Enzyme activators, determination

Potentiometry is another useful method for determining enzyme activity in cases where the reaction Hberates or consumes protons. This is the so-called pH-stat method. pH is kept constant by countertitration, and the amount of acid or base requited is measured. An example of the use of this method is the determination of Hpase activity. The enzyme hydroly2es triglycerides and the fatty acids formed are neutralized with NaOH. The rate of consumption of NaOH is a measure of the catalytic activity. 

Substrate (compound to be determined) Enzyme (activity to be determined) Products (sensed species) Buffer and optimum PH Indicating electrode Range... 

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

In determining enzyme activities, it is usually assumed that at a fixed set of so-called saturating substrate concentrations a sufficiently accurate value of F, ax is obtained. Bisubstrate kinetic analyses of UDP-glucu-ronyltransferase [assayed with bilirubin (P5) and p-nitrophenol (V6), respectively] indicate that a true measure of the amount of enzyme can be obtained only by suitable extrapolation procedures. This restriction applies in particular to bilirubin (A2, HIO, T8) and other aglycons (M15, V6) because of substrate inhibition. UDP-glucuronic acid was inhibitory at concentrations only about 10-fold higher than the apparent Km value (HIO) this was most pronounced at relatively short incubation times. Mg was noninhibitory at concentrations equal to 20 times the apparent Km values (F3, HIO). 

Klippel, A. Escobedo, J.A. Hirano, M. Williams, L.T. The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. Mol. Cell. Biol., 14, 2675-2685 (1994)... 

To determine enzymic activity, p-nitrophenyl a-L-arabinofurano-side,29 phenyl a-L-arabinofuranoside,3031 and beet L-arabinan are used as substrates. Most of the a-L-arabinofuranosidases so far reported act on each of these three substrates, but there are some enzymes that act exclusively on either the low-molecular-weight or high-molecular-weight substrates. 

To explore whether the presence of glucose in the reaction mixture of the AO activity measure could negatively affect the enzymatic activity of AO by means of an inhibitory effect, we conducted several experiments in which we added different amounts of glucose to the reaction mixture in order to determine enzyme activity. The results, shown in Table 4, clearly demonstrate that the presence of glucose at any concentration had no effect on AO activity. 

IL tissue was treated with cholera toxin and adenylate cyclase activity was determined. Enzyme activity was determined in the presence of the indicated concentrations of (-)-sulpiride alone (Q) or in combination with apomorphine (3 v-M, 10 utA, A. or 30 ( M, A) (left). In a separate experiment, enzyme activity was determined in the presence of the indicated concentrations of apomorphine alone (O) or in combination with (-)-sulpiride (3 mM, a. 30 mM, A) (right). In both experiments, data represent the mean SE (n — 3). 

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. 

Zhou HH. CYP2C19 genotype determines enzyme activity and inducibility of S-mephenytoin hydroxylase. Clin Chim Acta 2001 313 203-8. 

Kinetic methods are necessary to determine enzyme activities since the enzyme is a catalyst and affects only the reaction rate. 

The process whereby the analyzer uses reflectance readings to determine enzyme activity can be generalized in the following steps ... 

The second technique employed for substrate determination is the measurement of the rate of an enzymaticallv catalyzed reaction, as is used to determine enzyme activity. This may take one of three forms. First, the time required for the reaction to produce a preset amount of product or to consume a preset amount of substrate may be measured. Second, the amount of product formed or substrate consumed in a given time may be measured (see Experiment 35 for glucose determination). These are single-point measurements (called end-point measurements) and require well-defined reaction conditions. They are easy to automate or may be performed manually. A third procedure is continuous measurement of a product or substrate concentration as a function of time to give the slope of the reaction rate curve, Ac/Ar. These are the so-called true rate measurements. The measurements must generally be made during the early portion of the reaction where the rate is pseudo first order. 

Fig. 1.5 Scheme for the analytical procedure to determine enzyme activity. S substrate P product Pq product in control A, B, C coupling reagents Z analyte Zq analyte in control s, p, z are the corresponding molar concentrations... 

Because some clinical assays are done many times a day, the procedures for running the assays have been automated and computerized. The determined enzyme activity levels are usually reported in terms of enzyme international units (lU), which define enzyme activity as the amount of enzyme that will convert a specified amount of substrate to a product within a certain time. 

What observations may be used in experiments to determine enzyme activity ... 

The kinetic method employed for substrate determination is the measurement of the rate of an enzymatically catalyzed reaction, as is used to determine enzyme activity. Rate methods are generally more rapid than the endpoint method. Complete conversion reactions, on the other hand, are less subject to interference from enzyme inhibitors or activators as long as sufficient time is allowed for completion conversion. 

Changes in amino acid sequence of proteins (primary structure). Changes in protein function, measured directly by determining enzyme activity or antigenicity (secondary and tertiary structure). ... 

---