Quantitation of Enzymes and Their Substrates

The quantitation of enzymes and substrates has long been of critical importance in clinical chemistry, since metabolic levels of a variety of species are known to be associated with certain disease states. Enzymatic methods may be used in complex matrices, such as serum or urine, due to the high selectivity of enzymes for their natural substrates. Because of this selectivity, enzymatic assays are also used in chemical and biochemical research. This chapter considers quantitative experimental methods, the biochemical species that is being measured, how the measurement is made, and how experimental data relate to concentration. This chapter assumes familiarity with the principles of spectroscopic (absorbance, fluorescence, chemi-and bioluminescence, nephelometry, and turbidimetry), electrochemical (poten-tiometry and amperometry), calorimetry, and radiochemical methods. For an excellent coverage of these topics, the student is referred to Daniel C. Harris, Quantitative Chemical Analysis (6th ed.). In addition, statistical terms and methods, such as detection limit, signal-to-noise ratio (S/N), sensitivity, relative standard deviation (RSD), and linear regression are assumed familiar Chapter 16 in this volume discusses statistical parameters. 

Bianalytical Chemistry, by Susan R. Mikkelsen and Eduardo Corton ISBN 0-471-54447-7 Copyright 2004 John Wiley Sons, Inc. 

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A wide variety of instrumental methods have been used to quantitate enzymes and their substrates. The choice of method depends primarily on the physical properties of the species being measured, and this is generally the product of the enzymatic or indicator reaction. In this section, instrumental detection methods are broadly classified as optical, electrochemical or other , where other techniques include radiochemical and manometric methods. 

Quantitative Analysis of Selectivity. One of the principal synthetic values of enzymes stems from their unique enantioselectivity, ie, abihty to discriminate between enantiomers of a racemic pair. Detailed quantitative analysis of kinetic resolutions of enantiomers relating the extent of conversion of racemic substrate (c), enantiomeric excess (ee), and the enantiomeric ratio (E) has been described in an excellent series of articles (7,15,16). 

A direct comparison of catalysis of olefin epoxidation with a homogeneous chemical catalyst (Mn salen), an enzyme (CPO), and an antibody resulted in sufficiently high enantioselectivity for all three catalysts, a higher turnover number for the enzyme, and a slightly higher substrate/catalyst ratio for the homogenous catalyst. Criteria for comparison should be quantitative and include catalyst lifetime as well as volumetric productivities, but have been found to depend on the different needs of laboratory synthetic chemists, who need a broadly specific catalyst quickly, versus those of process chemists, who often control catalyst availability and can allow narrow specificity (provided their substrate is accepted) but need high productivity. 

In vitro Metabolism. Numerous variables simultaneously modulate the in vivo metabolism of xenobiotics therefore their relative importance cannot be studied easily. This problem is alleviated to some extent by in vitro studies of the underlying enzymatic mechanisms responsible for qualitative and quantitative species differences. Quantitative differences may be related directly to the absolute amount of active enzyme present and the affinity and specificity of the enzyme toward the substrate in question. Because many other factors alter enzymatic rates in vitro, caution must be exercised in interpreting data in terms of species variation. In particular, enzymes are often sensitive to the experimental conditions used in their preparation. Because this sensitivity varies from one enzyme to another, their relative effectiveness for a particular reaction can be sometimes miscalculated. 

We have now extended these studies to synthetic phospholipids that contain short chain fatty acyl groups and which are water soluble, such as dibutyryl and dihexanoyl phosphatidylcholine (PC). These phospholipids are monomeric below their critical micelle concentration (cmc), yet activate the enzyme. In order to carry out kinetic studies, the long chain phospholipid substrate must generally be solubilized by a detergent such as Triton X-100 which serves as an inert matrix. Further understanding of the mechanism of the activation by short-chain phospholipids requires first a quantitation of the solubilization of these compounds by detergent ... 

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