Kinetic chromogenic assay

Because the preceding chromogenic assay rely on choline quantitation, the hydrolysis of substrates with headgroups other than choline cannot be followed. To circumvent this problem, another useful protocol was devised whereby the phosphorylated headgroup produced by the PLCBc hydrolysis is treated with APase, and the inorganic phosphate (Pi) that is thus generated is quantitated by the formation of a blue complex with ammonium molybdate/ascorbic acid 5 nmol of phosphate may be easily detected. This assay, which may also be performed in a 96-well format, has been utilized to determine the kinetic parameters for the hydrolysis of a number of substrates by PLCBc [37,38]. 

Endotoxin determination employed a kinetic chromogenic Limulus assay (COAMATIC Chromogenics AB, Molndal, Sweden). 

Panteghini M, Bonora R, Pagani F. Measurement of pancreatic lipase activity in serum by a kinetic colorimetric assay using a new chromogenic substrate. Aim Clin Biochem 2001 38 365-70. 

The LAL assay is at present the most widely accepted assay for endotoxin measurements, having been adopted as the standard assay for endotoxin detection by the United States Food and Dmg Administration (FDA) in 1980. The more recent kinetic chromogenic versions of the LAL assay are very sensitive and have a broad measurement range (0.01 -100 Endotoxin Units (EU)/ml w 1 pg/ml-10 ng/ml). The detection limit for airborne endotoxin measurements is approximately 0.05 EU/m (5 pg/m ). Since different test batches may give different results an internal standard must be used. The U.S. FDA... 

Before an immobilized enzyme can be used for an industrial process, it is essential to characterize it in terms of its catalytic and kinetic properties. A quantitative assay must be developed to measure the activity, kinetic parameters, and stability of the enzyme. In a coupling reaction, H202 rapidly reacts with phenol and 4-aminoantipyrine (electron donor) in the presence of peroxidase to produce a quinoneimine chromogen (Equation E12.2, Figure El 1.2), which is intensely colored with a maximum absorbance at 510 nm. (This is the same as the product formed in the analysis of cholesterol in Experiment 11.)... 

The simplest, but least accurate, method of assaying DPO activity is to record the final color yield when the enzyme is incubated with a suitable chromogenic substrate such as catechol, DOPA, or 4-methylcatechol. DOPA is the most frequently used substrate in colorimetric assays because it yields a dark brown/black end-product. In this reaction, catecholase catalyzes the conversion of DOPA to dopaquinone and then to the red dopachrome, which subsequently polymerizes to yield dark brown melanin-type pigments. Unfortunately, this simple procedure has serious limitations, as it measures the end-product of a sequence of reactions rather than the true initial reaction rate. Furthermore, because different substrates yield different final colors, valid kinetic comparisons between substrates are not possible. Nevertheless, this simple assay technique has proved adequate for useful comparative studies of the levels of enzymic browning in different fruit varieties and similar problems (Vamos-Vigyazo, 1981 Machiex et al., 1990). 

Inhibitor kinetics The IC50 determinations described herein provide a simple method of comparing protease inhibitors. However, it may be desirable to characterize the interactions of inhibitors with the enzyme in more detail. Both the chromogenic and fluorescence assays can be read in kinetics mode on the machines described to give real-time rate measurements for use in determining kinetic parameters. 

Now you see it, now you don t. Pre-steady-state experiments using chymotrypsin and a chromogenic substrate (N-acetyl-L-phenylalanine p-nitrophenyl ester) show a burst of product at very short times (Figure 9.4). The Conceptual Insights module on enzyme kinetics explains this result. What results would you see if the product detected by the assay was the free N-terminal component of the substrate instead of the C-terminal component Hint Use the pre-steady-state reaction simulation to simulate the experiment. Select different times following mixing and observe the amount of each product). 

Chromogenic enzyme kinetic assay/photometry ELISA... 

Mass spectrometry can characterize enzyme kinetics, mechanism, and selectivity [27]. Notably, MS enables kinetic analyses of enzymatic reactions where it is not possible to carry out simple spectrophotometric assays - for example, in the absence of chromogenic substrates [28, 29]. 

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