First-order Inactivation Rate Constants Research Articles

Efficacy of sodium chlorite in inactivating Vibrio parahaemolyticus attached to polyethylene terephthalate surfaces.

The inactivation of Vibrio parahaemolyticus cells attached to a polyethylene terephthalate (PET) disc in a sodium chlorite (NaClO2) solution was kinetically studied in a weakly acidic pH range of 4.0 - 6.5. The logarithmic reduction in the survival ratio depended on the concentration-time product. All inactivation curves showed a linear reduction phase, and the reduction in viable cells was greater than 4-log. No significant desorption of attached cells was observed during the inactivation treatment. The first-order inactivation rate constant (k) increased by approximately 4.5-fold for every 1.0 unit fall in pH. At all pH values, the k values calculated for the attached cells were approximately half of those for the unattached cells. These findings indicate that a weakly acidic NaClO2 solution is effective in inactivating bacteria attached to hard surfaces.

Kinetics of the Inactivation of Vibrio parahaemolyticus in Weakly Acidic Sodium Chlorite Solution.

The kinetics of the inactivation of Vibrio parahaemolyticus in sodium chlorite (NaClO2) solution was studied in the weakly acidic pH range of 4.0 to 6.5 and at various temperatures. The logarithmic reduction of the survival ratio depended on the concentration-time product, and all the inactivation curves showed a linear reduction phase. The first-order inactivation rate constant (k) increased by approximately twice for every 0.44 unit fall in pH. During the inactivation experiments, no formation of chlorine dioxide occurred. These data indicated that undissociated HClO2 was the active species governing the inactivation of V. parahaemolyticus. It was also shown that the use of weakly acidic NaClO2 solutions containing high concentrations of ionized ClO2- gave slower kinetics of the inactivation, whereas it could achieve the significant reduction of viable cells of more than 4-log. The k value showed an Arrhenius-type temperature dependence in the temperature range of 5 to 40℃. The apparent activation energy for the inactivation of V. parahaemolyticus was estimated to be 43.5 kJ/mol. The k value increased by approximately 1.8 times for every 10℃ rise in temperature.

Inactivation of Vibrio parahaemolyticus Unattached and Attached to a Solid Surface in pH-Controlled Sodium Hypochlorite Solutions.

The inactivation of Vibrio parahaemolyticus cells that were unattached or attached to a polyethylene terephthalate (PET) disc in pH-controlled sodium hypochlorite (NaOCl) solutions was studied under turbulent conditions. No significant desorption of attached cells occurred at the free available chlorine (FAC) concentrations from 0.1 to 1.0 mg/l. The number of viable cells was estimated by microbial calorimetry. The logarithmic relative reduction of viable cells was proportional to the product of the FAC concentration and time. In the pH range of 5.6 to 9.3, the first-order inactivation rate constants for unattached and attached cells increased with decreasing solution pH. It was found that the rate constants for unattached cells were approximately 6 to 7 times higher than those for attached cells at all pH values examined. It was confirmed that attached cells were more resistant to NaOCl solutions than unattached cells even when accessibility of attached cells to HOCl/OCl- was enhanced under turbulent conditions.

Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation.

ObjectivesEfficient enzymatic saccharification of plant cell wall material is key to industrial processing of agricultural and forestry waste such as straw and wood chips into fuels and chemicals.ResultsSaccharification assays were performed on steam-pretreated wheat straw under ambient and O2-deprived environments and in the absence and presence of a lytic polysaccharide monooxygenase (LPMO) and catalase. A kinetic model was used to calculate catalytic rate and first-order inactivation rate constants of the cellulases from reaction progress curves. The addition of a LPMO significantly (P < 0.01, Student’s T test) enhanced the rate of glucose release from 2.8 to 6.9 h−1 under ambient O2 conditions. However, this also significantly (P < 0.01, Student’s T test) increased the rate of inactivation of the enzyme mixture, thereby reducing the performance half-life from 65 to 35 h. Decreasing O2 levels or, strikingly, the addition of catalase significantly reduced (P < 0.01, Student’s T test) enzyme inactivation and, as a consequence, higher efficiency of the cellulolytic enzyme cocktail was achieved.ConclusionOxidative inactivation of commercial cellulase mixtures is a significant factor influencing the overall saccharification efficiency and the addition of catalase can be used to protect these mixtures from inactivation.Electronic supplementary materialThe online version of this article (doi:10.1007/s10529-015-1989-8) contains supplementary material, which is available to authorized users.

