Enzyme saturation and inhibition kinetics studied from multiple progress curves recorded spectrophotometrically from single reaction mixtures for ADP-ribose pyrophosphatase.

Saturation and inhibition kinetics data for rat liver ADP-ribose pyrophosphatase (EC 3.6.1.13) were obtained from progress curves initiated by the addition of substrate and recorded spectrophotometrically until the end point was reached. The hydrolysis of ADP-ribose was coupled to either alkaline phosphatase and adenosine deaminase or AMP deaminase. The validity of the approach was shown because: (i) the coupled hydrolysis of ADP-ribose was essentially irreversible; (ii) ADP-ribose pyrophosphate was stable at 37 degrees C in the conditions needed for the assay; and (iii) accumulated reaction products did not inhibit detectably in the conditions of the assay. In addition, several identical progress curves could be successively recorded by repetition of the addition of substrate. In that way it was possible to carry out complete inhibition studies by increasing the inhibitor concentration between successive substrate additions. Studying the inhibition by high D-ribose concentrations, meaningful results could be obtained at four different inhibitor concentrations in a single reaction mixture, which represented a great saving of enzyme preparation with respect to what would be needed in an equivalent initial rate study.     

Lipase reaction in AOT-isooctane reversed micelles: effect of water on equilibria

The effect of water on equilibria for hydrolytic reaction in reversed micelles has been investigated using lipase as a model enzyme. The effect of water on equilibria has been ignored for hydrolase reactions in an aqueous phase. In a reversed micellar system, however, the equilibrium of the lipase reaction was changed when water was added during the hydrolytic reaction. Furthermore, equilibrium fractional conversion is affected by the initial water concentration, being shifted to higher values with higher water concentrations, with other reaction conditions being held constant, indicating that the reaction should be regarded as a two-substrate process. Equations corresponding to a two-substrate, second-order reversible model are derived and used for further analysis. The progress curves predicted from the rate equations agree very well with the experimental results under various reaction conditions. The values of the molar ratio of water to surfactant (R) which maximize the initial reaction rate and maximum fractional conversion is predictable from the derived rate equations and the resulting relationship between R and the kinetic constants.     

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters.

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction

The direct detection, using the polymerase chain reaction (PCR), of Listeria monocytogenes added to cows' milk was inhibited at some milk concentrations. This inhibitor was moderately heat-stable. Inhibition could be prevented by the addition of Bovine Serum Albumin (BSA) or proteinase inhibitors to the PCR and the evidence suggests that the inhibitor was plasmin.     

The oscillating peroxidase-oxidase reaction in an open system. Analysis of the reaction mechanism.

1. The oscillations in the peroxidase-oxidase reaction in an open system with NADH as the hydrogen donor are caused by the reaction starting and stopping at critical concentrations of the substrates O2 and NADH. The existence of such critical concentrations is typical of branched chain reactions. 2. The critical concentrations of O2 and NADH that determine the initiation of the reaction are mutually dependent. 3. The branching reactions that determine these critical concentrations involve compounds I and II. 4. Superoxide may be involved in the branching reactions by reacting with NADH and ferriperoxidase. At pH 5.1 the rate constant for the latter reaction is determined as 1.5 . 10(5) M-1 . s-1, whereas for the former reaction only an upper limit for the rate constant of 3.5 . 10(4) M-1 . s-1 could be estimated. These relatively low rate constants suggest that alternative branching reactions may also be involved.     

