The reaction mechanism of Photosystem I reduction by plastocyanin and cytochrome c6 follows two different kinetic models in the cyanobacterium Pseudanabaena sp. PCC 6903

Plastocyanin (Pc) and cytochrome c₆ (Cyt) have been purified to homogeneity from the cyanobacterium Pseudanabaena sp. PCC 6903, which occupies a unique divergent branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. The two metalloproteins have similar molecular masses (9–10 kDa), as well as almost identical isoelectric points (ca. 8) and midpoint redox potentials (ca. 350 mV, at pH 7). Their reaction mechanism of electron transfer to Photosystem I (PS I) has been analyzed by laser-flash absorption spectroscopy. The kinetic traces with Pc correspond to monophasic kinetics, whereas those with Cyt are better fitted to biphasic curves. The observed pseudo first-order rate constant (k_obs) with Pc and that for the slower phase with Cyt exhibit saturation profiles at increasing donor protein concentrations, thereby suggesting that the two metalloproteins are able to form transient complexes with PS I. The ionic strength dependence of the rate constants for complex formation makes evident the electrostatic nature of intermediate complexes. The experimental findings indicate that the PS I reduction kinetics in Pseudanabaena follow a type II mechanism with Pc and a type III mechanism with Cyt, according to the different kinetic models proposed previously [(Hervás M, Navarro JA, Díaz A, Bottin H and De la Rosa MA (1995) Biochemistry 34: 11321–11326)]. From an evolutionary point of view, this reinforces our previous observation that PS I was first adapted to operate efficiently with positively charged Cyt rather than with Pc.     

Pressure and temperature effects on human red cell cation transport

Summary The effects of hydrostatic pressure and temperature on the three components of K⁺ uptake in human red cells have been investigated, using ouabain and bumetanide to distinguish between the pump, passive diffusion and cotransport. The pressure sensitivity for passive diffusion has been shown to depend on the counter-ion present. The order of this effect, Cl^–>Br^–>NO ₃ ^– >I^–, is the same as for the ionic partial modal volumes and the Hofmeister series. We have analyzed our experimental results thermodynamically, and propose a model for the activated transition-state complex of the potassium ion which involves the loss of water molecules from the secondary hydration shell, cosphere II.     

Fluorescence polarization studies of different forms of angiotensin-converting enzyme

The interaction of three forms of bovine angiotensin-converting enzyme (ACE) with the competitive peptide inhibitor lisinopril with a fluorescent label was studied by the fluorescence polarization technique. The dissociation constants K _d of the enzyme-inhibitor complexes in 50 mM Hepes-buffer, pH 7.5, containing 150 mM NaCl and 1 M ZnCl₂ at 37°C were (2.3 ± 0.4)·10^–8, (2.1 ± 0.3)·10^–8, and (2.1 ± 0.2)·10^–8 M for two-domain somatic ACE, single-domain testicular ACE, and for the N-domain of the enzyme, respectively. The interaction of the enzyme with the inhibitor strongly depended on the presence of chloride in the medium, and the apparent dissociation constant of the ACE-chloride complex was (1.3 ± 0.2)·10^–3 M for the somatic enzyme. The dissociation kinetics of the complex of the inhibitor with somatic ACE did not fit the kinetics of a first-order reaction, but it was approximated by a model of simultaneous dissociation of two complexes with the dissociation rate constants (0.13 ± 0.01) sec^–1 and (0.026 ± 0.001) sec^–1 that were present at approximately equal initial concentrations. The dissociation kinetics of the single-domain ACE complexes with the inhibitor were apparently first-order, and the dissociation rate constants were similar: (0.055 ± 0.001) and (0.041 ± 0.001) sec^–1 for the N-domain and for testicular ACE, respectively.     