Virus Sensitivity Index of UV disinfection

A new concept of Virus Sensitivity Index (VSI) is defined as the ratio between the first-order inactivation rate constant of a virus, ki, and that of MS2-phage during UV disinfection, kr. MS2-phage is chosen as the reference virus because it is recommended as a virus indicator during UV reactor design and validation by the US Environmental Protection Agency. VSI has wide applications in research, design, and validation of UV disinfection systems. For example, it can be used to rank the UV disinfection sensitivity of viruses in reference to MS2-phage. There are four major steps in deriving the equation between Hi/Hr and 1/VSI. First, the first-order inactivation rate constants are determined by regression analysis between Log I and fluence required. Second, the inactivation rate constants of MS2-phage are statistically analysed at 3, 4, 5, and 6 Log I levels. Third, different VSI values are obtained from the ki of different viruses dividing by the kr of MS2-phage. Fourth, correlation between Hi/Hr and 1/VSI is analysed by using linear, quadratic, and cubic models. As expected from the theoretical analysis, a linear relationship adequately correlates Hi/Hr and 1/VSI without an intercept. VSI is used to quantitatively predict the UV fluence required for any virus at any log inactivation (Log I). Four equations were developed at 3, 4, 5, and 6 Log I. These equations have been validated using external data which are not used for the virus development. At Log I less than 3, the equation tends to under-predict the required fluence at both low Log I such as 1 and 2 Log I. At Log I greater than 3 Log I, the equation tends to over-predict the fluence required. The reasons for these may very likely be due to the shoulder at the beginning and the tailing at the end of the collimated beam test experiments. At 3 Log I, the error percentage is less than 6%. The VSI is also used to predict inactivation rate constants under two different UV disinfection scenarios such as under sunlight and different virus aggregates. The correlation analysis shows that viruses will be about 40% more sensitive to sunlight than to UV254. On the other hand, virus size of 500 nm will reduce their VSI by 10%. This is the first attempt to use VSI to predict the required fluence at any given Log I. The equation can be used to quantitatively evaluate other parameters influencing UV disinfection. These factors include environmental species, antibiotic-resistant bacteria or genes, photo and dark repair, water quality such as suspended solids, and UV transmittance.

An algebraic model for the kinetics of covalent enzyme inhibition at low substrate concentrations

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme–inhibitor complex (no “tight binding”). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (kinact). However, the inhibition constant (Ki), and therefore also the second-order inactivation rate constant kinact/Ki, is underestimated by the traditional method by up to an order of magnitude.

Virus removal and inactivation by iron (hydr)oxide-mediated Fenton-like processes under sunlight and in the dark

Advanced oxidation processes (AOPs) have emerged as a promising alternative to conventional disinfection methods to control microbial water quality, yet little is known about the fate of viruses in AOPs. In this study, we investigated the fate of MS2 coliphage in AOPs that rely on heterogeneous Fenton-like processes catalyzed by iron (hydr)oxide particles. Both physical removal of viruses from solution via adsorption onto particles as well as true inactivation were considered. Virus fate was studied in batch reactors at circumneutral pH, containing 200 mg L(-1) of four different commercial iron (hydr)oxide particles of similar mesh sizes: hematite (α-Fe2O3), goethite (α-FeOOH), magnetite (Fe3O4) and amorphous iron(iii) hydroxide (Fe(OH)3). The effect of adsorption and sunlight exposure on the survival of MS2 was considered. On a mass basis, all particles exhibited a similar virus adsorption capacity, whereas the rate of adsorption followed the order FeOOH > Fe2O3 > Fe3O4 ≈ Fe(OH)3. This adsorption behavior could not be explained by electrostatic considerations; instead, adsorption must be governed by other factors, such as hydrophobic interactions or van der Waals forces. Adsorption to three of the particles investigated (α-FeOOH, Fe3O4, Fe(OH)3) caused virus inactivation of 7%, 22%, and 14%, respectively. Exposure of particle-adsorbed viruses to sunlight and H2O2 resulted in efficient additional inactivation, whereas inactivation was negligible for suspended viruses. The observed first-order inactivation rate constants were 6.6 × 10(-2), 8.7 × 10(-2), 0.55 and 1.5 min(-1) for α-FeOOH, α-Fe2O3, Fe3O4 and Fe(OH)3 respectively. In the absence of sunlight or H2O2, no inactivation was observed beyond that caused by adsorption alone, except for Fe3O4, which caused virus inactivation via a dark Fenton-like process. Overall our results demonstrate that heterogeneous Fenton-like processes can both physically remove viruses from water as well as inactivate them via adsorption and via a particle-mediated (photo-)Fenton-like process.

