Substrate determination, enzymatic analysis

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

Figure 4.1 Overview of strategy for design of an HPLC method to determine enzymatic activity. The reaction tube contains a mix preparation to measure the activity of an ATP pyrophosphohydrolase, which catalyzes the formation of AMP and PPj from ATP. The mix contains the substrate, ATP the buffer, Tris-HCl and magnesium, a metal cofactor. The addition of a sample from the enzyme fraction initiates the primary reaction and also several secondary reactions. Samples of the incubation mixture are withdrawn at intervals (r( and r2), and the reaction is terminated by injection of the samples onto the HPLC column. A representative analysis of each sample is shown. The amount of each component can be calculated from the area of its peak and is graphed as a function of reaction time.

Enzymatic analysis involves the determination of sample constituents, which can be both substrates and inhibitors of enzymes, and the determination of enzyme activity occurring in the sample. Such analyses are carried out on a wide variety of materials, particularly foodstuffs and their precursor raw materials. 

FIGURE 16.12 Enzymatically synthesized amylose"-type nb/lcb glucans ( ) with a significant amount of the substrate glucose-1-PO4 separated on Sephacryl S-SOO/S-IOOO (60 + 9S x 1.6 cm) 3-ml fractions were collected for further analysis normalized (area = 1.0) eluogram profiles (ev) constructed from an off-line determined mass of carbohydrates for each of the pooled fractions flow rate 0.42 ml/ min V,xd = 126 ml, V , = 273 ml eluent O.OOS M NaOH. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

Similarly to quantitative determination of high surfactant concentrations, many alternative methods have been proposed for the quantitative determination of low surfactant concentrations. Tsuji et al. [270] developed a potentio-metric method for the microdetermination of anionic surfactants that was applied to the analysis of 5-100 ppm of sodium dodecyl sulfate and 1-10 ppm of sodium dodecyl ether (2.9 EO) sulfate. This method is based on the inhibitory effect of anionic surfactants on the enzyme system cholinesterase-butyryl-thiocholine iodide. A constant current is applied across two platinum plate electrodes immersed in a solution containing butyrylthiocholine and surfactant. Since cholinesterase produces enzymatic hydrolysis of the substrate, the decrease in the initial velocity of the hydrolysis caused by the surfactant corresponds to its concentration. Amounts up to 60 pg of alcohol sulfate can be spectrometrically determined with acridine orange by extraction of the ion pair with a mixture 3 1 (v/v) of benzene/methyl isobutyl ketone [271]. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

The POCL reaction has been used for trace determinations of hydrogen peroxide, most commonly in environmental and clinical analysis [26-52], The latter applications often include various enzyme systems [34-52], where a number of substrates can be indirectly determined by measuring the hydrogen peroxide that is produced as a by-product in the enzymatic reaction. 

If an investigator remains concerned about the substrate purity following repurification procedures and subsequent analysis, then a number of other approaches may be considered. For example, different lots of substrate could be analyzed with the enzyme to learn if identical kinetic parameters are obtained. If one has some idea as to the identity of the possible contamination, then the impurity can be added to the stock substrate solution at a known amount and the accuracy of the kinetic parameters in the presence of the adduct can be assessed. Since the researcher already knows the degree of sensitivity of the various chemical, enzymatic, and/or spectral methods used to assess substrate purity, this known addition provides a means for determining the maximum amount of impurity present. Combined with the observations seen with the known addition of the impurity, such information will provide an idea on the level of accuracy of the kinetic parameters. 

Total protein, albumin, urea (standard methods) and middle molecules (MM) were determined in citrated plasma [6]. The trypsin-like activity (TLA) of plasma was measured using the chromogenic peptide substrate (Z-glycyl-glycyl-L-arginine-4-nitroanilide) [7]. Evaluation of anti-enzymatic potential in plasma was based on concentrations of the main protease inhibitors -proteinase inhibitor (ttj-PI) and aj-macroglobulin (a -M). Student s t-test was used for statistical analysis. 

Kinetic analysis was used to characterize enzyme-catalyzed reactions even before enzymes had been isolated in pure form. As a rule, kinetic measurements are made on purified enzymes in vitro. But the properties so determined must be referred back to the situation in vivo to ensure they are physiologically relevant. This is important because the rate of an enzymatic reaction can depend strongly on the concentrations of the substrates and products, and also on temperature, pH, and the concentrations of other molecules that activate or inhibit the enzyme. Kinetic analysis of such effects is indispensable to a comprehensive picture of an enzyme. 

Reliable enzymatic assays for SeMet are not available as specific SeMet metabolizing enzymes have not been identified and enzymes such as glutamine transaminase react with Met equally as well as with SeMet (Blazon et al., 1994). However, with some enzymes reaction rates for SeMet and Met differ sufficiently to be of some use in SeMet analysis. For example, SeMet is a better substrate than Met for the a,y-elimination by i.-methionine y-lyase of Pseudomonas putida (Esaki et al., 1979). The adenosyl methionine transferase from rat liver reacts with L-SeMet at 51% of the rate with L-Met, and with the corresponding D-isomers at only 13 and 10% of the rate of L-Met (Pan and Tarver, 1967). Other adenosyl methionine transferases, such as that from yeast, react with SeMet more rapidly and with higher stereoselectivity than with Met, providing an indirect means for SeMet determination (Mudd and Cantoni, 1957 Sliwkowski, 1984 Uzar and Michaelis, 1994). 

The optimum synthesis of enzymatic biodiesel was determined by the ridge max analysis (SAS, 1990). The method of ridge analysis computes the estimated ridge of maximum response for increasing radii from the center of original design. The ridge max analysis (Table 9.6) indicated that maximum molar conversion was 99.4 4.6% at 12.4h, 38.0°C, 42.3% enzyme amount, 3.5 1 substrate molar ratio, and 7.2% added water content at the distance of the coded radius 0.8. 

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