Rate measurement enzymatic

It is during this steady state period that the rates of enzymatic reactions are traditionally measured and the parameter measured is the initial rate v of product formation - that is the formation of the first few percent of the products so that the substrate is not depleted and the products(s) have not yet accumulated. 

Figure 15-2 Absorption spectra of NAD+ and NADH. Spectra of NADP+ and NADPH are nearly the same as these. The difference in absorbance between oxidized and reduced forms at 340 nm is the basis for what is probably the single most often used spectral measurement in biochemistry. Reduction of NAD+ or NADP+ or oxidation of NADH or NADPH is measured by changes in absorbance at 340 nm in many methods of enzyme assay. If a pyridine nucleotide is not a reactant for the enzyme being studied, a coupled assay is often possible. For example, the rate of enzymatic formation of ATP in a process can be measured by adding to the reaction mixture the following enzymes and substrates hexokinase + glucose + glucose-6-phosphate dehydrogenase + NADP+. As ATP is formed, it phosphorylates glucose via the action of hexokinase. NADP+ then oxidizes the glucose 6-phosphate that is formed with production of NADPH, whose rate of appearance is monitored at 340 nm.

The precise relationship between potential hydrolysis rates measured with externally added substrates and the rates at which complex microbial communities in marine systems hydrolyze and ultimately remineralize a spectrum of organic macromolecules actually available as substrates is unknown. Extracellular enzymatic hydrolysis is frequently regarded as the rate-limiting step in remineralization of organic carbon (e.g., King, 1986 ... 

Enzyme kinetics is concerned with measuring the rates of enzymatic reactions, and with factors that affect the rates. 

If it is not possible to obtain an end point or complete conversion to product, substrate concentrations can be determined from the rate dependence of the enzyme reaction under conditions where [S] KM (Sect. 8.2.2). The sensitivity of the method is not as great as can be achieved by using equilibrium end point measurements, since accuracy depends on analysis of the initial rate of enzymatic turnover. 

Many different measurements of enzymes are undertaken to acquire different types of information. For example, the presence of genes for an enzyme has been used to infer whether organisms are capable of performing particular functions, the expression of those genes or the appearance of the enzyme protein is used to indicate if and under what conditions the gene is functioning, while assay of the activity of the enzyme has been used to infer rates of particular processes (see Section 2). In fact, virtually all measurements classified as molecular and most rate measurements of uptake are, in fact, measurements of enzymes. Because strictly molecular methods (i.e., the capacity to perform a reaction see Chapter 30 by Zehr and Jenkins, this volume) and N uptake (i.e., the net result of enzymatically mediated processes see Chapter 6 by Mulholland and Lomas, this volume) are discussed elsewhere in this book in this chapter, we will focus primarily on measurements of enzyme activities. 

Acetyl DL-methionine which is used as a substrate for amino acylase activity determination was prepared by acetylation of DL-methionine with acetic anhydride in acetic acid [5]. The rate of enzymatic hydrolysis was determined by measuring the liberated amino acid by ninhydrin method [6] where ascorbic acid was used as oxidizing agent instead of sodium cyanide. The activity curve of pure amino acylase enzyme is shown in Fig. 1 as a continuous line. For determining the effect of metal ions on the activity of amino acylase the following procedure was adopted. 

Figure 11.3 depicts the cell used by the automatic potentlometrlc Instrument devised by Malmstadt and Pardue [6] for ths enzymatic determination of glucose based on reaction-rate measurements. The chemistry involved Is... 

The rapid growth of instruments with built-in microprocessors or computers has been the result of a number of reasons. First, the enormous growth of the number of clinical analyses required, which increased by 10-20% yearly between 1970 and 1980. Second, the need for better performance from analysers whose precision could not be improved or even preserved without computerization. Even the basic concept behind some analysers (e.g. centrifugal analysers) or enzymatic rate measurements with multi-detection are not feasible without recourse to computerization. 

