Irreversible inhibition of jack bean urease by pyrocatechol

Pyrocatechol was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 25 degrees C. The inhibition was monitored by an incubation procedure in the absence of substrate and reaction progress studies in the presence of substrate. It was found that pyrocatechol acted as a time- and concentration dependent irreversible inactivator of urease. The dependence of the residual activity of urease on the incubation time showed that the rate of inhibition increased with time until there was total loss of enzyme activity. The inactivation process followed a non-pseudo-first order reaction. The obtained reaction progress curves were found to be time-dependent. The plots showed that the rate of the enzyme reaction in the final stages reached zero. From protection experiments it appeared that thiol-compounds such as L-cysteine, 2-mercaptoethanol and dithiothreitol prevented urease from pyrocatechol inactivation as well as the substrate, urea, and the competitive inhibitor boric acid. These results proved that the urease active site was involved in the pyrocatechol inactivation.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence,reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 °C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Inactivation of jack bean urease by N-ethylmaleimide: pH dependence, reversibility and thiols influence

N-Ethylmaleimide (NEM) was studied as an inactivator of jack bean urease at 25 degrees C in 20 mM phosphate buffer, pHs 6.4, 7.4, and 8.3. The inactivation was investigated by incubation procedure in the absence of a substrate. It was found that NEM acted as a time and concentration dependent inactivator of urease. The dependence of urease residual activity on the incubation time showed that the activity decreased with time until the total loss of enzyme activity. The process followed a pseudo-first-order reaction. A monophasic loss of enzyme activity was observed at pH 7.4 and 8.4, while a biphasic reaction occurred at pH 6.4. Moreover, the alkaline pH promoted the inactivation. The presence of thiol-compounds, such as L-cysteine, glutathione or dithiothreitol (DTT), in the incubation mixture significantly slowed down the rate of inactivation. The interaction test showed that the decrease of inactivation was an effect of NEM-thiol interaction that lowered NEM concentration in the incubation mixture. The reactivation of NEM-blocked urease by DTT application and multidilution did not result in an effective activity regain. The applied DTT reacted with the remaining inactivator and could stop the progress of enzyme activity loss but did not cause the reactivation. This confirmed the irreversibility of inactivation. Similar results obtained at pH 6.4, 7.4 and 8.4 indicated that the mechanism of urease inactivation by NEM was pH-independent. However, the pH value significantly influenced the process rate.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Inhibition of jack bean urease by p-benzoquinone: elucidation of the role of thiols and reversibility of the process

p-Benzoquinone (pBQ) was studied as an inhibitor of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 °C. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. The influence of the inhibitor concentration and the preincubation time on the enzyme activity was elucidated. It was found that increase in pBQ concentration resulted in a linear decrease of urease activity. The dependence of the enzyme activity on the preincubation time showed that the rate of inhibition rapidly decreased at the beginning of the process in order to achieve the constant value. The inhibition became time independent in the studied time range. This observation is characteristic of a slow binding mechanism of inhibition. The protective experiment proved that the urease active site is involved in the binding of pBQ. High effectiveness of thiol protectors against pBQ inhibition indicates the strategic role of the active site sulfhydryl group in the blocking process. There were two methods used for reactivation of pBQ-inhibited urease. The dilution of the urease-pBQ complex in urea solution did not result in a regain of enzyme activity. Alternatively, the addition of dithiothreitol into the urease-pBQ mixture caused the instant and efficient reactivation of the enzyme. The experiments showed that the nature of the urease-pBQ complex is irreversible but the application of a specific thiol reagent can release the active enzyme from the complex.     

Multi-step analysis of Hg2+ ion inhibition of jack bean urease

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).     

Kinetics of jack bean urease inhibition by 2,3-dichloro-1,4-naphthoquinone. Elucidation of the mechanism: redox cycling and sulfhydryl arylation

