Kinetic method, enzymatic analysis

Although kinetic methods of analysis are of special significance to clinical and pharmaceutical analysis in relation to enzymatic methods, they are equally applicable to nonenzymatic processes. Table 7 shows the most salient contributions reported in this respect over the past few years. Most of the clinical determinations shown have been performed on blood and/or urine samples and have involved inorganic or organic ions in similar proportions. On the other hand, pharmaceutical determinations have chiefly been concerned with organic substances. The Sandell-Kolthoff reaction has by far been the most commonly employed for the analysis of biological samples, so much... 

Methods in which some property related to substrate concentration (such as absorbance, fluorescence, chemiluminescence, etc.) is measured at two fixed times during the course of the reaction are known as two-point kinetic methods. They are theoreticahy the most accurate for the enzymatic determination of substrates. However, these methods are technically more demanding than equifibrium methods and all the factors that affect reaction rate, such as pH, temperature, and amount of enzyme, must be kept constant from one assay to the next, as must the timing of the two measurements. These conditions can readily be achieved in automatic analyzers. A reference solution of the analyte (substrate) must be used for calibration. To ensure first-order reaction conditions, the substrate concentration must be low compared to the K, (i.e., in the order of less than 0.2 X K, . Enzymes with high K , values are therefore preferred for kinetic analysis to give a wider usable range of substrate concentration. 

C. B. Elliott, Application of Flow-Injection Analysis to Enzymatic Fluorescence Kinetic Methods. Diss. Ahstr. Int. B, 43 (1983) 3966. 

Because resolution is a kinetic phenomenon, an expression for the relative rates of conversion of the two enantiomers is desirable for a quantitative analysis of resolution. As the development and use of such an expression requires an understanding of the kinetics of enzymatic reactions, we defer considering this to Chapter 20 which deals with biochemical methods of enhancing reaction rates and selectivities. 

Catalytic techniques are more significant than non-catalytic ones in the broad scope of applications of kinetic methods due to their superior sensitivity and selectivity. The general principles behind the techniques outlined in the following sections are described in more detail elsewhere in this encyclopedia. Thus, this article is exclusively concerned with reaction rate methods of catalyzed reactions in homogeneous media. No mention is made of enzymatic analysis, which is treated elsewhere. 

Substrate concentration is obtained using a method based on kinetics by measuring the reaction rate. To reduce the time required per assay, the requirement for the quantitative conversion of substrate is abandoned. Since kinetic methods are less susceptible to interference than the endpoint method, they are advantageous for automated methods of enzymatic analysis. 

Theory The method of LDH assay is based on kinetic analysis. In a kinetic enzymatic assay a unit of enzyme activity is defined as the quantity of enzyme that brings about a certain absorbance increase in 30 seconds or 1 minute at a fixed temperature (for instance 25 0.2°C) ... 

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. 

Instruments of this type may also be used quite effectively to evaluate kinetics of time-dependent changes in foods, be they enzymatic or reactive changes of other types. The computerized data-acquisition capabilities of these instruments allow precise measurement of absorbance or fluorescence changes, often over very brief time periods ( milliseconds). This is particularly useful for analysis of fluorescence decay rates, and in measurement of enzymatic activity in situ. A number of enzyme substrates is available commercially which, although non-fluorescent initially, release fluorescent reaction products after hydrolysis by appropriate enzymes. This kinetic approach is a relatively underused capability of computerized microspectrophotometers, but one which has considerable capability for comparing activities in individual cells or cellular components. Fluorescein diacetate, for example, is a non-fluorescent compound which releases intensely fluorescent fluorescein on hydrolysis. This product is readily quantified in individual cells which have high levels of esterase [50]. Changes in surface or internal color of foods may also be evaluated over time by these methods. 

Schramm, V.L. (1999) Enzymatic transition state analysis and transition-state analogs, in Schramm, V. L. and Purich, D. L. (eds.), Methods in Enzymology 308, Enzyme kinetics and Mechanism, Part E, Academic Press, San Diego, pp. 301-354. 

For determination of creatinine, the nonspecific Jaffe method, although subject to perturbation by many interfering substances of endogenous and exogenous origin, is the most widely used. However, a batchwise kinetic procedure and flow injection analysis have shown the possibility to determine creatinine in human urine samples by this reaction, free from any systematic error. Enzymatic assays have higher specificities, but still suffer... 

---