Fluorescence kinetic-based measurements

Fluorescence Kinetic-Based Measurements. Our studies of the reaction rate determination of thiamine (vitamin Bl) will be used to demonstrate the unique capabilities of rapid acquisition of spectra in kinetic measurements. The kinetic method is based on the oxidation of thiamine by Hg + in basic solutions to highly fluorescent thiochrome (16) The initial rate, taken as the change in fluorescence signal at 444 nm that occurs in a fixed time after mixing the sample and reagents, is directly proportional to the thiamine concentration. 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

For measurement of steady-state fluorescence, the rapid acquisition of spectra is a convenience but not essential. However, the rapid acquisition of spectral information is essential for measurement of transient species or where the luminescence signal continually changes with time. This will be demonstrated first for kinetic-based luminescence measurements and then chemiluminescence measurements. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

To increase the speed of the TIRF-based kinetic techniques, the perturbation can be optical rather than chemical. If the evanescent wave intensity is briefly flashed brightly, then some of the fluorophores associated with the surface will be photobleached. Subsequent exchange with unbleached dissolved fluorophores in equilibrium with the surface will lead to a recovery of fluorescence, excited by a continuous but much attenuated evanescent wave. The time course of this recovery is a measure of the desorption kinetic rate k2. This technique1-115) is called TIR/FRAP (or TIR/FPR) in reference to fluorescence recovery after photobleaching (or fluorescence photobleaching recovery). 

The absorption cross sections of the Sn Si transition are determined from the kinetic analyses based on the two-photon absorption measurement. In contrast, tetraphenylporphin scarcely show the S2 state fluorescence. In order to elucidate the effect of the imino hydrogens on the radiationless transitions, deuterium isotope effect on the relaxation processes from the lowest excited singlet state of metal free porphyrins has been investigated. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

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