Enzyme assay chromogenic substrates

C4. Christensen, U., Requirements for valid assays of clotting enzymes using chromogenic substrates. Thromb. Haemostasis 43, 169-174 (1980). 

Various assays are used to detect and quantify proteins. Some assays use a light-producing reaction or radioactivity to generate a signal. Other assays produce an amplified colored signal with enzymes and chromogenic substrates. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

The substance to be assayed—e.g., the hormone thyroxine in a serum sample—is pipetted into a microtiter plate (1), the walls of which are coated with antibodies that specifically bind the hormone. At the same time, a small amount of thyroxine is added to the incubation to which an enzyme known as the "tracer" (1) has been chemically coupled. The tracer and the hormone being assayed compete for the small number of antibody binding sites available. After binding has taken place (2), all of the unbound molecules are rinsed out. The addition of a substrate solution for the enzyme (a chromogenic solution) then triggers an indicator reaction (3), the products of which can be assessed using photometry (4). 

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). 

Iwanaga, S., Morita, T., Harada, T., Nakamura, S., Niwa, M., Takada, K., Kimura, T., Sakakibaru, S. Chromogenic substrates for horseshoe crab clotting enzyme. Its application for the assay of bacterial endotoxins. Haemostasis 7 (1978) 183-188. 

The assay was performed by first preincubating selected experimental agents and the enzyme for 10 minutes. Thereafter, the assay was initiated by adding the substrate to obtain a final volume of 100 p,l. The initial velocity of chromogenic substrate hydrolysis was measured by the change in absorbance at 405 nm at 25°C during the linear portion of the time course. Inhibition constants, K, for factor Xa are summarized in Table 1. 

For many enzymes of clinical or commercial importance, chromogenic substrates or cofactors have been developed to allow enzyme assays to be analysed photometrically. For example the compounds tosyl-glycyl-prolyl-lysyl-4-nitroacetanilidoacetate, or tosyl-glycyl-arginyl-4-nitroacetanilide can be used to assay the dinically important serine proteases plasmin and thrombin. 

Enzyme-linked immunosorbent assays (ELISA) are by far the most common immunological assay used in biochemical and clinico-chemical laboratories (Crowther 1995). The method is just as specific as RIA and it can achieve comparable sensitivities under optimal conditions. It can be carried out in various ways, but the common feature is that detection relies on turnover of a chromogenic substrate by an... 

Fig. 7 Chromogenic enzyme assays (A) pyroGlu-Pro-Arg-pNA is a peptide substrate for activated protein C and tissue factor XIa. Protolysis releases the chromophore pora-nitroaniline (pNA), which is monitored by absorbance measurement at 405 nm. (B) ortho-Nitrophenyl-o-galactopyranoside (oNPG) is an artificial substrate for galactosidase that metabolizes lactose. The cleavage of oNPG releases the yellow ortho-nitrophe-nol with maximum absorbance at 420 nm.

In Fig. 4, one can see the familiar UV absorbance curve for an intact chromogenic substrate and of the free pNA at the same concentration. By monitoring the formation of pNA at 405 nm one is ensured minimal interference from the reactants (F5). A linear relationship exists between the concentration of the enzyme in the assay system and the release of either the... 

Majority of phosphatase assays employed in HTS today rely on the ability of these enzymes to catalyze dephosphorylation of artificial chromogenic or flnorogenic snbstrates. Their popnlarity is dne to simplicity, high sensitivity, and robnsmess of these assays. The most popular chromogenic substrate is para-nitrophenyl phosphate (pNPP) (Fig. 3a). It is stable in aqueous solutions at different pH values and is readily hydrolyzed by majority of phosphatases [20-22]. Hydrolysis of pNPP results in generation of para-nitrophenol, which has pii =7.16 and once deprotonated becomes yellow-colored para-nitrophenolate ion with absorbance at 405 nm. It can be monitored continuously at pH values above 7 assays with lower pH values are performed in the end-point format, requiring alkalinization of solution prior to measuring absorbance. 

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