Auxiliary enzymes, assays with

A protocol for continuous enzyme assay that involves one or more auxiliary enzymes to convert a product of the primary reaction in a second or auxiliary reaction that produces a change in absorbance or fluorescence. As noted below, coupled enzyme assays, while convenient, are fraught with experimental limitations that must be overcome in order to obtain valid initial velocity data. 

The components of the coupling system should neither inhibit nor activate the primary enzyme. Moreover, care must be exercized to ascertain that the auxiliary enzyme (s) is not contaminated with other minor enzyme activities capable of influencing the primary enzymatic activity. The results from any coupled enzyme assay must exactly match the results obtained with other valid initial rate assays to ensure that the presence of the auxiliary system in no way affects the activity of the primary enzyme. This is typically accomplished by comparing data obtained from the coupled assay with stopped-time assay results to ensure that similar results are obtained. 

End-point assays can also use coupled enzyme systems in which the product of the test reaction provides the substrate for the subsequent auxiliary and indicator reactions. The choice of pH is less critical than with the measurement of enzyme activity frequently a compromise pH is used and... 

Historically, saccharogenic, amyloclastic, and chromolytic starch methods were the assays of choice for determining AMY activity. These assays are now completely displaced in favor of ones with well-defined substrates with shorter glu-cosyl chains. The use of defined AMY substrates and auxiliary and indicator enzymes in the AMY assay has improved the reaction stoichiometry and has led to more controlled and consistent hydrolysis conditions. Substrates used include small oligosaccharides and 4-nitrophenyl (4-NP)-glycoside substrates. 

Kulys et al. (1986b) studied urea determination by difference measurement between two antimony electrodes covered with exchangable membranes (Fig. 68). Urease was attached in the pores of a macroporous membrane (thickness, 10 pm, pore diameter, 0.1 pm) by glutaraldehyde. This layer was covered with a monoacetylcellulose membrane. The membrane for the auxiliary electrode was prepared analogously, but using BSA instead of urease. The assay of urea was carried out with a differential amplifier which simultaneously differentiated the time course of the potential difference between enzyme and auxiliary electrode (kinetic method). Thus, a response time of only 20 s was possible. 

This enzyme catalyses one of the intermediate steps of glycolysis. The reaction is not considered to be a rate-lunitmg step, but it may be used as an indicator reaction for the estimation of phosphofructokinase activity which is thought to be rate limiting. Serum levels of aldolase are elevated in diseases of skeletal muscle and in the presence of tumours in many tissues. Aldolase may be assayed by an ultraviolet method using triose phosphate isomerase as an auxiliary reaction and glycerol-3-phosphate dehydrogenase as an indicator reaction [219]. The trioses can be trapped as formed with the aid of... 

Serum levels of this enzyme are elevated in diseases of skeletal muscle, cardiac muscle, and brain. It may be assayed by a u.v. meOiod uring ADP formation, pyruvate kinase in an auxiliary reaction and lactate dehydrogenase in an indicator reaction [336]. An alternate ultraviolet assay uses ATP formation, hexokinase in an auxiliary reaction, and glucose-6-phosphate dehydrogenase in the indicator reaction [337]. A colorimetric assay utilises creatine formation, complexing with diacetyl and colour complex formation with a-naphthol [338—339]. 

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