Initial Rate Measurements Research Articles

Single time-point analysis of product and substrate inhibition.

When enzyme inhibition by either the product or excess substrate occurs, it is possible to determine the characteristic kinetic parameters based on [P]/t measurements, even when a large proportion of the substrate is converted. The advantages of various approaches are discussed. Most of them allow a good estimation of the V and Km values. Conversely, the determination of Kp (product inhibition) and Ki (inhibition by excess substrate) can be more challenging. In the first case, determination of the type of inhibition requires more complex experiments that are beyond the scope of the present contribution. In the second, the inhibition constant Ki can only be roughly estimated. In an experimental approach, we compared the results obtained either with initial rate measurements or with 50 to 60% conversion of the substrate. Similar values of V and Km were obtained. Measurements involving the conversion of a large proportion of substrate are particularly advantageous when the assay method is difficult or time-consuming, or when obtaining the substrate presents experimental difficulties or involves substantial costs.

Reaction Kinetics using a Chemputable Framework for Data Collection and Analysis

AbstractAutomated chemistry platforms have been widely explored, but many focus on fixed tasks for chemical synthesis or analysis. However, a typical synthetic chemistry workflow utilizes both, such as kinetic measurements for reaction development and optimization. Due to their repetitive and time‐consuming nature, kinetic measurements are often omitted, which limits the mechanistic investigation of reactions. Herein, we present a “Chemputer” platform with on‐line analytics (UV/Vis, NMR) which automates routine kinetic measurements. The system's capabilities are showcased by exploring an inverse electron‐demand Diels–Alder using initial rate measurements, a metal complexation using variable time normalization analysis (VTNA), and formation of a series of tosylamide derivatives using Hammett analysis. Over 60 individual experiments are presented which required minimal intervention, highlighting the significant time savings of automation. Owing to the modular design of the platform, which facilitates rapid integration of commercial analytical tools, our approach is widely accessible and adjustable to the reaction under investigation. The platform is operated using the chemical programming language, XDL, hence experimental procedures and results are stored in a precise, computer‐readable format. We propose that widespread adoption of this reporting protocol in the chemical community could build a database of validated kinetic data beneficial for Machine Learning.

Reaction Kinetics using a Chemputable Framework for Data Collection and Analysis.

Automated chemistry platforms have been widely explored, but many focus on fixed tasks for chemical synthesis or analysis. However, a typical synthetic chemistry workflow utilizes both, such as kinetic measurements for reaction development and optimization. Due to their repetitive and time-consuming nature, kinetic measurements are often omitted, which limits the mechanistic investigation of reactions. Herein, we present a "Chemputer" platform with on-line analytics (UV/Vis, NMR) which automates routine kinetic measurements. The system's capabilities are showcased by exploring an inverse electron-demand Diels-Alder using initial rate measurements, a metal complexation using variable time normalization analysis (VTNA), and formation of a series of tosylamide derivatives using Hammett analysis. Over 60 individual experiments are presented which required minimal intervention, highlighting the significant time savings of automation. Owing to the modular design of the platform, which facilitates rapid integration of commercial analytical tools, our approach is widely accessible and adjustable to the reaction under investigation. The platform is operated using the chemical programming language, XDL, hence experimental procedures and results are stored in a precise, computer-readable format. We propose that widespread adoption of this reporting protocol in the chemical community could build a database of validated kinetic data beneficial for Machine Learning.

Higher precision in initial rates may be achievable: A test of a pseudo-statistical method

There has been a concerted effort at establishing the best method for the measurement of initial rates for various purposes, including the calculation of kinetic parameters, the maximum velocity (Vmax), and the Michaelis-Menten constant (KM). The objectives of this research are: 1) to derive equations without KM for the determination of the Vmax in particular and vice versa; 2) to determine the KM and Vmax with other equations other than the Michaelian equation; and 3) to subject the calculated and extrapolated kinetic parameters to pseudo-statistical remediation where necessary as a test of their viability and usefulness. The study was experimental and theoretical. It is supported by the Bernfeld method of enzyme assay. By graphical means, the Vmax and KM values for galactosidase respectively range between 163 and 185 mM/min and between 2.07 and 2.77 mg/L; the range by calculations is 177 and 214 mM/min and 2.45 and 3.311 mg/L, subject to pseudo-statistical remediation. Overall, the ranges of Vmax and KM values for alpha-amylase from both the graphical method and calculation are, respectively, 1.095 to 1.018 mM/min and 18.15 to 20.554 g/L. The equations for the determination of the KM and Vmax, which are respectively invariant with respect to each other, were rederived. The initial rates must not be characterised by a mixture of conditions that validate different quasi-steady-state assumptions if the true KM and Vmax are of interest. The new pseudo-statistical method for the remediation of error in all measurements, if necessary, is viable, useful, and robust.