Biosorption of nonylphenol on dead biomass of Rhizopus arrhizus encapsulated in chitosan beads

The nonylphenol (NP) biosorption and desorption potential for fungal biomass used under batch conditions was investigated using kinetics and isotherm models. Fungal biomass of Rhizopus arrhizus TISTR 3610 exhibited preferential uptake of NP, an endocrine disrupting chemicals. Sporangiospores, asexual spores, were immobilised in chitosan beads. The biosorption data of NP on the moist heat inactivated R. arrhizus–chitosan beads were analyzed using four popular adsorption isotherms and, by using non-linear least-regression with the solver add-in in Microsoft Excel, correlated in order with the Fritz–Schluender > Redlich–Peterson > Freundlich > Langmuir isotherms. The pseudo first-order kinetics was found to have the best fit with the experimental data. The diffusivity of NP in the R. arrhizus–chitosan beads was calculated using the shrinking core model, and the diffusivity values were in the ranges of 2.3736 × 10⁻⁴–1.8950 × 10⁻⁴ cm² s⁻¹. Desorption to recover the adsorbed NP from the beads was performed in methanol and was best described using a pseudo second-order kinetic model.     

Size selectivity of aqueous pores in astomatous cuticular membranes isolated from<Emphasis Type="Italic"> Populus canescens</Emphasis> (Aiton) Sm. leaves

Little is known about the permeability of plant cuticles to ionic molecules with hydration shells that render them lipid insoluble and limit their diffusion to narrow aqueous pores. Therefore, the permeation of cuticular membranes to ionised calcium salts with anhydrous molecular weights ranging from 111 to 755 g mol^–1 was studied. Penetration was a first-order process and rate constants (k) (proportional to permeability) decreased exponentially with molecular weight. Plots of log k vs. molecular weight had slopes of –2.11×10^–3 and –2.80×10^–3, respectively, depending on the year in which the cuticular membranes were isolated. This corresponds to decreases in permeability by factors of about 7 to 13 when molecular weight increased from 100 to 500 g mol^–1. This size selectivity is small compared to the dependence on molecular weight of solute mobility in Populus cuticles. A decrease in mobility of neutral molecules by more than 3 orders of magnitude has been reported [A. Buchholz et al. (1998) Planta 206:322–328] for the same range of molecular weights. Hence, discrimination of large ionic species diffusing in aqueous pores (polar pathway) is much smaller than that for neutral solutes diffusing in cutin and waxes (lipophilic pathway). This indicates that formulating large solutes as ionic species would be advantageous.Abbreviation CM Cuticular membrane     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Reconstituted Cl− pump protein: A novel ion(Cl−)-motive ATPase

Cl^– absorption by theAplysia californica foregut is effected through an active Cl^– transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl^–-stimulated ATPase and ATP-dependent Cl^– transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg²⁺, Cl^–, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl^– accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl^– accumulation and electrical potential while an outwardly-directed valinomycininduced K⁺ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl^– accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl^– accumulation, intraliposomal negative potential difference, and also Cl^–-stimulated ATPase activity. Both aspects of Cl^– pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl^–-ATPase actively transports Cl^– by an electrogenic process.     

Salt effects on urea hydrolysis and nitrification during leaching through laboratory soil columns

Summary We studied the transport and transformation of urea under steady-state conditions in two soils and at three water salinities (1.0, 5.0, and 10.0 dS/m) using laboratory soil columns. A mathematical model that considers diffusion, convection, adsorption and first-order kinetic transformation of nitrogen was used to describe measured effluent concentration of the two nitrogen species. Increasing salt levels in the applied water decreased the hydrolysis of urea in the two soils studied with first-order rate coefficients decreasing from 0.015 to 0.009 h^–1 in the fine sandy loam, and from 0.075 to 0.015 h^–1 in a silty loam. Similarly, the nitrification rate decreased by 50% and 70% in the two soils as salinity increased. The rate coefficients measured in the leaching studies were much smaller than measured in incubation-type experiments. Calculated half-lives for urea and NH ₄ ⁺ provided a method interpreting the kinetic rate coefficients as a function of the experimental conditions.     