Chemical reactivity of labile sulfur of iron-sulfur proteins The reaction of triphenyl phosphine

The reaction of triphenyl phosphine to iron-sulfur proteins from adrenal cortex mitochondria, spinach chloroplasts, and Clostridium pasteurianum was investigated. As ethanol concentrations in the reaction mixture increased, the rate of the reaction decreased. In the simultaneous presence of 1 M KCl and 5 M urea, the reaction rate reached at maximum. Under these conditions the initial rates of the decolorization reaction by the phosphine were found to be 8.7, 0.88, and 1.8 nmol of ferrodoxin per min at 25°C for adrenal, spinach, and clostridial ferredoxins, respectively. The kinetic curves for the reaction of the phosphine sulfide formation, the loss of labile sulfur, and the deterioriation of visible absorption showed a similar pattern with a comparable rate. During this reaction, the complete reduction of ferric ions present in ferredoxin was observed with a fast rate under either aerobic or anaerobic conditions.These results suggest that the iron atoms in ferredoxin are first reduced by the intramolecular reductants in the presence of triphenyl phosphine with the concomitant formation of S₂^2?, which then reacts with triphenyl phosphine resulting in the formation of triphenyl phosphine sulfide.     

A theoretical treatment of damped oscillations in the transient state kinetics of single-enzyme reactions

An extension of the available kinetic theory for reactions in the transient state is presented which establishes that single-enzyme reactions may exhibit damped oscillations under the conditions of standard kinetic experiments performed by stopped-flow techniques. Such oscillations may occur for reasonable magnitudes of rate constants in the enzymic reaction mechanism and at physiological concentrations of enzyme and substrate. In the simplest reaction systems, the oscillations will be strongly damped and lead to progress curves resembling those of a reaction governed by standard exponential transients; statistical regression methods may then have to be applied for their detection and characterization. The observation that single-enzyme reactions may exhibit oscillatory behaviour points to a previously unrecognized possible source of the damped oscillations observed in metabolic systems such as the pathways of glycolysis or photosynthesis.     

Development of selected reaction monitoring‐MS methodology to measure peptide biomarkers in prostate cancer

Prostate cancer is a leading cause of cancer‐related death. The current modality of diagnosis, the measurement of serum PSA, not only suffers from lack of specificity, but does not distinguish clinical cases in which current treatment measures would be most successful, i.e. aggressive, life‐threatening tumors. A multiplexed MS methodology, selected reaction monitoring‐MS/MS coupled with stable isotope dilution (SID), was developed and tested in both cells lines and clinical tissue samples. Standard curves were generated for two peptides representing PSA and one peptide from each of two additional orthogonally validated biomarkers, AMACR and EZH2. The standard curves show high reproducibility, sensitivity, and good linearity. All four peptides were then measured in six clinically relevant cell lines and are in agreement with the biochemical characteristics of each individual cell line. The SID selected reaction monitoring‐MS/MS methodology was then transferred to tissue samples, in which the assay shows potential to differentiate benign disease from localized cancer and localized cancer from aggressive metastatic disease. These results establish the preliminary development of a rational targeted MS platform that strives to bridge the gap between discovery and validation of biomarkers for the detection of prostate cancer.     

The reaction of 2,4,6-trinitrobenzenesulphonic acid with amino acids, peptides and proteins

1. The kinetics of the reaction of 2,4,6-trinitrobenzenesulphonic acid with various amino acids, peptides and proteins were studied by spectrophotometry. 2. The reaction of the α- and -amino groups in simple amino acids was found to be second-order, and the unprotonated amino group was shown to be the reactive species. 3. By allowing for the concentration of unreactive −NH₃⁺ group, intrinsic reactivities for the free amino groups were derived and shown to be correlated with the basicities. 4. The SH group of N-acetylcysteine was found to be more reactive to 2,4,6-trinitrobenzenesulphonic acid than most amino groups. 5. The reactions of insulin, chymotrypsinogen and ribonuclease with 2,4,6-trinitrobenzenesulphonic acid were analysed in terms of three exponential rate curves, each referring to one or more amino groups of the proteins. 6. The reaction of lysozyme with 2,4,6-trinitrobenzenesulphonic acid was found to display an acceleration effect. 7. From the reaction of 2,4,6-trinitrobenzenesulphonic acid with glutamate dehydrogenase at several enzyme concentrations, it was possible to discern two sets of amino groups of different reactivity, and to show that the number of groups in each set was decreased by aggregation of the enzyme.     