The measurement of true initial rates is not always absolutely necessary to estimate enzyme kinetic parameters

In the chapters dealing with enzyme reactions, the authors of all Biochemistry textbooks and of even more specialized texts consider that the characteristic parameters (kcat and Km) must be determined under initial or steady-state rate conditions. This implies the transformation of a very limited proportion of substrate (at most 10–20%) or a continuous recording of the product or substrate concentration vs. time. Both options can present practical difficulties. Is it possible to get around these very stringent conditions? Here we show that in the most favourable cases up to 70% of the substrate can be converted resulting in systematic errors on the parameters (that can easily be taken account of) if the simple Henri-Michaelis–Menten equation is utilised. Alternatively, the integrated form of the same equation directly yields excellent estimates of the same parameters. Our observations should greatly facilitate the task of researchers who study systems in which measurements of the reaction progress are painstaking or when substrate concentrations close to the detection limit must be used. The general conclusion is that it is not always absolutely necessary to determine initial or steady-state rates to obtain reliable estimations of the enzyme kinetic parameters..

Production of Fructooligosaccharides Using a Commercial Heterologously Expressed Aspergillus sp. Fructosyltransferase

The catalytic properties of Seqenzym® FT, a fungal fructosyltransferase heterologously expressed in yeasts, were investigated at a temperature of 55 °C and pH 5.5. The initial rate measurements showed that the transfructosylation rate was only slightly inhibited by sucrose above the concentration of 1.5 M. A rather low level of hydrolytic side activity was observed even at sucrose concentrations as low as 0.25 M. In progress curve experiments, the mass yield of fructooligosaccharides (FOS) reached a maximum value of 57% at this sucrose concentration, although it dropped to about 35% later on. At high initial sucrose concentrations up to 2 M, the FOS yield reached a maximum value of approximately 63% at a sucrose conversion of approximately 90%. Although neither the yield nor the conversion changed much later on, the progress of the reaction was manifested by the gradual depletion of shorter chain FOS, 1-kestose and nystose, and the accumulation of 1-β-fructofuranosyl nystose. At initial sucrose concentrations of 2 M, the degree of polymerization expressed through the number of fructosyl units grew from 2.3 at a conversion degree of 87% to 3.1 at a conversion degree of 94%. Compared to other commercial preparations, Seqenzym® FT can better produce FOS with a higher degree of polymerization.

PH-Selective Reactions to Selectively Reduce Cancer Cell Proliferation: Effect of CaS Nanostructures in Human Skin Melanoma and Benign Fibroblasts.

An acidic extracellular pH value (pHe) is characteristic of many cancers, in contrast to the physiologic pHe found in most benign cells. This difference in pH offers a unique opportunity to design and engineer chemicals that can be employed for pH-selective reactions in the extracellular fluid of cancer cells. The viability of human skin melanoma and corresponding fibroblasts exposed to CaS dispersions is reported. The viability of melanoma cells decreases with CaS dispersion concentration and reaches 57% at 3%, a value easily distinguishable from melanoma control experiments. In contrast, the viability of benign fibroblasts remains nearly constant within experimental error over the range of dispersion concentrations studied. The CaS dispersions facilitate vinculin delocalization in the cytoplasmic fluid, a result consistent with improved focal adhesion kinase (FAK) regulation in melanoma cells. Thermodynamic considerations are consistent with the formation of from CaS in the presence of protons. The thermodynamic prediction is verified in independent experiments with solid CaS and acidic aqueous solutions. The amount of formed decreases with pH. An activation energy for the process of (30 ± 10) kJ/mol in the temperature range of 280 to 330 K is estimated from initial rate measurements as a function of temperature. The total Gibbs energy minimization approach was employed to establish the distribution of sulfides-including in the gas and aqueous phases-from the dissociation of CaS as a function of pH to mimic physiologically relevant pH values. Theoretical calculations suggest that partially protonated CaS in solution can be stable until the sulfur atom bonds to two hydrogen atoms, resulting in the formation of Ca2+ and , which can be solvated and/or released to the gas phase. Our results are consistent with a model in which CaS is dissociated in the extracellular fluid of melanoma cells selectively. The results are discussed in the context of the potential biomedical applications of CaS dispersions in cancer therapies.