Anaerobic degradation pathway and kinetics of domestic wastewater at low temperatures

The effect of temperatures below 20 °C (20, 15 and 10 °C) on the anaerobic degradation pathway and kinetics of domestic wastewater fractionated at different sizes was studied in a fluidized-bed batch reactor. The overall degradation pathway was characterized by a soluble fraction degrading according to zero-order kinetics and a colloidal fraction (between 0.45 and 4.5 μm) that first disintegrates into a particulate fraction smaller than 0.45 μm before finally degrading. The colloidal degradation processes follow a first-order kinetic. In contrast, suspended solids (bigger than 4.5 μm) degrade to soluble and colloidal fractions according to first-order kinetics. The colloidal fraction originating from suspended solids further degrades into soluble fraction. These soluble fractions have the same degradation kinetics as the original soluble fraction. The suspended solids degradation was highly affected by temperature, whereas the soluble fraction slightly affected and the colloidal fraction was not affected at all. On the other hand, the colloidal non-degradable fraction increased significantly with the decrease in temperature while the suspended solids slowly increased. The soluble non-degradable fraction was little affected by temperatures changes.     

Reactivation kinetics of guanidine hydrochloride-denatured creatine kinase measured using the substrate reaction

Guanidine hydrochloride-denatured creatine kinase (CK) can very quickly form a dimer with reactivity when the denaturant is diluted into the reaction system in the presence of DTT or EDTA. Tsou's method and its applied equation [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381–436; Yang and Zhou (1998), Biochim. Biophys. Acta 1388, 190–198] were used to measure the kinetic reactivation rate constants and the reactivation degree for reassociated CK dimers. Partial reactivation (about 50% at best) occurred following a monophasic course during the substrate reaction when compared with previous time interval measurements. The reactivation degree increased with increasing DTT (0.1–5 mM) and EDTA (0.1–1 mM) concentrations. The apparent forward rate constants do not change with DTT concentration, showing that the reactivation is a reversible first-order reaction, but not of complex formation type. However, the apparent forward rate constants do change with EDTA concentration, showing that the reactivation with EDTA is a reversible first-order reaction as well as of complex formation type. Excess DTT concentrations have an inhibitory effect, indicating that the excessive EDTA acts as a metal chealate not only for free Mg²⁺, but also for MgATP during the enzyme catalysis. This study shows that additional information about the reactivation of CK can be obtained from examining the substrate reaction. The possible refolding pathway of CK is discussed.     

Decomposition of urea by size-fractionated planktonic community in a eutrophic reservoir in Japan

Free bacterial populations were separated from an intact planktonic community in water of a eutrophic reservoir in Japan by filtration through Whatman GF/ C glass fiber filters (mean porosity 1.2 µm). Urea decomposition by the free bacterial populations and the intact planktonic community was determined in six different months.The separated free bacteria apparently did not take part in urea-decomposition in waters of the reservoir through the year: the number of free heterotrophic bacteria increased during the urea-decomposition experiments, however, the concentration of urea did not decrease. Whereas, in five cases out of six, urea was decomposed by the intact planktonic community. Probably, phytoplankton were responsible for most of the urea-decomposition. On the assumption that the decomposition of urea obeyed first-order kinetics, rate constants were calculated to be 0.00–0.63 day^–1 with a mean value of 0.21 day^–1.     

Activities of benthic nitrifiers in streams and their role in oxygen consumption

The in situ rates of oxygen consumption by benthic nitrifiers were estimated at 11 study sites in 4 streams. Two methods were used: an in situ respiration chamber method and a method involving conversion of nitrifying potential measurements to in situ rates. Estimates of benthic nitrogenous oxygen consumption (BNOC) rate ranged from 0–380 mmol of O₂ m^–2·day^–1, and BNOC contributed between 0–85% of the total benthic oxygen consumption rate. The activity of nitrifiers residing in the sediments was influenced by O₂ availability, temperature, pH, and substrate. Depending upon site, nitrification could approximate either first-order or zero-order kinetics with respect to ammonium concentration. The source of ammonium for benthic nitrifiers could be either totally from within the sediment or totally from the overlying water. Nitrate produced in the sediments could flux to the water above or be lost within the sediment. The sediments could act as a source (positive flux) or sink (negative flux) for both ammonium (–185 mmol·m^–2·day^–1 to +195 mmol·m^–2·day^–1) and nitrate (–135 mmol·m^–2·day^–1 to +185 mmol·m^–2·day^–1).This study provides evidence to suggest that measurements of down-stream mass flow changes in inorganic nitrogen forms may give poor estimates of in situ rates of nitrification in flowing waters.     

