Rate assays

There are many sources of errors in the plant. The principal ones are related to sampling (qv), mass flow rates, assaying, and deviations from steady state. Collecting representative samples at every stage of the flow sheet constitutes a significant task. Numerous methods and equipment are available (10,16,17). 

Enzyme Assays. An enzyme assay determines the amount of enzyme present in sample. However, enzymes are usually not measured on a stoichiometric basis. Enzyme activity is usually determined from a rate assay and expressed in activity units. As mentioned above, a change in temperature, pH, and/or substrate concentration affects the reaction velocity. These parameters must therefore be carefully controlled in order to achieve reproducible results. 

Spectrophotometry, a simple and rehable technique, is often used in rate assays. This method can be used when the substrate or the product of the reaction absorbs in the uv or the visible region. In other cases, a nonabsorbing system can be coupled to a system in which the substrate or product absorbs in the uv or visible region. 

Clinical chemistry Enzyme rate assays, colorimetric assays, end-point assays, immunoassays Upstone, 2000 ... 

The components of the coupling system should neither inhibit nor activate the primary enzyme. Moreover, care must be exercized to ascertain that the auxiliary enzyme (s) is not contaminated with other minor enzyme activities capable of influencing the primary enzymatic activity. The results from any coupled enzyme assay must exactly match the results obtained with other valid initial rate assays to ensure that the presence of the auxiliary system in no way affects the activity of the primary enzyme. This is typically accomplished by comparing data obtained from the coupled assay with stopped-time assay results to ensure that similar results are obtained. 

The rate equation to be used for kinetic analysis of enzyme depletion is that for simple noncompetitive inhibition. If the Henderson equation or similar types are not employed, keep in mind that the inhibitor concentration [I] is the free inhibitor concentration. Determination of Ki may not be feasible if the rate assay is insensitive and requires an enzyme concentration much greater than K[. Alternatively, Ki may be obtained by measuring the on-off rate constants of the E l complex, provided the rate constants for any conformation change steps involved are also known. 

The availability of completely pure enzyme affords the opportunity (a) to investigate mechanistic and regulatory properties in the absence of contaminating enzyme activities, and (b) to determine those kinetic parameters requiring an accurate knowledge of the enzyme concentration. Nevertheless, most kinetic studies do not require pure enzyme. What is required in that there be no (or, at least, minimal) contaminating activities that interfere with the initial-rate assay. A contaminating protein may act on the substrate(s), product(s), or effector(s). Hence,... 

R. G. Price, I. Pocsi, A. C. Richardson, and P. H. Whiting, Rate assays of IV-acetyl-P-D-glucosaminidase some problems and fundamental principles, Clin. Chem., 36 (1990) 1259-1260. 

Much interest for ion transport has its origin in the field of crown ether chemistry. Therefore, most model studies of ion channels have been more or less based on crown ether chemistries. Pioneering work has been undertaken by Fylcs, who not only synthesized varieties of gigantic molecules starting from crown ethers, " but established a method of the rate assay for ion transport across lipid bilayer membranes, a pH sCat technique. Vesicles having different inside and outside... 

Wastell H, Dale G, Bartlett (1984) A sensitive rate assay for biotinidase using a new derivative of biotin, biotinyl-6-aminoquinoline. Anal Biochem 140 69-73... 

Active-site titration versus rate assay... 

The assay of an enzyme solution, historically tied to a rate assay, depends on the correlation between the rate and a concentration (determined by some means, usually spectroscopic) of the purest possible sample of that enzyme. Since with even the most thoroughly characterised enzymes, such as a-chymotrypsin, a substantial proportion of the matter is inactive enzyme and water (up to about 30% in some cases), the concentration of active enzyme from rate assays is liable to a wide margin of error. 

If it is not possible to measure [Lg ] explicitly by analytical measurements, an irreversible inhibitor can be employed in conjunction with a rate assay using a substrate. Increasing... 

Analytical Determinations Involving Rate Assays. Rate assays must be used to determine the concentration of enzyme, of an activator, or of an inhibitor, and they may be used to determine the concentration of a compound which serves as a substrate for an enzyme. Substrate concentration may also be determined by a total change method. The advantages of a rate assay over a total change method are its speed and the requirement for less enzyme. 

Under most conditions the initial rate, Uo, of the reaction is directly proportional to enzyme concentration (8). In assays to determine the amount of enzyme in a sample the initial substrate concentration should be at least 10 times so that the reaction is zero order with respect to substrate concentration (Equation 3). At substrate concentrations less than 0.1 Km the reaction follows a first-order rate process with the rate directly proportional to substrate concentration. Enzyme rate assays to determine the amount of a compound as substrate in a sample should be run under these conditions. At substrate concentrations greater than 0.1 Km and less than 10 Km the reaction follows a mixed-order process intermediate between first and zero order. 

Enzymatic assay methods are classified as fixed-time assays, fixed-change assays, or kinetic (initial rate) assays. Kinetic assays continuously monitor concentration as a function of time pseudo-first-order conditions generally apply up to 10% completion of the reaction to allow the initial reaction rate to be determined. If the initial substrate concentration is > 10Km, then the initial rate is directly proportional to enzyme concentration. At low initial substrate concentrations (< 0.1 Km), the initial rate will be directly proportional to initial substrate concentration (cf. Chapter 2). For enzyme quantitation, a plot of initial rate against [E] provides a linear... 

It is clear that whatever the relative values of Sq and in Eqn. 6, as long as o> observed initial velocity is proportional to the concentration of functioning enzyme (Eq) present in solution. This is the basis of rate assays for enzymes. By working at Sq K, i.e. Vq = Pj ax> the observed velocity is not complicated by errors made in values of Sq. To obtain a value of Eq itself from such a velocity measurement requires at least an accurate knowledge of (for Sq /Tm... 

Brodbeck and Ebner found that the soluble lactose synthetase from milk can be separated into two protein components, A and B, which individually do not exhibit any catalytic activity however, their recombination restores full lactose synthetase activity. The B fraction has been crystallized from bovine skim milk and bovine mammary tissue, and was identified as a-lactalbumin. It was thus found that a-lactalbumin can be substituted for the B protein of lactose synthetase. Lactose synthetases from the milk of sheep, goats, pigs, and humans were also resolved into A and B proteins, and the fractions from these species were shown to be qualitatively interchangeable in the rate assay of lactose synthesis. Determination of the amino acid sequence of a-lactalbumin (B fraction) has shown a distinct homology in the sequence of amino acids of a-lactalbumin and hen s egg-white lysozyme, suggesting that lysozyme and a-lactalbumin have evolved from a common ancestral gene. 

When the electrode was removed from the sample solution and placed in a solution free from glucose, the output of the microbial electrode gradually increased and returned to initial level within 15 min at 30°C. (The current means the steady-state current hereinafter.) The response time of the microbial electrode was longer than that of the enzyme electrode. This may be caused by the time lag of the bacterial respiration. However, employment of a rate assay improved the response time and the... 

Fig. 1. The integration strategy for enzyme initial rate assay (Modified from Liu et al. (2009)).

After the integration strategy for enzyme initial rate assay is validated, a switch point should be determined for changing from the classical initial rate method to kinetic analysis of reaction curve. The estimation of Vm by kinetic analysis of reaction curve usually prefers substrate consumption percentages reasonably high. Therefore, the substrate consumption percentage that gives an enzyme activity from 90% to 100% of the upper limit of linear response by the classical initial rate method can be used as the switch point. 

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