Fluorescence lactate dehydrogenase

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. 

Lactate dehydrogenase (LDH) LDH released by cells with damaged membrane results in the conversion of a tetrazolium compound into a water-soluble fluorescent formazan dye Cell membrane Yes [32]... 

Reliable measurements of L-lactate are of great interest in clinical chemistry, the dairy and vine industry, biotechnology, or sport medicine. In particular, blood lactate levels are indicative of various pathological states, including shock, respiratory insufficiencies, and heart and liver diseases. Silica sol-gel encapsulation of the lactate dehydrogenase and its cofactor was employed as a disposable sensor for L-lactate51. The sensor utilized the changes in absorbance or fluorescence from reduced cofactor nicotinamide adenine dinucleotide (NADH) upon exposure to L-lactate. 

Some reactions that do not give chromophoric or fluorescent products may be coupled with another enzymatic reaction that does. Many of these coupled reactions are based on the formation or the disappearance of NADH. The formation of pyruvate may be linked with the conversion of NADH to NAD+ by lactate dehydrogenase ... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

LDH (lactate dehydrogenase Membrane integrity Easy to measure. Fluorescent methods sensitive... 

Enzyme catalyzed reactions have also been studied at the single molecule level. Earlier work involved measurement of beta-galactosidase activity in droplets after a 10-15 h incubation. In a recent study, detection of fluorescent product generated by individual molecules of lactate dehydrogenase after a 1 hr incubation has been achieved using capillary electrophoresis . The activity of individual molecules were reproducible but activity of different molecules showed a 5-fold range. The differences in activity were suggested to reflect differences in conformation. 

M31. McKenzie, D., and Henderson, A. R., An artifact in lactate dehydrogenase isoenzyme patterns, assayed by fluorescence, occurring in the serum of patients with end-stage renal disease requiring maintenance hemodialysis. Clin. Ckim. Acta 70, 33-336 (1976). 

Fia. 32. Reaction of lactate dehydrogenase with NAD and lactate at pH 6 monitored by nucleotide absorbance, nucleotide fluorescence, and protein fluorescence, The derivative plots on the right represent the initial transient approach to a steady-state rate of NADH production for nucleotide absorbance and fluorescence. To obtain the derivative plot for protein fluorescence, the fluorescence was measured as a fraction of the fluorescence of unliganded enzyme at each time, and the fractional fluorescence was then converted into fractional saturation of the enzyme with ligand by using the equation F " = 1 — a(l — x) (see Section II,D,1). Steady-state rate (s.s.) was 4.8 sec h... 

Most applications of native fluorescence detection involve the direct detection of a fluorescent analyte of interest. However, native fluorescence can also be used indirectly to detect the presence of nonfluorescent species. For example, the presence of a nonfluorescent enzyme can be detected based on its conversion of substrate into natively fluorescent products. This is often used in conjunction with electrophoretically mediated microanalysis, in which differences in buffer mobilities enable on-column mixing and reaction of enzymes with substrates. One such study exploited the intrinsic fluorescence of nicotinamide adenine dinucleotide (NADH) to investigate differences in the reactivity of individual molecules of lactate dehydrogenase. 

Figure 16.56. Equilibrium unfolding of three single-tryptophan mu> tants of lactate dehydrogenase monitored by trypto an fluorescence intensi. lYyptophan fluorescence was excited at 295 nm and measured at 34S nm. Revi d from Ref. 125.

Both NAD and NADP in their reduced forms have native fluorescence. By using a recording spectrophotofluorometer instead of a u.v. spectrophotometer, it has been claimed that sensitivity for NAD coupled reactions like lactate dehydrogenase [123] can be increased by one or two orders of magnitude. 

The ensyme lactate dehydrogenase is a tetramer. The association/dissociation properties of this en yme have been studied using fluorescence polarization. The trick is in tagging dansyl chloride to the polypeptides. When the monomers associate to form tetramers, the resulting complex is large. Thus polarization increases as association occurs. When the tetramer dissociates, depolarization occurs. It is also seen that depolarization and loss of activity occur hand in hand meaning that the tetramer is the active form while the monomer is inactive. 

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