Immobilized enzyme kinetics analyzed by flow-through microfluorimetry

Hofmann, Jürgen; Sernetz, M.

doi:10.1016/s0003-2670(00)81494-4

Cited by 59 publications

(27 citation statements)

References 6 publications

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“…The amount of substrate used for each reaction is in the nanogram range depending on the concentration used. Thus, the amount of both the enzyme and the substrate required are similar to what has been found with other microchipbased enzyme assays and are four orders of magnitude improved relative to the traditional plate assay [11,26,27]. Thus, the use of optically gated sample introduction on a microchip for enzyme assays not only successfully solves the problem of fluorescence interference that results from using FMG as the substrate, but also should lead to low-cost analysis because a small amount of the sample is required.…”

Section: Resultssupporting

confidence: 72%

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Ewing

2004

Analytical and Bioanalytical Chemistry

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The ability to perform enzyme assays on microchips is demonstrated using optically gated sample introduction. The hydrolysis of fluorescein mono- beta- d-galactopyranoside (FMG) by beta- d-galactosidase ( beta-Gal) is continuously monitored using a microchip for 5 to 10 min. The outcome of the reaction was analyzed by performing serial on-chip separations of fluorescent substrate, FMG, and product, fluorescein. Kinetic information about beta-Gal has been successfully obtained by varying the concentration of FMG. beta-Gal enzymes from two different sources including bovine liver and E. coli., have been examined and compared to each other and to results obtained using traditional assay methods. In addition, the competitive inhibition of beta-Gal by phenylethyl beta- d-thiogalactoside (PETG) and beta-lactose has been studied using this technique. PETG is found to have higher inhibition than lactose in the hydrolysis. This separation-based enzyme assay technique avoids the possible fluorescence interference between FMG and fluorescein, which is a problem with the traditional plate assay method. Additionally, the amount of the enzyme and substrate required with this technique is at least four orders of magnitude lower than the traditional plate assay method. By using optically gated sample introduction, microchips allow continuous serial injections and separations without any potential switch, thus making this technique ideal as a sensor for enzyme assays. This technique should therefore be valuable for high-throughput screening in the drug discovery industry.

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Section: Resultssupporting

confidence: 72%

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Ewing

2004

Analytical and Bioanalytical Chemistry

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“…Derivatives of 7-hydroxycoumarin such as 7-hydroxy-4-methylcoumarin, 6,8-difluoro-7-hydroxy-4-methylcoumarin and 7-hydroxycoumarin-3-carboxylic acid all have a hydroxyl, which can be modified to get various glycosidases substrates [35,90,[92][93][94]. These substrates have a similar response mechanism to that shown in Fig.…”

Section: Spectroscopic Probes With Glycosidase-cleavable Active Bondsmentioning

confidence: 95%

“…Unlike substrates based on fluorescein, resorufin-based substrates require only a single-step hydrolytic reaction to recover full fluorescence [90]. An example of the process is shown in Fig.…”

Section: Spectroscopic Probes With Glycosidase-cleavable Active Bondsmentioning

confidence: 99%

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Progress in Spectroscopic Probes with Cleavable Active Bonds

Chen¹,

Sun²,

2006

COC

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Spectroscopic probes may be defined as the molecules that can react with analytes (targets) accompanying the changes of their spectroscopic (chromogenic, fluorescent, or chemiluminescent) properties; based on such changes the targets can thus be determined. Spectroscopic probes have been extensively investigated and used widely in many fields because of their powerful ability to improve analytical sensitivity and to offer greater temporal and spatial sampling capability. In this review, special interest is devoted to a new type of spectroscopic probe that is constructed with a cleavable active bond as a linker. This type of spectroscopic probe, developed greatly in the past few years, has opened a novel alternate route to the specific determination of analytes with high hydrolytic reactivity, e.g., from metal ions to enzyme activity, and enabled many biological processes to be monitored in situ and in real-time. Theoretically, various photophysical processes, such as photoinduced electron transfer, photoinduced proton transfer, and fluorescence resonance energy transfer, can be used to design spectroscopic probes with cleavable active bonds for selective detection of analytes. Herein we review the progress and application of this type of spectroscopic probe, including spectroscopic response mechanism and the probes with active bonds cleavable by enzyme, metal ion and reactive oxygen species.

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“…1). The kinetic constants for this enzyme-substrate pair are K M ϭ 380 M and k cat ϭ 730 s Ϫ1 (19), which were also derived from single-enzyme molecule experiments (4). The strong inhibition of ␤-galactosidase by D-galactal was elucidated through bulk experiments (13,(20)(21)(22).…”

mentioning

confidence: 99%

Stochastic inhibitor release and binding from single-enzyme molecules

Gorris

Rissin

Walt

2007

Proc. Natl. Acad. Sci. U.S.A.

115

133

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Inhibition kinetics of single-␤-galactosidase molecules with the slow-binding inhibitor D-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultrasmall reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of ␤-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.␤-galactosidase ͉ enzyme kinetics ͉ fluorescence microscopy ͉ single molecule T he emergence of new assays for studying enzymes at the single-molecule level has profoundly extended our view of enzyme mechanisms. Whereas stochastic molecular behaviors are hidden by using bulk methods, single-molecule experiments have revealed that, in a population of enzymes, such as lactate dehydrogenase (1), phosphatase (2), or ␤-galactosidase (3), the catalytic rates of individual enzyme molecules are heterogeneous and do not interconvert quickly. Furthermore, it has been shown that the substrate turnover of individual ␤-galactosidase (4), cholesterol oxidase (5), or lipase (6) molecules undergoes dynamic fluctuations in sequential catalytic cycles. These two variations in enzyme activity are referred to as static and dynamic heterogeneity, respectively, and have both been attributed to different conformational states of the enzyme. Despite such heterogeneity, the Michaelis-Menten model derived from bulk enzyme experiments still holds for single-molecule experiments once a stochastic perspective is adopted (4,7,8).Traditionally, one of the most important tools to elucidate an enzyme's catalytic mechanism has been to study the enzyme in the presence of inhibitors. Here, we set out to correlate inhibition mechanisms established in bulk enzyme studies with singleenzyme molecule experiments. To date, inhibitors have been used in single-molecule studies to obtain information about conformational dynamics. Ha et al. (9) showed that it is possible to distinguish between free and inhibitor-bound states of a single-staphylococcal nuclease enzyme molecule. The binding of an inhibitor imposed a conformational constraint on the enzyme molecule resulting in a change of single-molecule polarization and intramolecular single-pair FRET. In this article, we report the direct observation of inhibitor release and binding from single-enzyme molecules by monitoring their substrate turnover.

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Immobilized enzyme kinetics analyzed by flow-through microfluorimetry

Cited by 59 publications

References 6 publications

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

A rapid enzyme assay for � -galactosidase using optically gated sample introduction on a microfabricated chip

Progress in Spectroscopic Probes with Cleavable Active Bonds

Stochastic inhibitor release and binding from single-enzyme molecules

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