The inhibition of jack bean urease by 2,3-dichloro-1,4-naphthoquinone (DCNQ) was studied at ambient temperature in 20?mM phosphate buffer, pH 7.8. The process was investigated by incubation procedure in the absence of substrate. It was found that DCNQ acted as a time- and concentration-dependent inactivator of urease. The time course of the reaction displayed a biphasic mode. Each phase followed a pseudo-first-order kinetics, however the inactivation rate at the first phase was significantly faster than at the next one. The biphasity indicated the complex mechanism of DCNQ action on urease. Quinones action on proteins has been elucidated as at least two processes: direct arylation of essential protein thiols and/or indirect oxidation of essential thiols by reactive oxygen species (ROS) realising during quinone reduction to semiquinones. The next evidence of the studied mechanism was provided by the reactivation experiment that showed the participation of reversible and irreversible processes in the inactivation. The application of dithiothreitol (DTT) into DCNQ blocked-urease solution resulted in an effective enzyme activity regain which quickly returned to 70?±?10%. The irreversible inactivation of urease was attributed to DCNQ arylation of thiol residues in the protein. On the other hand, it was assumed that the reversible inactivation was a result of the action of ROS such as H₂O₂. Presence of H₂O₂ in the incubation system was proved by an experiment with the use of catalase. The enzyme by the elimination of H₂O₂ decreased DCNQ inactivating influence on urease. The comparison of participation of the fast and slow phase in the inactivation with the percentage of the process reversibility was assumed that the fast period was a result of the arylation mechanism while the slow phase was related to the oxidative influence of H₂O₂.     

5-Hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) influence on jack bean urease activity: Elucidation of the difference in inhibition activity

The aim of this study was elucidation of the difference in inhibition influence of 5-hydroxy-1,4-naphthoquinone (juglone) and 2-hydroxy-1,4-naphthoquinone (lawsone) on jack bean urease activity. It was found that juglone acted as a strong, time and concentration dependent inactivator of urease. On the contrary, lawsone showed an inconsiderable inhibition influence. The reactivation of juglone modified urease showed the participation of reversible and irreversible contribution in the inactivation. In the presence of an excess of DTT, urease inactivated by juglone regained 70% of its activity. The reversible inactivation was attributed to oxidation of the essential urease thiols by reactive oxygen species (ROS) realizing during reduction of juglone to seminaphthoquinone. Presence of hydrogen peroxide in the incubation system was proved by direct determination and by application of catalase. The irreversible contribution in the inhibition was assumed as an arylation of urease thiol groups by juglone. The insignificant urease inhibition by lawsone was concluded as an effect of a low hydrogen peroxide generation and lawsone resistance for reaction with protein thiols. It was found that lawsone well reacted with l-cysteine, poorly with glutathione and hardly with urease thiols. The observed sequence was arranged according the rule the more complex thiol the less susceptible for reaction with lawsone. On the other hand, juglone displayed an excellent reactivity towards both thiols and urease. Thus, this indicated a significance of a steric hindrance which appeared when the hydroxyl group changing position from 5 in juglone (5-hydroxy-1,4-naphthoquinone) to 2 in lawsone (2-hydroxy-1,4-naphthoquinone).     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 10(-6) mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

STUDIES ON THE REGULATION OF GABA SYNTHESIS: SUBSTRATE-PROMOTED DISSOCIATION OF PYRIDOXAL-5''-PHOSPHATE FROM GAD

The association between glutamate decarboxylase (GAD) and its cofactor, pyridoxal-5′-phos-phate (pyridoxal-P), was studied using 20,0000 supernatant of rat brain. In this preparation GAD required added pyridoxal-P to maintain a linear reaction rate beyond 5 min of incubation. Following exhaustive dialysis the enzyme was more than 83% saturated with cofactor indicating that the cofactor was tightly bound to the enzyme. When incubations were performed in the presence of glutamate and without added pyridoxal-P there was a progressive inactivation of the enzyme which was dependent on the glutamate concentration. This lost activity was almost completely recovered by addition of pyridoxal-P to the dialyzed glutamate-inactivated enzyme. The results suggest that glutamate inactivates GAD by promoting the dissociation of pyridoxal-P from the enzyme thereby producing inactive apoen-zyme which can be reactivated by combining with available pyridoxal-P. This interpretation is supported by the finding that progress curves for the reaction were accurately described over a 30 min incubation period and 10-fold glutamate concentration range by an integrated rate equation which takes the glutamate-promoted dissociation of cofactor into account. The progressive inactivation could not be attributed to denaturation of the enzyme, impurities in the substrate, effects of pH, depletion of substrate, protein concentration, sulfhydryl reagents or product inhibition. The results presented here also show that certain precautions must be adopted to accurately measure GAD activity in the absence of added pyridoxal-P as has been widely done in studies of drug action. Specifically, measurements must be made at short times of incubation and low concentrations of glutamate to minimize the glutamate-promoted inactivation of the enzyme.     