Variation in continuous reaction norms: quantifying directions of biological interest

Thermal performance curves are an example of continuous reaction norm curves of common shape. Three modes of variation in these curves--vertical shift, horizontal shift, and generalist-specialist trade-offs--are of special interest to evolutionary biologists. Since two of these modes are nonlinear, traditional methods such as principal components analysis fail to decompose the variation into biological modes and to quantify the variation associated with each mode. Here we present the results of a new method, template mode of variation (TMV), that decomposes the variation into predetermined modes of variation for a particular set of thermal performance curves. We illustrate the method using data on thermal sensitivity of growth rate in Pieris rapae caterpillars. The TMV model explains 67% of the variation in thermal performance curves among families; generalist-specialist trade-offs account for 38% of the total between-family variation. The TMV method implemented here is applicable to both differences in mean and patterns of variation, and it can be used with either phenotypic or quantitative genetic data for thermal performance curves or other continuous reaction norms that have a template shape with a single maximum. The TMV approach may also apply to growth trajectories, age-specific life-history traits, and other function-valued traits.     

基于香草醛-高氯酸显色反应测定灵芝三萜的方法探讨与修正

本文报道了基于香草醛-高氯酸显色反应的分光光度法定量测定灵芝三萜的修正方法,并对该方法应用进行了探讨和优化。采用此方法检测了灵芝子实体中含量较高的几种三萜酸,结果表明若采用齐墩果酸为标准品检测灵芝三萜,检测结果远低于真实值。在光谱分析上,研究表明对紫外-可见光扫描吸收峰进行面积积分,获得的标准曲线的线性关系更优。     

Mathematics analysis of polymerase chain reaction kinetic curves

The paper reviews different approaches to the mathematical analysis of polymerase chain reaction (PCR) kinetic curves. The basic principles of PCR mathematical analysis are presented. Approximation of PCR kinetic curves and PCR efficiency curves by various functions is described. Several PCR models based on chemical kinetics equations are suggested. Decision criteria for an optimal function to describe PCR efficiency are proposed.     

Single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin

A study of the single turnover kinetics of the reaction between oxycytochrome P-450cam and reduced putidaredoxin was performed using the inhibitor metyrapone to trap the cytochrome immediately after release of the product, 5-exo-hydroxycamphor. EPR determinations of the concentrations of reduced putidaredoxin and ferric metyrapone-bound cytochrome at the same time points showed that there is no time lag between the oxidation of reduced putidaredoxin and the appearance of metyrapone-bound cytochrome. This implies that the rate constant for electron transfer is smaller than the rate constant for the later processes involved in product formation and release, lumped into a single step. Taking this restriction into account and doing computer simulation of absorbance versus time curves, previously obtained at various putidaredoxin concentrations using stopped-flow spectrophotometry, allowed bounds to be determined for rate constants of the processes within the reaction. At 4 degrees C in buffer at pH 7.4 with 0.50 M KCl, the rate constant for the bimolecular association of the two enzymes is between 3 and 20/microM.s; the rate constant for dissociation is between 12 and 600/s; the rate constant for electron transfer is between 60 and 100/s; and the rate constant for the later processes is at least 200/s.     

Feulgen nucleal reaction

Summary Electrophoretic means of separation revealed the presence of as many as five reaction products in Schiff-apurinic acid reaction at the maximum. They differed not only in their absorption maxima, but also in their ratios of apurinic acid phosphorus to fuchsin moiety. Some considerations on the reaction mechanism to account for the occurrence of these multiple reaction products have been made. The stoichiometry of Schiff-apurinic acid reaction was studied with respect to the main product responsible for the presentation of reaction color. A reaction product consisting of six or eight atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be formed, provided that the reagent of infinite concentration is used. From theoretical view point, a reaction product consisting of four atom moles of apurinic acid phosphorus per mole of fuchsin moiety is to be expected with the reagent of infinite concentration, provided that apurinic acid retains essentially the nucleotide sequence of its parent desoxyribonucleic acid except for some modification of the original purin nucleotide groups to react as aldehyde moieties, and provided that the reaction proceeds at a constant rate irrespective of the concentrations of the reagent.     

