Enzymatic analyses

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). 

Most biomolecules do not show any absorption in the visible or ultraviolet spectrum. In addition, they are usually present in the form of mixtures with other—similar—compounds that would also react to a chemical test procedure. These two problems can be avoided by using an appropriate enzyme to produce a colored dye selectively from the metabolite that is being analyzed. The absorption of the dye can then be measured. 

Koolman, Color Atlas of Biochemistry, 2nd edition 2005 Thieme All rights reserved. Usage subject to terms and conditions of license. 

All oxidoreductases (see p. 88) require coenzymes. The most important of these redox coenzymes are shown here. They can act in soluble form (S) or prosthetically (P). Their normal potentials E° are shown in addition to the type of reducing equivalent that they transfer (see p. 18). 

Many assay procedures depend on the use of specific NAD-dependent dehydrogenases, e.g.  

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. 

Enzymatic reactions that cannot be monitored directly by spectroscopic changes can be coupled to other reactions that do show such changes. One of the classic examples is the detection of glucose by an assay which depends on its conversion by hexo-kinase into glucose 6-phosphate, which is then coupled to an ancillary indicator reaction with NADP+ and glucose 6-phosphate dehydrogenase  

Alternative assays are available, and glucose can also be measured by the coupled reaction of glucose oxidase and peroxidase. 

Glucose + H20 + 02 — Gluconic acid + H202 (glucose oxidase) 

Enzymes are biological catalysts which the living cell uses to metabolize materials. Enzymes have an effect specific to substance, and allow reactions to occur with very small quantities of this substance. Now that 

Enzymatic analyses have the advantage of being specific for a transformation of a particular material, so that as a rule no separation is necessary, even in the event of small concentrations of this substance existing 

The following general points apply to enzymatic analyses  

The enzyme must act specifically, and must supply analytically determinable reaction products. (So far, it has only been possible to achieve this if several enzyme reactions are allowed to take place in succession, or if work is conducted in parallel with complementary enzymes). 

The enzyme must effect the complete transformation of the material, and the reaction must take place within a practicable period of time. 

Polyphenol oxidases (cf. 0.3.3) in mushrooms and potatoes require pressures of 800-900 MPa for inactivation. The addition of glutathione (5 mmol/1) increases the pressure sensitivity of the mushroom enzyme. In this case, the inactivation is obviously supported by the reduction of disulfide bonds. 

Up to a certain extent, enzymes need to be hydrated in order to develop activity. Hydration of e. g. lysozyme was determined by IR and NMR spectroscopy. As can be seen in Table 2.15, first the charged polar groups of the side chains hydrate, followed by the uncharged ones. Enzymatic activity starts at a water content of 0.2 g/g protein, which means even before a monomolec-ular layer of the polar groups with water has taken place. Increase in hydration resulting in 

3 - Peptide-CO Monomolecular hydration of polar groups Apolar side chains  

For preservation of food it is mandatory to inhibit enzymatic activity completely if the storage temperature is below the phase transition temperature Tg or Tg (cf. 0.3.3). With help of a model system containing glucose oxidase, glucose and water as well as sucrose and maltodextrin (10 DE) for adjustment of Tg values in the range of — 9.5 to — 32 °C, it was found that glucose was enzymatically oxidized only in such samples that were stored for two months above the Tg value and not in those kept at storage temperatures below T  

Enzymatic food analysis involves the determination of food constituents, which can be both substrates or inhibitors of enzymes, and the determination of enzyme activity in food. 

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Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

As the name implies, the odor of urine in maple syrup urine disease (brancbed-chain ketonuria) suggests maple symp or burnt sugar. The biochemical defect involves the a-keto acid decarboxylase complex (reaction 2, Figure 30-19). Plasma and urinary levels of leucine, isoleucine, valine, a-keto acids, and a-hydroxy acids (reduced a-keto acids) are elevated. The mechanism of toxicity is unknown. Early diagnosis, especially prior to 1 week of age, employs enzymatic analysis. Prompt replacement of dietary protein by an amino acid mixture that lacks leucine, isoleucine, and valine averts brain damage and early mortality. 

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. 

In an extensive study, Okamoto and co-workers [76-86] introduced a biochemical switching device based on a cyclic enzyme system in which two enzymes share two cofactors in a cyclic manner. Cyclic enzyme systems have been used as biochemical amplitiers to improve the sensitivity of enzymatic analysis [87-89], and subsequently, this technique was introduced into biosensors [90-93], In addition, cyclic enzyme systems were also widely employed in enzymic reactors, in cases where cofactor regeneration is required [94-107], Using computer simulations, Okamoto and associates [77,80-83] investigated the characteristics of the cyclic enzyme system as a switching device, and their main model characteristics and simulation results are detailed in Table 1.1, as is a similar cyclic enzyme system introduced by Hjelmfelt et al. [109,116], which can be used as a logic element. 

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

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. 

Volkmann S, Wohrl BM, Tisdale M, Moelling K. Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleoside H. JBiol Chem 1993 268 2674-2683. 

Wu LC, Cheng CM (2005) Flow-injection enzymatic analysis for glycerol and triacylglycerol. Anal Biochem 346 234-240... 

Ruijter GJG, Boer M, Weykamp CW, de Vries R, van den Berg I, Janssens-Puister J, Niezen-Koning K, Wevers RA, Poorthuis BJHM, van Diggelen OP (2005) External quality assurance programme for enzymatic analysis of lysosomal storage diseases a pilot study. J Inherit Metab Dis 28 979-990... 

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... 

Analysis of dopamine-jS-hydroxylase activity in plasma can not be used to make a definitive diagnosis, as approximately 4% of the population have very low plasma activities. Support for such a diagnosis can come from examining the noradrenaline dopamine ratio in plasma. This is markedly reduced in this condition. Deficiencies of the MAO isoforms have been confirmed from enzymatic analysis of fibroblasts (MAO-A) or platelets (MAO-B). 

As AADC is expressed in plasma, enzymatic analysis to elucidate further upon a suggestive CSF profile can be performed. 

Biosensors Enzymatic analysis of components and products from bioprocesses is widely utilized because this type of analysis is both selective and sensitive. To use enzymes for the automatic analysis and instrumentation of bioprocesses, various sensors using enzymatic reactions, the so-called "biosensors, have been... 

A postchromatographic -glucuronidase procedure has been also used for the analysis of phenolic glucuronides, such as those produced from trimethoprim (274). The enzymatic analysis of these glucuronides provided the production of the corresponding phenolic compounds, which were measured by both UV and electrochemical detection. 

Each oligosaccharide subjected to methylation or enzymatic analysis... 

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. 

Isolation, Subfractionation, and Enzymatic Analysis oi Beef Heart Mitochondria... 

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