Effect of microbial concentration on biodegradation rates of phenols

Summary Biodegradation rates of 12 phenols were measured with respect to acclimated microbial biomass ranging from 2.3×10⁴ to 2.3×10⁸ cells/l. Rates ranged between 0.02 mg l^–1 day^–1 for 1.6 mg/lp-bromophenol exposed to 2.3×10⁴ cells/l and 1.41 mg l^–1 day^–1 for 3.2 mg/lp-methylphenol exposed to 2.3×10⁸ cells/l. Generally, rates for all phenols were first-order in substrate concentration and zero-order in biomass concentration. Bromophenol biodegradation was preceded by lag periods of varying lengths and to a small extent the rate was dependent on microbial biomass. Results from this study suggest chemical biodegradation generally exhibits pseudo-first-and occasionally, second-order kinetics.     

Inactivation and conformational changes of aminoacyclase in trifluoroethanol solutions

The inactivation and unfolding of aminoacyclase (EC 3.5.1.14) during denaturation by different concentrations of trifluoroethanol (TFE) have been studied. A marked decrease in enzyme activity was observed at low TFE concentrations. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436] was applied to study the kinetics of the inactivation course of aminoacyclase during denaturation by TFE. The inactivation rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method. The inactivation reaction was a monophasic first-order reaction. The kinetics of the unfolding course were a biphasic process consisting of two first-order reactions. At 2% TFE concentration, the inactivation rate of the enzyme was much faster than the unfolding rate. At a higher concentration of TFE (10%), the inactivation rate was too fast to be determined by conventional methods, whereas the unfolding course remained as a biphasic process with fast and slow reactions occurring at measurable rates. The results suggest that the aminoacyclase active site containing Zn²⁺ ions is situated in a limited and flexible region of the enzyme molecule that is more fragile to the denaturant than the protein as a whole.     

The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

The autolysis of trypsin and α-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in `inert' vessels) are second-order for both trypsin and α -chymotrypsin, with rate constants of 13.0mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for trypsin (in 50mm-NaCl at pH8.0 at 25°C) and 10.2mol<sup>−1</sup>·dm<sup>3</sup>·s<sup>−1</sup> for α-chymotrypsin (in 0.1m-glycine at pH9.2 at 30°C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and α-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of α-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH–activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the `microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.     

Photon correlation spectroscopy of human IgG

The translational diffusion coefficient D _20,w ⁰ , of monomeric human immunoglobulin G (IgG) has been studied by photon-correlation spectroscopy as a function of pH and protein concentration. At pH 7.6, we find D _20,w ⁰ =3.89×10^–7±0.02 cm²/sec, in good agreement with the value determined by classic mehods. This value corresponds to an effective hydrodynamic radius R, of 55.1±0.3 Å. As pH is increased to 8.9; with the same ionic strength, the molecule appears to expand slightly (3.5% increase in hydrodynamic radius). The concentration dependence of the IgG diffusion constant is interpreted in terms of solution electrostatic effects and shows that long-range repulsive interactions are negligible in the buffer used. The diffusion coefficient for dimeric IgG has also been determined to be D_20,w=2.81×10^–7±0.04 cm²/sec at 1.6 mg/ml, which corresponds to a hydrodynamic radius of 75 Å. For light-scattering studies of protein molecules in the dimension range of 5–10 nm (M_r=10⁵–10⁷) we find monomeric horse spleen ferritin well suited as a reference standard. Ferritin is a spherical molecule with a hydrodynamic radius R of 6.9±0.1 nm and is stable for years in our standard Tris-HCl-NaCl buffer even at room temperature.     

