Fluorimetric enzyme assay

He W, Voznyi YV, Boer AM, Kleijer WJ, van Diggelen OP (1993) A fluorimetric enzyme assay for the diagnosis of Sanfilippo disease type D (MPS HID). J Inherit Metab Dis 16 935-941... 

Van Diggelen OP, Zhao H, Kleijer WJ, Janse HC, Poorthuis BJHM, van Pelt J, Kamerling JP, Galjaard H (1990) A fluorimetric enzyme assay for the diagnosis of Morquio disease type A (MPS IV A) Clin Chim Acta 187 131-140... 

Ceroid Lipofucinosis palmitoyl-protein thioesterase (PPT 1) granular osmiophilic deposits of acylated proteins CNLl (for PPTl) CNLx>10 1 12500 fluorimetric enzyme assay of PPT... 

Table 8.7 Examples of fluorimetric methods of enzyme assay...

Note All NAD+- and NADP - linked enzyme assays are also capable of being monitored fluorimetrically. The fluorescent compounds in each assay are shown in bold type. ... 

Enzymatic techniques have also been employed in the analysis of these compounds. The toxicity of carbamate insecticides is due to the inhibition of the enzyme acetylcholine esterase, so the determination of these compounds can be achieved by enzyme inhibition (2,83,119), bioassay (118,167), or enzyme-linked immunosorbent assay (ELISA) (168-171). In the detection of carbamates by fluorimetric enzyme inhibition, the effluent from a reversed-phase chromatographic column was incubated with cholinesterase, which was introduced via a postcolumn reagent delivery pump. Then, the resulting partially inhibited cholinesterase was reacted with N-methyl indoyl acetate to produce a fluorophore and a reduction in the baseline fluorescence (172). 

Catecholamines. The quantitative determination of dopamine and noradrenaline in tissue samples of 0.1-10 mg at levels in the order of 0.5 pmol has been described [84]. These methods are based on extraction, formation of the pentafluorpropionyl derivatives, and the use of the homologues, a-methyidopamine and a-methylnoradrenaline as internal standards in SIM. Higher sensitivity than obtainable with fluorimetric or enzymic assays is reported [462J. Applications have been to amine determination in specific regions of rat brain [84] and to measurement of heart ventricle concentrations [463]. A combination of assays of this type with the use of synthesis inhibitors or radioisotope labelled precursors allows direct estimation of brain amine turnover in animals. 

Guilbault GG, Sadar SH, McQueen R (1969) Fluorimetric enzymic method for the assay of mixtures of organic acids. Anal Chim Acta 45 1-12... 

Another approach of using fluorimetric assays for screening purposes is the use of coupled enzyme systems. McElroy et al. presented an assay for glutamate... 

GTPCH (EC 3.5.4.16) converts the substrate GTP to 7,8-dihydroneopterin triphosphate (H2NTP) and formate. GTPCH activity is determined by measuring neopterin, the completely oxidized and dephosphorylated H TP-product of the enzyme reaction. Conversion of H2NTP to neopterin is carried out after the enzymatic reaction in presence of iodine at pH 1.0, followed by dephosphorylation with alkaline phosphatase at pH 8.5-9.0. Neopterin is detected fluorimetrically at 350/440 nm upon HPLC separation. The assay is based with some modifications on the methods published by Viveros et al. and Hatakeyama and Yoneyama [15,16]. 

CG Knight. Fluorimetric assays of proteolytic enzymes. Meth Enzymol 248 18— 34, 1995. 

Among different coumarin derivatives used, 7-Amino-4-trifluoromethylcoumarin (ATFMC) revealed the most promising characteristics as an efficient fluorescent emitter. AFTMC is used in the synthesis of a substrate for fluorimetric assay of proteolytic enzymes and for use as a laser dye. We have recently investigated the chemiluminescence reactions of some peroxyoxalate esters, hydrogen peroxide and AFTMC. In this paper we report the solvent effects on the kinetics of the chemiluminescence process of the peroxyoxalate chemiluminescence in the presence of AFTMC. 

The preparation of a sodium (4-methylumbelliferyl Of-D-iV-acetylneuraminate) substrate and its use in a sensitive fluorimetric assay of neuraminidase from human leucocytes, Vibrio cholerae, and cultured fibroblasts have been reported. The fibroblast and leucocyte neuraminidases showed maximum activity at pH4.2-4.4 and values of 0.13 and 0.22 mM, respectively the enzyme activity was considerably reduced in cultured fibroblasts of patients with mucolipidosis types I, II, and III. V. cholerae neuraminidase (pH optimum 4.6, 1.5 mM) showed activation by calcium chloride, H4edta, sodium... 

CYP inhibition assays include ones that utilize liver microsomes, isolated/cultured hepatocytes, and human cDNA-expressed enzymes. Typically, LC-MS/MS quantification is used for the microsome and hepatocyte methods while fluorimetric assays are used for the human cDNA-expressed enzymes. Typically, a weak inhibitor is defined as having a fei>20 rmolN, whereas a potent inhibitor has a ki[Pg.882]

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