About the role of iron on the alteration of simplified nuclear glasses in deaerated solutions at 50°C

The effect of Fe on the alteration of two SiO2-B2O3-Na2O based glasses (Fe2O3-free or containing 3.1 mol.% Fe2O3) was studied by measurement of initial alteration rates and long-term leaching experiments at 50°C in initially pure water or in a 10−2 mol.L−1 FeCl2 solution. The glass alteration kinetics were monitored by periodical leachate analyses; and morphology, structure and composition of solid alteration products were characterized at the end of long-term experiments (i.e. after 323 days).Iron(III) initially incorporated in the glass strengthens the glass network, leading to a decrease of the initial dissolution rate (from 0.15 to 0.026 g.m−2.d−1 at 50°C). The environment of Fe(III) incorporated into the gel depends on the pH of the leaching solution. A change of Fe(III) coordination from fourfold (i.e. charge-compensated by Na+ ions) to sixfold is expected when the pH decreases. Whatever its coordination, Fe3+ hinders the gel restructuring into a passivating barrier. Thus, the addition of Fe in the glass composition ultimately leads to higher alteration extent (≈1.5 times greater after 323 days of leaching in our experiments).In experiments where Fe cations are provided by the leaching medium (mainly at the +II oxidation state), precipitation of Fe-silicates is systematically observed, as well as an increase in glass alteration compared to experiments led in initially pure water (by a factor slightly less than 2). A drastic change in the average pH of experiments (from ≈9 in initially pure water to ≈6 in FeCl2 solution) is also observed. Among mechanisms proposed in the literature to explain the detrimental effect of Fe-silicates precipitation, it seems that the decrease in pH resulting from this precipitation is the main cause of increase in glass alteration observed in this study.

Direct Measurement of Initial Rate of Enzyme Reaction by Electrokinetic Filtration Using a Hydrogel-plugged Capillary Device.

A novel electrokinetic filtration device using a plugged hydrogel was developed to directly measure the initial rate of enzyme reactions. In the proposed method, the enzyme reaction proceeded only for a short time when the substrate was passed through a thin layer of enzyme trapped by the hydrogel without any lag times for mixing and detection. In experimental conditions, alkaline phosphatase (enzyme) was filtrated at a cathodic-side interface of the plugged hydrogel by molecular sieving effect, providing the thin enzyme zone whose thickness was approximately 100 μm. When 4-methylumberiferyl phosphate (substrate) was electrokinetically introduced into the device after trapping the enzyme, 4-methylumberiferone (product) was generated by the enzyme reaction for only 1.26 s as the substrate passed through the trapped enzyme zone. As a result, the initial rate of the enzyme reaction could be directly calculated to 31.0 μM/s by simply dividing the concentration of the product by the tunable reaction time. Compared to the initial rate obtained by mixing the enzyme and substrate solutions, the value of the maximum velocity of the enzyme reaction was 30-fold larger than that in the mixing method due to the preconcentration of the enzyme by trapping. The Michaelis-Menten constant in the proposed method was 2.7-fold larger than that in the mixing method, suggesting the variation of changes in the equilibrium of complex formation under the experimental conditions.

Growth Inhibition by Amino Acids in Saccharomyces cerevisiae.

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5–5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates.

A simple linearization method unveils hidden enzymatic assay interferences

Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

Determination of iodine content in Fijian foods using spectrophotometric kinetic method

The spectrophotometric kinetic method was validated for the determination of iodine in different food samples. The method is based on the iodide catalysed reduction of Ce4+ to Ce3+ by As3+. The absorbance of the kinetic indicator reaction was measured at 370 nm for exactly 1 min at 37 °C. The change in absorbance per min, as a measure of initial rate, was plotted against the different iodine concentrations to achieve a linear calibration equation with the R2 value of 0.9998 which showed excellent reproducibility. The limit of detection (LOD) was 1.54 ng/mL and the limit of quantification (LOQ) was 4.90 ng/mL. The incineration of the food organic matter was achieved by ashing the food samples at 600 °C using KOH and ZnSO4 in steps for 3 h. Trace levels of iodine (ng) were determined successfully using the validated spectrophotometric kinetic method for the 9 food samples (36 sub-samples). The Fiji seaweeds, lumiwawa (brown seaweed) showed the highest iodine content being 6373.30 ± 0.39 ng/g followed by sea grapes (green seaweed) 1162.81 ± 0.61 ng/g, lettuce 114.81 ± 0.08 ng/g, English cabbage 108.40 ± 0.06 ng/g, Chinese cabbage 104.01 ± 0.06 ng/g, pumpkin 101.24 ± 0.08 ng/g, long bean 97.61 ± 0.10 ng/g, banana 76.18 ± 0.10 ng/g and tomato 40.32 ± 0.04 ng/g. The coefficient of variation for the sample analysis was <5.31% with a mean and standard deviation of 2.55 ± 0.17% for the food samples analysed. The recovery analysis of iodine from standard samples ranged from 99.84 ± 0.91% to 100.24 ± 5.92% with an excellent average recovery of 100.06 ± 3.16%. The analytical coefficient of variation was calculated to be 0.34% for the food samples analysed. This shows exceptional system analytical stability of the method used in this study.

