Enzymatic Analysis Methods

Many analyses in biological and clinical chemistry, and in the environmental, food and forensic sciences, involve the study of exceedingly complex multicomponent sample matrices. Moreover, the analytes under study are frequently present in trace amounts. Analytical techniques which combine exceptional selectivity with high sensitivity are thus required. Preeminent among these methods are those where the necessary selectivity is 

The catalytic activity of enzymes provides for an enormous range of analytical techniques. Analytes are not restricted to conventional organic molecules, but include virtually all chemical species, including gases and metal ions. The ability of a single enzyme molecule to catalyze the reaction of numerous substrate molecules also provides an amplification effect which enhances the sen.sitivity 

Antibodies have been used in analysis for over 60 years, and offer an unexpectedly wide range of techniques and applications. In some cases, the specific combination of an antibody with the corresponding antigen or hapten can be detected directly (e.g., by nephelometry), but more often such reactions are monitored by a characteristic label such as a radioisotope, fluorophore, etc. Since antibody reactions do not have a built-in amplification effect, these labels are frequently necessary to provide sufficient analytical sensitivity. Antibodies of different classes vary greatly in stability, but some are relatively robust proteins, and this contributes significantly to the range of methods available. 

Enzymes and antibodies are proteins, with the ability to bind appropriate ligands very strongly and specifically. Enzymes frequently need cofactors or metal ions for (full) activity. Antibodies are multifunctional molecules, whose ability to bind to cell surfaces and to other proteins, at sites distinct from the antigen/hapten-binding sites, extends the range of labeling and detection methods available in immunoassays. 

This article can only attempt a general survey of enzyme and immunoassay methods, with references to more detailed reviews and books in specific areas. Many important clinical, veterinary, and forensic analyses in which enzymes themselves are the analytes are not discus.sed this article considers only those methods where enzymes (and antibodies) are analytical reagents. 

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Probably the most important carbohydrate polymer in food, certainly from a nutritional standpoint, is starch, and Chapter E2 presents three units that can be used for starch isolation and characterization. UNITE2.1 condenses many different starch isolation methods into one scheme for starch isolation from virtually any plant source. In this protocol, the author notes the care that must be taken to prevent starch degradation during isolation. unite2.2 presents a simple enzymatic analysis method that can be used for estimation of starch in food. Finally, unit E2.3 describes a colorimetric method to determine starch amylose content. Again, different combinations of the units in this chapter can be used to characterize starch from any source. 

Michal, G., Mollering, H., and Siedel, J., Bergmeyer s Methods in Enzymatic Analysis 1,... 

Aebi, H. (1974). Catalase. In Methods of Enzymatic Analysis. FLU. Bergmeyer (ed), pp. 673-684. Verlag Chemie, Weinheim, Germany. 

Huber, S.J. and B. Hock. 1986. Atrazine in water. Pages 438-451 in H.U. Bergmeyer, J. Bergmeyer, and M. Grassl (eds.). Methods of Enzymatic Analysis. Vol. XII. Drugs and Pesticides, Third Edition. VCH Publishers, Deerfield Beach, FL. 

Enzymes play an important role in biochemical analysis. In biological material—e. g in body fluids—even tiny quantities of an enzyme can be detected by measuring its catalytic activity. However, enzymes are also used as reagents to determine the concentrations of metabolites—e.g., the blood glucose level (C). Most enzymatic analysis procedures use the method of spectrophotometry (A). 

H. U. Bergmeyer (1983) Methods of Enzymatic Analysis, 3rd ed., Springer-Verlag, Berlin. 

AOAC International. (1995). AOAC Method 991.43, Total, Insoluble and Soluble Dietary Fiber in Food—Enzymatic-Gravimetrie Method, MES-TRIS Buffer. Official Methods of Analysis, 16th ed., Gaithersburg, MD. 

Bergmeyer, H.U. Beutler, H.O. In Methods of Enzymatic Analysis Bergmeyer, H.U., Ed. 3rd ed. Verlag Chemie Weinheim Deerfield Beach, FL, 1985 Vol. VIII. 

Beutler HO (1984) D-Fructose. In Bergmeyer HU, Bergmeyer J, Grassl M (eds) Methods of Enzymatic Analysis, 3rd edn, Vol VI. Verlag Chemie, Weinheim, Deerfield Beach/Florida, Basel, pp 321-327... 

Kurz, G. and Wallenfels, K. 1974. D-Galactose. UV-assay with galactose dehydrogenase. In Methods of Enzymatic Analysis, 2nd ed. H. U. Bergmeyer (Editor). Academic Press, New York. 

Methods of Enzymatic Analysis, H. Bergmeyer, Editor. Contains methods for enzyme purification and assay, in several volumes. 

Bergmeyer, H. U. and Brent, E. (1974a) Enzymatic analysis of sucrose, in Methods of Enzymatic Analysis (ed. H.U. Bergmeyer), Academic Press, London, pp. 1176-9. 

Brand, K. and Hess, B. (1983) Methods for animal tissue. In Methods for Enzymatic Analysis (ed. Bergmeyer, H.U.), 3rd edn. Verlag Chemie, Weinheim. 

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