When we measure the rate (also called the velocity) of an enzymatic reaction at varying substrate concentrations, we see that the rate depends on the substrate concentration, [S]. We measure the initial rate of the reaction (the rate measured immediately after the enzyme and substrate are mixed) so that we can be certain that the product is not converted to substrate to any appreciable extent. This velocity is sometimes written or Vq to indicate this initial velocity, but it is important to remember that aU the calculations involved in enzyme kinetics assume that the velocity measured is the initial velocity. We can graph our results as in Figure 6.8. In the lower region of the curve (at low levels of substrate), the reaction is first order (Section 6.3), implying that the velocity, V, depends on substrate concentration [S]. In the upper portion of the curve (at higher levels of substrate), the reaction is zero order the rate is independent of concentration. The active sites of aU of the enzyme molecules are saturated. At infinite substrate concentration, the reaction would proceed at its maximum velocity, written kJnax-... 

It is obvious from the discussion above that any kinetic-based analytical procedure must take into account the degree of approximation made in the various rate equations with respect to the period of measurement, the relative initial concentrations of reactants, and, in some cases, the reversibility of the reactions. Care must be taken, for example, in using a pseudo-first-order method when the initial concentration of the unknown varies over several orders of magnitude the error introduced in assuming the validity of the pseudo-first-order approximation of Equation 18.8 is a function of [A]q. Although the reaction mechanisms and rate equations for enzymatic and other catalyzed reactions in general are somewhat more complex, similar assumptions and simplifications (and, therefore, restrictions in validity) apply to the rate-measurement techniques employed in the analytical use of these systems. 

There are several methods for the determinations of kinetic parameters, most of them based on initial rate measurements at varying substrate concentrations alternatively they can be determined from the time course of the enzymatic reaction. 

The rate of enzymatic reaction increases with substrate concentration for example, in the case of a cyanide electrode coupled with immobilized jS-gluco-sidase enzyme, a response time of 20 s for 10 moll amygdalin and Imin for 10 " moll amygdalin is obtained. Rather than waiting until an equilibrium potential is reached, the rate of potential change (AE/At) can be measured, the result being proportional to substrate concentration. 

The rate of enzymatic reactions can also be established by potentiometric measurement of product formation using an ion selective electrode. The most important ion selective electrode is the glass electrode for pH measurement. Despite their outstanding selectivity for H+ ions, glass electrodes are used only seldom in enzyme electrodes because their sensitivity is affected by the buffer capacity of the sample matrix or sensor filler solution. 

The role of enzyme-mediated hydrolysis (depolymerization) of PL has been investigated (Schakenraad et al., 1990). This study concluded that the m or route of degradation of PL is most likely via simple (non-enzymatic) hydrolysis. However, the results of the study showed that the possibility of some enzyme-mediated hydrolysis could not be ruled out as a minor pathway. The conclusion that the process is not enzyme mediated is supported by another study which showed that the degradation rates measured in-vivo (sheep, dogs, and rats) were essentially the same as measured in-vitro (Leenslag et al, 1987). 

The rates of adsorption and chain scission are affected by physicochemical properties of the substrate, such as the molecular weight, chemical composition, crystallinity, and surface area, and also by the inherent characteristics of the enzyme which can be measured in terms of its activity, stability, concentration, amino acid composition, and conformation. Moreover, environmental conditions such as pH and temperature also influence the activity of enzymes. The presence of stabilizers, activators, or inhibitors released from the polymer during the degradation process or additives that are leached out may also affect enzyme activity. Chemical modification of biopolymers may also affect the rate of enzymatic resorption since, depending on the degree of chemical modification, it may prevent the enzyme from recognizing the polymeric substrate. The rate of enzymatic resorption is limited by an enzyme saturation point. Beyond this enzyme concentration, no further increase in the rate of resorption is observed even when more enzyme is added. 

In the world market, more than 90% of commercial biosensors are enzymatic, in particular, those that measure glucose, used by diabetics [79]. This kind of biosensors is very useful because if immobilized enzymes are sensitive to certain pollutants, these analytes can be easily measured. For example, biosensors based on the inhibition of acetylcholinesterase detect phosphorus insecticides and other inhibitors [80]. A comprehensive analysis showed that the developed enzymatic biosensors demonstrated reproducible, stable, and fast responses to the substrates to be measured. Unfortunately, the application of these biosensors can be restricted because of the dramatic decrease in the sensor response at increasing buffer capacity and ionic strength, pH-dependence of the enzyme kinetics, and cosubstrate limitation of the measured enzymatic reaction rate (the glucose sensor) [79]. Recently, Soldatkin et al. reported a complete review of some biopolymers used in enzymatic... 

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