The [Rh(Xantphos)]+ catalyzed hydroboration of diphenylacetylene using trimethylamine-borane

The rhodium(I) complex [Rh(κ3-P,O,P-Xantphos)(η2-PhC≡CPh)][BArF4] (ArF = 3,5-(CF3)2C6H4) is an effective catalyst for the cis-selective hydroboration of the alkyne diphenylacetylene using the amine-borane H3B·NMe3. Detailed mechanistic studies, that include initial rate measurements, full simulation of temporal profiles for a variety of catalyst and substrate concentrations, and speciation experiments, suggest a mechanism that involves initial coordination of alkyne and a saturation kinetics regime for amine-borane binding. The solid-state molecular structure of a model complex that probes the proposed resting state is also reported, [Rh(κ3-P,O,P-Xantphos)(NCMe)(η2-PhC≡CPh)][BArF4].

How to extend range linearity in enzyme inhibition-based biosensing assays

Bioassays based on enzyme inhibition are analytical tools widely employed for inhibitor analysis. Beside the conventional analytical techniques such as chromatography and mass spectrometry, these bioassays are cost-effective, easy to use, and suitable for in situ measurement but they are often characterised by a quite narrow linear range.Herein, we report a novel graphical method based on integrated Michaelis-Menten equation, valid for all types of reversible inhibition, which provides an extended linear range. The suitability of this innovative approach was demonstrated in the case of fluoride quantification using a colorimetric bioassay based on acetylcholinesterase inhibition. The “half time reaction”, estimated by the progress curve of cholinesterase inhibition, was plotted versus the fluoride inhibitor concentration, observing an extended linear range up to 5 mM, instead of 0.6 mM using initial rate measurements. The applicability of this new concept was further demonstrated in the case of catalase enzyme inhibited by cyanide.Furthermore, it was demonstrated that fixed substrate conversion at level of 10–50% allows determination of inhibitor concentration in a wide linear range with high precision and in short time of analysis. This novel theoretical and practical approach allows for the extension of the linear range without any further experiments, with several advantages including low reagent consumption, reduced waste generation and time of measurement.

Investigation on adsorption kinetics of heavy metals by rice husk

Removal of heavy metal ions and their derivative forms from the environment has become a priority to safeguard the quality of the ecosystem. In this context, the present study is based on the removal of Cd(II), Cr(III), Cu(II), Ni(II), Pb(II) and Zn(II) from synthetic industrial effluents using heat-treated rice husk, which can be used to develop cost-effective and ecofriendly effluent treatment procedures. The characterisation experiments reveal that Si available in rice husk in high percentage and many chemical functional groups present on the surface are involved in the adsorption mechanism. Metal ions commonly present in effluents of metal finishing industries were specially considered for their removal. The optimum firing temperature of rice husk, determined using 10.0 mg L-1 solutions of each metal ion, was 100 oC. The effect of other experimental parameters, such as contact time and pH were also investigated using rice husk fired at the optimised temperature of 100 oC. Kinetics studies conducted for the interaction of the above metal ions and rice husk lead to the validity of pseudo second order kinetics with high regression coefficients. The initial rate of adsorption ( ho ) of metal ions varies in the order of Pb(II) > Zn(II) > Cd(II) > Cu(II) > Ni(II) > Cr(III). According to the diffusion models investigated, Weber and Morris intraparticle diffusion model supports the data for rate constant of particle diffusion, which varies in the same order as observed in initial adsorption rate measurements ( ho ).

A general correction to catalytic rates determined for nonprocessive exo-depolymerases acting on both substrate and product in the initial-rate measurement

We recently reported on the kinetics of the polygalacturonase TtGH28 acting on trimer and dimer substrates. When the starting substrate for hydrolysis is the trimer, the product dimer is also subject to hydrolysis, resulting in discrepancies when either the concentration of dimer or monomer product is used for analysis of trimer hydrolysis. Here, we derive a method for determining catalytic rates of exo-hydrolases acting on trimer (and higher order) substrates when products may also be substrates for hydrolysis and show how this correction may be applied for TtGH28.