Determination of thiopental in urine sample with high-performance liquid chromatography using iodine-azide reaction as a postcolumn detection system

The reaction between iodine and azide ions induced by thiopental was utilized as a postcolumn reaction for chromatographic determination of thiopental. The method is based on the separation of thiopental on an Nova-Pak CN HP column with an acetonitrile-aqueous solution of sodium azide as a mobile phase, followed by spectrophotometric measurement of the residual iodine (lambda=350 nm) from the postcolumn iodine-azide reaction induced by thiopental after mixing an iodine solution containing iodide ions with the column effluent containing azide ions and thiopental. Chromatograms obtained for thiopental showed negative peaks as a result of the decrease in background absorbance. The detection limit (defined as S/N=3) was 20 nM (0.4 pmol injected amount) for thiopental. Calibration graphs, plotted as peak area versus concentrations, were linear from 40 nM. The elaborated method was applied to determine thiopental in urine samples. The detection limit (defined as S/N=3) was 0.025 nmol/ml urine. Calibration graphs, plotted as peak area versus concentrations, were linear from 0.05 nmol/ml urine. Authentic urine samples were analyzed, thiopental was determined at nmol/ml urine level.     

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.     

Crystalline desoxyribonuclease; digestion of thymus nucleic acid; the kinetics of the reaction

A study was made of the enzymatic properties of crystalline desoxyribonuclease. The general effect of the crystalline enzyme on its specific substrate, thymus nucleic acid, was found to be essentially the same as described by previous workers for the digestive action of crude preparations of the enzyme. The digestive action consists mainly in splitting thymus nucleic acid into fragments approaching the size of tetranucleotides. The digested nucleic acid is diffusible through collodion or cellophane membranes and is non-precipitable with strong acid, alcohol, or proteins. The digestion of thymus nucleic acid by desoxyribonuclease is accompanied by the liberation of one atom equivalent of free acid per four atoms of nucleic acid phosphorus. Crystalline desoxyribonuclease acts very slowly, if at all, in the absence of magnesium (or manganese) ions. The optimal concentration of magnesium ion required increases with the increase in concentration of the substrate but is independent of the enzyme concentration. The optimal pH range for the action of crystalline desoxyribonuclease is 6.0 to 7.0. A study was made of the kinetics of the digestion of thymus nucleic acid as manifested mainly by the gradual formation of acid-soluble split products. At low concentrations of nucleic acid, the process approximates closely a reaction of the first order, the unimolecular constant being independent of the concentration of desoxyribonuclease in the digestion mixture. At relatively higher concentrations of substrate, however, the initial rate of reaction decreases rapidly with the increase in concentration of substrate, and the reaction as a whole is represented by non-symmetric S-shaped curves apparently too complicated for a simple rational interpretation.     

Preparative scale enantioseparation of flurbiprofen by lipase-catalysed reaction

Effects of reaction media, alcohols and water on the enzyme activity of the immobilised Candida antarctica lipase were investigated for the separation of racemic flurbiprofen by an esterification reaction catalysed by immobilised enzyme in organic media. The S-enantiomer of flurbiprofen was directly resolved by the immobilised lipase esterification reaction in acetonitrile. Ping-Pong Bi–Bi kinetics were found to fit the initial reaction well of all the experimental runs. Model parameters for the reaction kinetics were evaluated from experiments at relatively low substrate concentrations, have shown to be applicable for preparative separation scale at high concentrations. Finally, the gram-scale production of single enantiomer with the optical purity of 93% e.e. was obtained.