Enzyme assay procedure

Enzyme Assays. Procedures for the HPLC assay of prephenate aminotransferase (32), the spectrophotometric assay of shikimate dehydrogenase... 

The conditions used in an enzyme assay depend on what is to be accomplished by the assay. There are two primary applications of an enzyme assay procedure. First, it may be used to measure the concentration of active enzyme in a preparation. In this circumstance, the measured rate of the enzyme-catalyzed reaction must be proportional to the concentration of enzyme stated in more kinetic terms, there must be a linear relationship between initial rate and enzyme concentration (the reaction is first order in enzyme concentration). To achieve this, certain conditions must be met (1) the concentrations of substrate(s), cofactors, and other requirements must be in excess (2) the reaction mixture must not contain inhibitors of the enzyme and (3) all environmental factors such as pH, temperature, and ionic strength should be controlled. Under these conditions, a plot of enzyme activity (p-rnole product formed/minute) vs. enzyme concentration is a straight line and can be used to estimate the concentration of active enzyme in solution. 

Fumaric acid has been found to be a good marker to detect the addition of D,L-malic acid to apple juice (Junge Spandinger, 1982). With the advent of the enzymic assay procedure for D-malic acid the method fell out of use. However, in 1995, a number of samples of apple juice in Germany were found to contain elevated levels of fumaric acid, which was attributed to the addition of L-malic acid. 

Enzyme Assay Procedure. The catalytic potency of the immobilized g-galactosidase was determined in a plug flow reactor ( 9). Glucose liberated by the catalytic activity of 3-galactosi-dase on lactose was determined by the glucose oxidase-chromogen method (21 ) with some modifications. 

Assay each of the fractions preserved in steps 10-5 to 10-38 for protein concentration and enzyme activity. Use the Lowry method described in Chapter 2 for protein determinations. Use the enzyme assay procedures described in steps 10-52 to 10-57 to determine enzyme activity. 

The Zymark robotic laboratory automation system Although detail procedures differ in each laboratory, the basic elements of binding and enzyme assays are similar. The generalized procedure shown in Table 1.10 highlights the common steps and indicates which Zymate laboratory systems are required. These procedures are performed using common laboratory glassware such as test tubes or in multiple tube devices such as microtitre plates. 

Figure 8.14 The effective analytical range of an enzyme assay. The assay of D-amino acid oxidase (EC 1.4.3.3), using the method detailed in Procedure 8.5, shows a valid analytical range up to a maximum reaction rate of 0.10 absorbance change per minute.

Coupled enzyme assays provide a good alternative to chemical modification and permit a kinetic technique to be employed. In a coupled assay, the rate at which the product is formed is measured by using the product of the reaction as a substrate for a second enzyme reaction which can be monitored more easily. Coupled assays offer great flexibility in enzyme methodology while still retaining all the advantages of continuous monitoring techniques and are illustrated by Procedure 8.5. 

A wide range of coupled assays involving ATP, NAD(P)H and FMN can be developed using these two enzymes and provide increased levels of sensitivity over other coupled assays (Procedure 8.7). 

In one case, a small peptide with enzyme-like capability has been claimed. On the basis of model building and conformation studies, the peptide Glu-Phe-Ala-Ala-Glu-Glu-Phe-Ala-Ser-Phe was synthesized in the hope that the carboxyl groups in the center of the model would act like the carboxyl groups in lysozyme 17). The kinetic data in this article come from assays of cell wall lysis of M. lysodeikticus, chitin hydrolysis, and dextran hydrolysis. All of these assays are turbidimetric. Although details of the assay procedures were not given, the final equilibrium positions are apparently different for the reaction catalyzed by lysozyme and the reaction catalyzed by the decapeptide. Similar peptide models for proteases were made on the basis of empirical rules for predicting polypeptide conformations. These materials had no amidase activity and esterase activity only slightly better than that of histidine 59, 60). 

Preparation and analysis of SDS extracts from digester sludge. The particulates from a 30-ml sample were removed by centrifugation (15,00Qg) at 4 C for 20 min. The particulates were washed three times with 100 mM Tris buffer pH 7.0 and resuspended in 15 ml of buffer. The extraction procedure consisted of agitating the sample with a Fisher model 346 rotator at 25 C in the presence of 0.1% SDS for 1 h. The particulate material was then removed by centrifugation at 15,000 g at 4 C for 20 min, and the supernatant was used to perform the enzyme assays. 

Perhaps of first concern in determining the overall design of a particular assay is the actual method used for product identification (or for substrate depletion) per unit time. Many different methods have been utilized (e.g., radiometric, spectrophotometric, fluorometric, pH-stat, polarimetric, etc.) No matter which method is used, the product has to be clearly identified (or substrate, if substrate depletion is being measured). With stopped-time assays, it may be necessary to separate product(s) from substrate(s) prior to determination of the amounts of the metabolite(s) present (as well as demonstration that product(s) and substrate(s) are truly separated). If so, the investigator should be able to demonstrate that the assay procedure clearly measures true initial rates (see below). Closely related to these issues are concerns about purity (See Substrate Purity Enzyme Purity Water Purity, etc.) and stability (See Substrate Stability Enzyme Stability, etc.. If the components of the assay mixture are not stable over the time course of the experiment (or, if certain side reactions occur), then corrections have to be made in analyzing the rate behavior. 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. PTPS activity is assayed by measuring the biopterin produced upon enzymatic incubation at 37°C for 120 min in a final volume of 110 pi in the dark (due to the light sensitivity of pterins), followed by chemical oxidation. To stabilize the produced BH4, DHPR and NADH are present in the enzyme assay. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the biopterin that was present in the lysate, and a blank reaction without cell lysate to detect the biopterin that is generated from the incubation (substrate) buffer. 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

It is of key importance for any study of the kinetic properties of an enzyme that the assay procedure should measure the initial rate of the reaction for enzymes such as DPOs that suffer product inactivation, this may pose difficult problems (Mayer et al., 1966). 

In all types of assay, the use of enzyme extracts that are too strong or too dilute can lead to poor results. Thus, it is essential to conduct a preliminary range-finding experiment for each enzyme and each assay procedure. 

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