Carbon–Carbon σ-Bond Transfer Hydrogenation with DMF Catalyzed by Cobalt Porphyrins

Cobalt porphyrins were found to catalyze the transfer hydrogenation of the carbon–carbon σ bond of [2.2]paracyclophane (PCP) with the solvent DMF serving as the hydrogenating agent. Successful trapping experiments with benzene solvent and the kinetic isotope effect (4.9) suggested the presence of benzyl radical intermediates in undergoing hydrogen atom transfer from DMF as the rate-limiting step. The rate law was established by initial rate measurements to be rate = kobs[CoII(ttp)][PCP].

Glass dissolution rate measurement and calculation revisited

Aqueous dissolution rate measurements of nuclear glasses are a key step in the long-term behavior study of such waste forms. These rates are routinely normalized to the glass surface area in contact with solution, and experiments are very often carried out using crushed materials. Various methods have been implemented to determine the surface area of such glass powders, leading to differing values, with the notion of the reactive surface area of crushed glass remaining vague. In this study, around forty initial dissolution rate measurements were conducted following static and flow rate (SPFT, MCFT) measurement protocols at 90 °C, pH 10. The international reference glass (ISG), in the forms of powders with different particle sizes and polished monoliths, and soda-lime glass beads were examined. Although crushed glass grains clearly cannot be assimilated with spheres, it is when using the samples geometric surface (Sgeo) that the rates measured on powders are closest to those found for monoliths. Overestimation of the reactive surface when using the BET model (SBET) may be due to small physical features at the atomic scale—contributing to BET surface area but not to AFM surface area. Such features are very small compared with the thickness of water ingress in glass (a few hundred nanometers) and should not be considered in rate calculations. With a SBET/Sgeo ratio of 2.5 ± 0.2 for ISG powders, it is shown here that rates measured on powders and normalized to Sgeo should be divided by 1.3 and rates normalized to SBET should be multiplied by 1.9 in order to be compared with rates measured on a monolith. The use of glass beads indicates that the geometric surface gives a good estimation of glass reactive surface if sample geometry can be precisely described. Although data clearly shows the repeatability of measurements, results must be given with a high uncertainty of approximately ±25%.

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA.

Relationships between lumbar inter-vertebral motion and lordosis in healthy adult males: a cross sectional cohort study.

BackgroundIntervertebral motion impairment is widely thought to be related to chronic back disability, however, the movements of inter-vertebral pairs are not independent of each other and motion may also be related to morphology. Furthermore, maximum intervertebral range of motion (IV-RoMmax) is difficult to measure accurately in living subjects. The purpose of this study was to explore possible relationships between (IV-RoMmax) and lordosis, initial attainment rate and IV-RoMmax at other levels during weight-bearing flexion using quantitative fluoroscopy (QF).MethodsContinuous QF motion sequences were recorded during controlled active sagittal flexion of 60° in 18 males (mean age 27.6 SD 4.4) with no history of low back pain in the previous year. IV-RoMmax, lordotic angle, and initial attainment rate at all inter-vertebral levels from L2-S1 were extracted. Relationships between IV-RoMmax and the other variables were explored using correlation coefficients, and simple linear regression was used to determine the effects of any significant relationships. Within and between observer repeatability of IV-RoMmax and initial attainment rate measurements were assessed in a sub-set of ten participants, using the intra-class correlation coefficient (ICC) and standard error of measurement (SEM).ResultsQF measurements were highly repeatable, the lowest ICC for IV-RoMmax, being 0.94 (0.80–0.99) and highest SEM (0.76°). For initial attainment rate the lowest ICC was 0.84 (0.49–0.96) and the highest SEM (0.036). The results also demonstrated significant positive and negative correlations between IV-RoMmax and IV-RoMmax at other lumbar levels (r = −0.64–0.65), lordosis (r = −0.52–0.54), and initial attainment rate (r = −0.64–0.73). Simple linear regression analysis of all significant relationships showed that these predict between 28 and 42 % of the variance in IV-RoMmax.ConclusionsThis study found weak to moderate effects of individual kinematic variables and lumbar lordosis on IV-RoMmax at other intervertebral levels. These effects, when combined, may be important when such levels are being considered by healthcare professionals as potential sources of pain generation. Multivariate investigations in larger samples are warranted.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-0975-1) contains supplementary material, which is available to authorized users.