Inactivation of o-diphenoloxidase in the pyrocatechol oxidation reaction

The inactivation kinetics of o-diphenoloxidase isolated from potato tubers was studied in the process of pyrocatechol oxidation. The enzyme when saturated with the substrate is inactivated with the inactivation rate constant kin = 0.5-1.0 min-1; kin depends on the initial concentration of pyrocatechol. The ultimate yield of the enzymic reaction product increases linearly with the initial concentration of the enzyme. Introduction of ethylene-diaminosulphate, a substance which condenses with o-quinones, does not increase the operation stability of o-diphenoloxidase. The data obtained evidence for inactivation of o-diphenoloxidase either at the level of the enzyme-substrate complex or due to bimolecular reaction with the substrate.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves

The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Inhibition of urease by bismuth(III): Implications for the mechanism of action of bismuth drugs

Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)₃ are competitive inhibitors of jack bean urease with K _i values of 1.74 ± 0.14 and 1.84 ± 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K _i value of 1.17 ± 0.09 mM. A ¹³C NMR study showed that Bi(Cys)₃ reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 ± 0.17 and 11.94 ± 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi³⁺ to the active site cysteine (Cys³¹⁹) as the mechanism of enzyme inactivation.     

Inhibition of beta-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new beta-lactam antibiotic.

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.     

Inhibition of the microsomal N-hydroxylation of 2-amino-6-nitrotoluene by a metabolite of methimazole

Inhibition studies were used to investigate the identity of the microsomal enzyme(s) responsible for the NADPH-dependent N-hydroxylation of 2-amino-6-nitrotoluene. The N-hydroxylation reaction was inhibited by several cytochrome P-450 inhibitors as well as by methimazole, a substrate for flavin-containing monooxygenase. Heat inactivation of flavin-containing monooxygenase had no effect on the rate of the reaction but abolished the inhibition by methimazole. These results indicate that the flavin-containing monooxygenase-mediated metabolism of methimazole produced an inhibitor of the cytochrome P-450-catalyzed N-hydroxylation reaction. When glutathione was included in the incubation the inhibition by methimazole was abolished, presumably due to the reduction of oxygenated metabolites of methimazole. These results show that methimazole inhibition does not necessarily implicate flavin-containing monooxygenase in microsomal N-hydroxylation reactions.     

Inactivation of jack bean urease by allicin

Allicin--diallyl thiosulfinate--is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20 mM phosphate buffer, pH 7.0 at 22 degrees C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).     

Fluorimetric urease inhibition assay on a multilayer microfluidic chip with immunoaffinity immobilized enzyme reactors

We fabricated a three-layer polydimethylsiloxane (PDMS)-based microfluidic chip for realizing urease inhibition assay with sensitive fluorescence detection. Procedures such as sample prehandling, enzyme reaction, reagent mixing, fluorescence derivatization, and detection can be readily carried out. Urease reactors were prepared by adsorption of rabbit immunoglobulin G (IgG) and immunoreaction with urease-conjugated goat anti-rabbit IgG. Acetohydroxamic acid (AHA) as a competitive inhibitor of urease was tested on the chip. Microfluidically generated gradient concentrations of AHA with substrate (urea) were loaded into urease reactors. After incubation, the produced ammonia was transported out of reactors and then reacted with o-phthalaldehyde (OPA) to generate fluorescent products. Urease inhibition was indicated by a decrease in fluorescence signal detected by microplate reader. The IC₅₀ value of AHA was determined and showed good agreement with that obtained in microplate. The presented device combines several steps of the analytical process with advantages of low reagent consumption, reduced analysis time, and ease of manipulation. This microfluidic approach can be extended to the screening of inhibitory compounds in drug discovery.     

Inactivation of Jack Bean Urease by Allicin

Allicin—diallyl thiosulfinate—is the main biologically active component of freshly crushed garlic. Allicin was synthesized as described elsewhere and was tested for its inhibitory ability against jack bean urease in 20?mM phosphate buffer, pH 7.0 at 22°C. The results indicate that allicin is an enzymatic inactivator. The loss of urease activity was irreversible, time- and concentration dependent and the kinetics of the inactivation was biphasic; each phase, obeyed pseudo-first-order kinetics. The rate constants for inactivation were measured for the fast and slow phases and for several concentrations of allicin. Thiol reagents, and competitive inhibitor (boric acid) protected the enzyme from loss of enzymatic activity. The studies demonstrate that urease inactivation results from the reaction between allicin and the SH-group, situated in the urease active site (Cys592).