Escherichia coli survival in waters: Temperature dependence

Knowing the survival rates of water-borne Escherichia coli is important in evaluating microbial contamination and making appropriate management decisions. E. coli survival rates are dependent on temperature, a dependency that is routinely expressed using an analogue of the Q10 model. This suggestion was made 34 years ago based on 20 survival curves taken from published literature, but has not been revisited since then. The objective of this study was to re-evaluate the accuracy of the Q10 equation, utilizing data accumulated since 1978. We assembled a database of 450 E. coli survival datasets from 70 peer-reviewed papers. We then focused on the 170 curves taken from experiments that were performed in the laboratory under dark conditions to exclude the effects of sunlight and other field factors that could cause additional variability in results. All datasets were tabulated dependencies “log concentration vs. time.” There were three major patterns of inactivation: about half of the datasets had a section of fast log-linear inactivation followed by a section of slow log-linear inactivation; about a quarter of the datasets had a lag period followed by log-linear inactivation; and the remaining quarter were approximately linear throughout. First-order inactivation rate constants were calculated from the linear sections of all survival curves and the data grouped by water sources, including waters of agricultural origin, pristine water sources, groundwater and wells, lakes and reservoirs, rivers and streams, estuaries and seawater, and wastewater. Dependency of E. coli inactivation rates on temperature varied among the water sources. There was a significant difference in inactivation rate values at the reference temperature between rivers and agricultural waters, wastewaters and agricultural waters, rivers and lakes, and wastewater and lakes. At specific sites, the Q10 equation was more accurate in rivers and coastal waters than in lakes making the value of the Q10 coefficient appear to be site-specific. Results of this work indicate possible sources of uncertainty to be accounted for in watershed-scale microbial water quality modeling.

Lactoferrin Inhibits Porphyromonas gingivalis Proteinases and Has Sustained Biofilm Inhibitory Activity

Porphyromonas gingivalis is a bacterial pathogen associated with chronic periodontitis that results in destruction of the tooth's supporting tissues. The major virulence determinants of P. gingivalis are its cell surface Arg- and Lys-specific cysteine proteinases, RgpA/B and Kgp. Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in saliva and gingival crevicular fluid, is believed to play an important role in innate immunity. In this study, bovine milk LF displayed proteinase inhibitory activity against P. gingivalis whole cells, significantly inhibiting both Arg- and Lys-specific proteolytic activities. LF inhibited the Arg-specific activity of purified RgpB, which lacks adhesin domains, and also inhibited the same activity of the RgpA/Kgp proteinase-adhesin complexes in a time-dependent manner, with a first-order inactivation rate constant (k(inact)) of 0.023 min(-1) and an inhibitor affinity constant (K(I)) of 5.02 μM. LF inhibited P. gingivalis biofilm formation by >80% at concentrations above 0.625 μM. LF was relatively resistant to hydrolysis by P. gingivalis cells but was cleaved into two major polypeptides (53 and 33 kDa) at R(284) to S(285), as determined by in-source decay mass spectrometry; however, these polypeptides remained associated with each other and retained inhibitory activity. The biofilm inhibitory activity of LF against P. gingivalis was not attributed to direct antibacterial activity, as LF displayed little growth inhibitory activity against planktonic cells. As the known RgpA/B and Kgp inhibitor N-α-p-tosyl-l-lysine chloromethylketone also inhibited P. gingivalis biofilm formation, the antibiofilm effect of LF may at least in part be attributable to its antiproteinase activity.