Chemical evolution of dehydrogenases: Amino acid pentacyanoferrate (II) as possible intermediates

Dehydrogenation of ascorbic acid and reduced nicotinamide adenine dinucleotide (NADH) with methylene blue using complexes of the type [Fe(II)(CN)₅ (L)] ^n– (wheren=3 or 4; L=glycine, histidine, imidazole, and triglycine) as catalyst have been studied at pH 9.18. Similar kinetic behavior was observed for the dehydrogenation of ascorbic acid as well as for NADH; both reactions showed first order dependency on the substrates. First order dependence was observed only at lower concentrations of methylene blue; at higher concentrations of methylene blue, the reactions were independent of methylene blue. The order with respect to catalyst varied between 0.3–0.5. A tentative mechanism which conforms to the observed kinetics has been proposed. It is believed that on the primitive earth when the reducing potential of the atmosphere was not high enough, lower oxidation state iron complexes like [Fe(II)(CN)₅(L)] ^n– might have been involved in dehydrogenase-type activity.     

Death rate in a small air-lift loop reactor of vero cells grown on solid microcarriers and in macroporous microcarriers

The death rate of Vero cells grown on Cytodex-3 microcarriers was studied as a function of the gas flow rate in a small air-lift loop reactor. The death rate may be described by first-order death-rate kinetics. The first-order death-rate constant as calculated from the decrease in viable cells, the increase in dead cells and the increase in LDH activity is linear proportional to the gas flow rate, with a specific hypothetical killing volume in which all cells are killed of about 2·10^–3 m³ liquid per m³ of air bubbles. In addition, an experiment was conducted in the same air-lift reactor with Vero cells grown inside porous Asahi microcarriers. The specific hypothetical killing volume calculated from this experiment has a value of 3·10^–4 m³ liquid per m³ of air bubbles, which shows that the porous microcarriers were at least in part able to protect the cells against the detrimental hydrodynamic forces generated by the bubbles.     

Kinetic study of hydrolysis of xylan and agricultural wastes with hot liquid water

We investigated the kinetics of hot liquid water (HLW) hydrolysis over a 60-min period using a self-designed setup. The reaction was performed within the range 160–220 °C, under reaction conditions of 4.0 MPa, a 1:20 solid:liquid ratio (g/mL), at 500 rpm stirring speed. Xylan was chosen as a model compound for hemicelluloses, and two kinds of agricultural wastes–rice straw and palm shell–were used as typical feedstocks representative of herbaceous and woody biomasses, respectively. The hydrolysis reactions for the three kinds of materials followed a first-order sequential kinetic model, and the hydrolysis activation energies were 65.58 kJ/mol for xylan, 68.76 kJ/mol for rice straw, and 95.19 kJ/mol for palm shell. The activation energies of sugar degradation were 147.21 kJ/mol for xylan, 47.08 kJ/mol for rice straw and 79.74 kJ/mol for palm shell. These differences may be due to differences in the composition and construction of the three kinds of materials. In order to reduce the decomposition of sugars, the hydrolysis time of biomasses such as rice straw and palm shell should be strictly controlled.     

Ethanol acetylation by mycelium-bound carboxylesterase of Aspergillus oryzae: estimation of thermodynamic parameters and integral productivity

The results collected at different temperatures for ethanol acetylation by cell-bound carboxylesterase from lyophilized cells of Aspergillus oryzae have been used to investigate the kinetics and thermodynamics of this esterification in n-heptane. The occurrence of reversible unfolding followed by irreversible denaturation of the enzyme has been proposed to explain the increase in the starting rate of ethyl acetate formation with temperature observed up to 55 °C and the consequent fall beyond this threshold. The Arrhenius model has been used to estimate the apparent activation enthalpies of both the acetylation reaction (H = 29–33 kJ mol^–1) and reversible enzyme unfolding (H _u = 56–63 kJ mol^–1). The results of residual activity tests performed with cells previously exposed at different temperatures for variable times enabled us also to estimate the first-order rate constant of irreversible denaturation (2.40 × 10^–3 h^–1 < k _d < 8.11 × 10^–3 h^–1) as well as the related thermodynamic parameters (H _d = 22 kJ mol^–1; S _d = –0.29 kJ mol^–1 K^–1). This last phenomenon proved particularly slow for the system under consideration, probably because the biocatalyst link to the mycelium was able to improve its thermostability. In view of future continuous application, the effects of operating time, starting substrate concentration and temperature on the theoretical integral productivity of a fixed-bed column filled with this biocatalyst have been investigated.