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]. 

Lee-Vaupel M, Conzelmann E (1987) A simple chromogenic assay for arylsulfatase A. Clin Chim Acta 164 171-180... 

Lise the equation below to calculate the units of activity per mg of gel. The number 6.58 in the denominator is the absorption coefficient for the quinoneimine chromogen assay product. 

S Roychoudhury, RE Kaiser, DN Brems, WK Yeh. Specific interaction between (3-lactams and soluble penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus development of a chromogenic assay. Antimicrob Agent Chemother 40 2075-2079, 1996. 

Acid chloride handles on various templates 49-51 were acylated with equimolar mixtures of 19 different appropriately protected amines to prepare non-peptidic polyamide libraries. The three templates possess different types of symmetry and offer varying degrees of 3D spatial diversity. Screening for trypsin inhibition employing a chromogenic assay identified the xanthane analog 52 (K, = 9.4 pM) as a moderately active, but structurally novel, inhibitor of trypsin (see Fig. 7) [37]. 

Table 1 Mass spectra characterization and associated IC50 values for selected experimental agents determined using the chromogenic assay. H NMR for products and intermediates supplied by author...

The chromogenic assay procedure was performed according to the method of Reynolds (1). IC50 values were determined by plotting log concentration versus inhibition values in the range from 10 to 90% inhibition and are provided in Table 2. 

Table 2 Chromogenic assay test results of selected experimental indicating their effectiveness as sPLA2 inhibitors...

A population of 24 compounds was randomly chosen from the 64 million possible hexapeptides and optimised for trypsin inhibition using a genetic algorithm [21], Biological testing was performed with a chromogenic assay... 

Staphylococcus aureus lipase epPCR/ shuffling chromogenic assay 12-fold increase in phospholipase activity 38... 

The chromogenic assay may be used as a screen for inhibitors in the endpoint format described here, although relatively high substrate costs may limit... 

Activity was determined by the ability of rSLPI to inhibit a known amount of trypsin, in a chromogenic assay using Tosyl-gly-pro-lys-4-nitranilide acetate (Chromozym PL, Boehringer-Mannheim). 

A sensitive and versatile chromogenic assay for POase is based on the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB) (Ngo and Lenhoff, 1980) in the presence of substrate. MBTH is the donor which, after oxidation, reacts with DMAB to form a cationic in-... 

Chromogenic assays are colorimetric assays which use a substrate containing a chromophore. The substrate itself is designed such that it is colorless, and during the enzymatic reaction the chromophore is released and the color change is measured by a plate reader. This technique is applied for certain enzyme reactions for which a conversion of assays to fluorescent readout for HTS is Hkely to be problematic or even impossible, e.g., when it would require labehng of a small substrate with a large fluorophore. 

Recently, an assay using chromophoric substrate (Chromozym TH) was developed [7], When Chromozym TH is added, the absorbance at 405nm will increase, and the linear section from 15 to 30 sec is used to calculate the activity (international unit, one unit is defined as the specific activity required to convert 1 pM substrate/min/mg of enzyme), Fig. (7). The increasing absorbance is resulted from the p-nitroanilide released off the substrate during hydrolyzation. As compared with the fibrin plate method, this chromogenic assay is more rapid, convenient and reproducible. The specificity of the assay, however, is lower than that of fibrin-plate method because the chromogenic substrate could be hydrolyzed by some other proteases, for instance thrombin. 

A number of studies have investigated interferences with the LAL assay and have attempted to optimize the assay (Douwes et al., 1995 Hollander et al., 1993 Thorne et al., 1997). These studies have demonstrated that results may vary depending upon the sample matrix, the extraction method and the assay method. Other constituents present in the sample may interfere with the LAL assay and cause inhibition or enhancement of the test or aggregation and adsorption of endotoxins, resulting in under- or over-estimation of the concentration. Techniques such as spiking with known quantities of purified endotoxin and analysis of dilution series of the same sample have been described to deal with these interferences (Hollander et al., 1993 Milton et al., 1990 Milton et al., 1992 Whitakker, 1988). Studies in the laboratories of the authors of this chapter have demonstrated within-Iaboratory coefficients of variation between 15 % and 20 % in routine assay work. When extra care is taken to optimize precision this can be reduced to under 5 % in the endpoint chromogenic assay (Thorne, unpublished data). Several interlaboratory comparison studies have been performed and demonstrate much greater variability. One in-depth comparison of two laboratories experienced in the LAL assay. 

The use of aeeelerated degradation studies is illustrated in Table 7 by referenee to Faetor VIII concentrate stored at —20°C potency was tested using the Ph Eur chromogenic assay... 

Mahoney DF, Baisden DK, Yao RC. A peptide-binding chromogenic assay for glycopeptide antibiotics of the vancomycin class. J Ind Microbiol 1989 4 43-47. 

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