Human Virus and Bacteriophage Inactivation in Clear Water by Simulated Sunlight Compared to Bacteriophage Inactivation at a Southern California Beach

Few quantitative data exist on human virus inactivation by sunlight and the relationship between human and indicator viruses under sunlit conditions. We investigated the effects of sunlight on human viruses (adenovirus type 2, poliovirus type 3) and bacteriophages (MS2, Q-Beta SP, Fi, M13, PRD1, Phi-X174, and coliphages isolated from Avalon Bay, California). Viruses were inoculated into phosphate buffered saline or seawater, exposed to a laboratory solar simulator for ≤12 h, and enumerated by double agar layer or cell culture to derive first-order inactivation rate constants (k(obs), h(-1)). The viruses most resistant to sunlight were adenovirus type 2 (k(obs)= 0.59 ± 0.04 h(-1)) and bacteriophage MS2 (k(obs)= 0.43 ± 0.02 h(-1)), which suggests MS2 may be a conservative indicator for sunlight resistant human viruses in clear water when sunlight inactivation is the main removal mechanism. Reasonable agreement was observed between somatic coliphage inactivation rates measured in the solar simulator (k(mean) = 1.81 h(-1)) and somatic coliphages measured in the surf zone during a field campaign at Avalon Bay during similar sunlight intensity (k = 0.75 h(-1) at log-RMSE minimum; k(range) = 0.54 h(-1) to >1.88 h(-1); Boehm, A. B. et al. Environ. Sci. Technol. 2009, 43, (21), 8046-8052). Hence, measuring sunlight inactivation rates of viruses in the laboratory can be used to estimate inactivation in the environment under similar sunlight and water quality conditions.

Kinetics of inactivation of indicator pathogens during thermophilic anaerobic digestion

Thermophilic anaerobic sludge digestion is a promising process to divert waste to beneficial use, but an important question is the required temperature and holding time to achieve a given degree of pathogen inactivation. In this study, the kinetics of inactivation of Ascaris suum and vaccine strain poliovirus type 1 (PVS-1), selected as indicators for helminth ova and enteric viruses respectively, were determined during anaerobic digestion at temperatures ranging from 51 to 56 °C. Inactivation of both indicator organisms was fast with greater than two log reductions achieved within 2 h for A. suum and three log reductions for PVS-1, suggesting that the current U.S. regulations are largely conservative. The first-order inactivation rate constants k followed Arrhenius relationship with activation energies of 105 and 39 KJ mol −1 for A. suum and PVS-1, respectively indicating that A. suum was more sensitive to temperature. Although inactivation was fast, the presence of compounds in the sludge that are known to be protective of pathogen inactivation was observed, suggesting that composition-dependent time–temperature relationships are necessary.

Comparison of the Disinfection Effects of Vacuum‐UV (VUV) and UV Light on Bacillus subtilis Spores in Aqueous Suspensions at 172, 222 and 254 nm

The efficacy of UV and vacuum-UV (VUV) disinfection of Bacillus subtilis spores in aqueous suspensions at wavelengths of 172, 222 and 254 nm was evaluated. A Xe(2)* excilamp, a KrCl* excilamp and a low-pressure mercury lamp were used as almost monochromatic light sources at these three wavelengths. The first-order inactivation rate constants at 172, 222 and 254 nm were 0.0023, 0.122 and 0.069 cm(2) mJ(-1), respectively. Therefore, a 2 log reduction of B. subtilis spores was reached with fluences (UV doses) of 870, 21.6 and 40.4 mJ cm(-2) at these individual wavelengths. Consequently, for the inactivation of B. subtilis spores, VUV exposure at 172 nm is much less efficient than exposure at the other two wavelengths, while exposure at 222 nm is more efficient than that at 254 nm, which is probably because triplet energy transfer from DPA to thymine bases at 222 nm is higher than that at 254 nm. This research indicated quantitatively that VUV light is not practicable for microorganism disinfection in water and wastewater treatment. However, in comparison with other advanced oxidation processes (e.g. UV/TiO(2), UV/H(2)O(2) or O(3)/H(2)O(2)) the VUV-initiated photolysis of water is likely more efficient in generating hydroxyl radicals and more effective for the inactivation of microorganisms.

An ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37-59 degrees C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constants k(in), k(in)*, and k(in)(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO* -DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.

