Fluorescence assays, stopped-flow

P 75] A static enzyme assay experiment was carried out using a stopped-flow method [161]. This is commonly used for monitoring reaction kinetics. P-Galacto-sidase was used as model enzyme to convert the substrate fluorescein mono-p-D-galactopyranoside (FMG) via hydrolysis into fluorescein. As buffer solution 10 mM potassium phosphate at pH 7.2 with 1 mM ascorbic acid was used to minimize photobleaching. The enzymatic reaction is accompanied by a change in fluorescence intensity which can be monitored with a microscope. 

Figure 8. Tracings of stopped-flow fluorescence assays of the hydrolym of Dns-(Gly)g-ia-Phe, 2.5 X catalyzed by zinc and cadmium carboxypeptidase A, 5 X J0-5M, at pH = 7.5 and 25 C in 0.03M Tris-l.OM NaCl (48). Enzyme tryptophans were excited at 285 nm and their emission was measured by means of a bandpass filter peaking at 360 nm.

Figure 10 Superimposition of rapid chemical quench (open circles O) and stopped-flow fluorescence (blue) assays. In (a) tryptophan emission was detected, and in (b) the fluorescence change from 2-AP was monitored. Insets show the dNTP binding-induced conformational change In the presence of dideoxy-terminated DNA substrate. Adapted with permission from A. K. Showalter B. J. Lamarche M. Bakhtina M. I. Su K. H. Tang M. D. Tsai, Chem. Rev. 2006, 106, 340-360. Copyright 2006 American Chemical Society.

It is generally accepted that thio-substituted analogues (Sp-dNTPaS) should perturb mainly the chemical step rather than conformational steps. Use of thio-substituted analogues in stopped-flow assays show that the rate of the fast phase remains unperturbed, while the rate of the slow fluorescence transition is significantly reduced (Figure 11(a)).This reinforces the association of the rate of the slow fluorescence transition with the rate of chemistry. 

Figure 13 Comparison of Pol /3 catalyzed single-nucleotide incorporation at 10% and 35% glycerol, pH 8.3. (a) 2-AP fluorescence stopped-flow assays show that both phases of the fluorescence change are slowed down at increased glycerol concentration, (b) In contrast, rapid chemical quench assays demonstrate that the rate of nucleotide incorporation remains unaffected by the altered glycerol concentration. Adapted with permission from M. Bakhtina M. P. Roettger S. Kumar ...

Figure 15 Forward conformational closing (dark blue) and reverse conformational opening (pink) as monitored by stopped-flow 2-AP fluorescence assays. Adapted with permission from M. Bakhtina M. P. Roettger M. D. Tsai, Biochemistry 2009, Submitted. Copyright 2009 American Chemical Society.

Previous stopped-flow fluorescence assays investigating matched dNTP incorporation showed that both the fast and the slow fluorescence transitions demonstrated a hyperbolic dependence on dNTP concentra-tion. " " " Similarly, the dNTP dependence of both the fast and the slow fluorescence phases during mismatched dNTP incorporation in stopped-flow has been examined. The observed rate constants for the fast and the slow phases, individually plotted as a function of dNTP concentration, reveal that both phases demonstrate a hyperbolic dependence on dNTP concentration (parameters obtained for k2, K, k o, and d,app as described in Section 8.10.4.2.3 and reported in Table 1). The observed hyperbolic dependence of the fast phase on mismatched dNTP largely indicates that this phase originates from a conformational change induced by mismatched dNTP binding. 

An integrated pTAS system for the detection of bacteria including lysis, DNA purification, PCR and fluorescence readout has also been published recently [113]. A microfluidic plastic chip with integrated porous pol5mier monoliths and silica particles for lysis and nucleic acid isolation was used for detection (Fig. 8). A custom-made base device provided liquid actuation and off-chip valving by stopping liquid flow from the exits of the chip, utilizing the incompressibility of liquids. Detection of 1.25 x 10 cells of B. subtilis was demonstrated with all assay steps performed on-chip. 

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