen-bonding parameter appears to be the only parameter yet evaluated capable of correlating the water requirement for enzyme activity in microaqueous systems, suggesting that water affects protein structure through enthalpic rather than entropic processes in nonaqueous systems. Insufficient data are available for miscible and biphasic systems, but it is proposed that enzyme activity may correlate with the average solubility parameters of miscible systems and of the aqueous phase in biphasic systems.