Thermal Inactivation of Salmonella in Whole Muscle and Ground Turkey Breast

The effect of the physical structure of turkey meat (ground and whole muscle) on the thermal resistance of Salmonella was evaluated. Irradiated whole and ground turkey breasts were exposed to a marinade containing eight serovars of Salmonella at ~108 CFU/ml for 20 min. Inoculated samples then were subjected to isothermal heating at 55, 60, or 62.5°C, for varying times. Salmonella counts before and after the thermal lag time (time to reach the target temperature) were not significantly different (α = 0.05). The first-order inactivation rate constants in whole muscle were ~50% lower than those in ground muscle of the same composition, at each temperature, indicating that the Salmonella inactivation rate was greater (P < 0.05) in ground samples than in whole-muscle samples. These results suggest that internalization of Salmonella in whole-muscle product leads to enhanced thermal resistance.

Identification of Surface Epitopes of Human Coagulation Factor Va That Are Important for Interaction with Activated Protein C and Heparin

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

The 1,4-naphthoquinone scaffold in the design of cysteine protease inhibitors

A series of 1,4-naphthoquinone derivatives diversely substituted at C-2, C-3, C-5 and C-8, prepared by reaction of amines, amino acids and alcohols with commercial 1,4-naphthoquinones, has been evaluated against papain and bovine spleen cathepsin B. These 1,4-naphthoquinone derivatives were found to be irreversible inhibitors for both cysteine proteases, with second-order rate constants, k 2, ranging from 0.67 to 35.4 M −1 s −1 for papain, and from 0.54 to 8.03 M −1 s −1 for cathepsin B. Some derivatives display a hyperbolic dependence of the first-order inactivation rate constant, k obs, with the inhibitor concentration, indicative of a specific interaction process between enzyme and inhibitor. The chemical reactivity of the compounds towards cysteine as a model thiol is dependent on the naphthoquinone LUMO energy, whereas papain inactivation is not. The 1,4-naphthoquinone derivatives are inactive against the serine protease, porcine pancreatic elastase.

Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP+ was characterized in aqueous solutions at 36-47 degrees C under treatment with low frequency (27 kHz, 60 W/cm2) and high frequency ultrasound (880 kHz, 1 W/cm2). To this end, we measured three effective first-order inactivation rate constants: thermal k(in)* , total (thermal and ultrasonic) kin, and ultrasonic kin (US). The values of the constants were found to be higher for the free enzyme than for its complexes GP-DH-GP and GPDH-NADP+ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (Ea) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (deltaH*, deltaS*, and deltaG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of Ea and deltaH* values in comparison with the corresponding values for thermal inactivation. The Ea, deltaH*, and deltaS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the deltaH* and deltaS* values was observed for all the inactivation processes for free GPDH and its complexes.

Interactions of Nitroaromatic Compounds with the Mammalian Selenoprotein Thioredoxin Reductase and the Relation to Induction of Apoptosis in Human Cancer Cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.

Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor

The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K (M) of oxygen must be known. The determination of this constant was done using progress curve analysis (K (M) = 260 micromol l(-1)). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35 degrees C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5 degrees C to 9 min at 35 degrees C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.

Cysteine-scanning Mutagenesis of Serotonin Transporter Intracellular Loop 2 Suggests an α-Helical Conformation

Like other proteins involved in neurotransmitter transport, serotonin transporter (SERT) activity is regulated by multiple intracellular signal transduction pathways. The second intracellular loop (IL2) of SERT contains consensus sequences for cGMP-dependent protein kinase and protein kinase C. A 24-residue region of SERT including IL2, from Ile-270 through Ser-293, was analyzed by cysteine-scanning mutagenesis and chemical modification. 2-(Aminoethyl)methanethiosulfonate hydrobromide (MTSEA) failed to inhibit serotonin transport or binding of the cocaine analog 2beta-carbomethoxy-3beta-(4-[125I]iodophenyl)tropane (beta-CIT) in intact cells expressing these mutants, but it inactivated beta-CIT binding in membrane preparations. From the pattern of sensitivity, IL2 appears to extend from Trp-271 through Ile-290, a significantly longer region than that initially predicted by hydropathy analysis. Six mutants reacted with MTSEA much faster than the others, and the pattern of the more reactive mutations suggested that IL2 is in an alpha-helical conformation. Some of the mutants had significantly elevated transport rates, suggesting a possible mechanism for the regulation of SERT activity.