Enzyme Measurements

Enzymes, measured in clinical laboratories, for which kits are available include y-glutamyl transferase (GGT), alanine transferase [9000-86-6] (ALT), aldolase, a-amylase [9000-90-2] aspartate aminotransferase [9000-97-9], creatine kinase and its isoenzymes, galactose-l-phosphate uridyl transferase, Hpase, malate dehydrogenase [9001 -64-3], 5 -nucleotidase, phosphohexose isomerase, and pymvate kinase [9001-59-6]. One example is the measurement of aspartate aminotransferase, where the reaction is followed by monitoring the loss of NADH ... 

Fig. 8. Plot of data from patients having Hver diseases A or B or unknown X (a) on two blood enzymes (b) scores of points on the first two eigenvectors obtained from an eight-dimensional enzyme space and (c) eigenvector plot of the variance weighted data. Variance weights ranged from 3.5 to 1.2 for the eight blood enzymes measured. A weight of 1.0 indicates no discrimination information (22).

Clinical Criteria for Clinical Enzyme Methods. While it is possible to measure the activity of several thousand enzymes in body samples, in most cases it is practical to measure only a few while obtaining clinically useful results. In order to select the enzyme measurements which will be most useful, the following guidelines are suggested ... 

A second test for buildup of a free inactivator is to measure product formation in the presence of an excess of compound until the progress curve reaches a plateau, and to then add a second aliquot of enzyme. As described earlier in this section, the addition of a second aliquot of enzyme should result in renewed product formation, which will wane with time as the new molecules of enzyme are inactivated. The rate of inactivation of the second aliquot of enzyme (measured as kobs) should be the same as that of the first aliquot of enzyme in the experiment, if the compound is functioning as a true mechanism-based inactivator. If, instead, inactivation is due to buildup of an inhibitory species, then the second value of lcohs should be greater than the first value. This experiment can also be performed by preincubating the enzyme with compound and initiating the reaction with cognate substrate, as... 

Establish the validity of using two specific erythrocyte enzyme measurements as sensitive indicators of very low body burdens of lead and other heavy metals... 

The inputs into the system are the pH and the concentration of enzyme, measured across all forms, both active and inactive. From these pieces of data, the system is required to provide an estimate of the reaction rate. Let us assume that the total concentration of enzyme is 3.5 mmol dnr3 and that the pH is 5.7 and use these values to estimate the rate of reaction. 

Fed diets containing fish oil vs. no fish oil diet for 5 months, then given single ip injection of BaP. Hepatic transformation enzymes measured 14 days postinjection... 

Dupont Y. 1984. A rapid filtration technique for membrane fragments or immobilized enzymes measurements of substrate binding or ion fluxes with a few millisecond time resolution. Anal Biochem 142 504. 

The most common assay uses 3a-hydroxysteroid dehydrogenase to form the 3-keto bile acid that is trapped by, for example, hydrazine hydrate, causing the reaction to go to completion. The co-factor NAD is reduced stoichiometrically and can be measured by ultraviolet absorption or more commonly by fluorescence at an activation of 345 nm and emission of 450 nm. Use of this enzyme measures all bile acids with a 3a-hydroxyl but not cholesterol, which has a 3p-hydroxyl, and does not measure bile acids with a sulphate or glucuronide group conjugated to the 3a-hydroxyl. 

Figure 2. Enzymic measurement of a-L-arabinan in fruit-juice concentrates, (a) Effect of time of incubation of sample with arabinofuranosidase and ndo-arabinanase. Diluted pear juice concentrate (1 10 0.1 ml) or sugar beet arabinan solution (0.1 ml) was incubated with an aliquot (0.1 ml) of arabinofuranosidase (5 units) plus ndo-arabinanase (0.2 units) at 35 C and pH 4.0. Aliquots were analysed for arabinose. (b) Arabinose determination using the NAD-Galactose Dehydrogenase method. Arabinose solution (0.2 ml, 50 digrams) was incubated with Tris-HCl buffer (2.5 ml, pH 8.6), NAD (0.1 ml, 10 m ml) and galactose dehydrogenase (20/i, 140 milliunits) at 35°C. Absorbance (340 nm) was measured after various time intervals.

Before enzyme measurements, approximately 300-500 pi of demineralized water is added and the mixture is sonicated for 10 s on ice. Depending on the ultrasound homogenizer, the time required may vary slightly. Protein is determined according to Lowry et al. [43]. The final protein content for enzyme measurements is adjusted to approximately 5 mg/ml with demineralized water. 

Lawrence and Sanderson proposed another micro-method for measuring chymosin and other proteolytic enzymes. Measurement of concentration was based on the rate of radial diffusion of the enzyme through a thin layer of caseinate-agar gel. The limit of diffusion was marked by a zone of precipitated casein (Emstrom and Wong 1974). Holmes et al (1977) developed a microdiffusion assay for residual proteolytic enzymes in curd and whey that is more sensitive than the method of Lawrence and Sanderson or the clotting-time assay of Reyes (1971). 

The same type of substrates may also serve for ultraviolet spectrophotometry, since acyl thiocholines show a maximum at 226 fi. In practice, it is more convenient to follow the change in optical density at about 250 y 12). The method, which works only with purified enzymes, measures substrate concentrations of 5 X 10-6 M. 

Carbamates effect the reversible carbamylation of acetylcholinesterase, permitting accumulation of acetylcholine at cholinergic neuroeffector junctions (muscarinic effects), at the myoneural junctions of skeletal muscle, and in the autonomic ganglia (nicotinic effects). CNS function is also impaired. However the relatively large dissociation constant of the carbamyl-enzyme complex indicates that it dissociates more readily than does the organophosphate-enzyme complex, mitigating the toxicity of the carbamate pesticides. The reversibility of the carbamyl-enzyme complex affects (limits) the utility of blood enzyme measurements as a diagnostic tool. 

Repeatability - enzyme measurements over time can be used to determine the reversibility or progression of renal lesions... 

The substrates, either 1 mg/ml amylose (c.l. 405) or amylopectin (c.l. 21), were incubated with 5 units/ml maize-branching enzyme (measured as by stimulation of phosphorylase a Guan and Preiss. 1993). The decrease in absorbance by the iodine/glucan complex was measured at 660 nm. 

Figure 4.10 Fits to kinetic data from [135] on the operation of citrate synthase from rat kidney. Data (flux as a function of substrate concentrations) were obtained from Figures 2, 3, 4, 5, 6, 7, and 9 of [135], Initial fluxes (p.mol of COASH (or CIT) synthesized per minute per ug of enzyme) measured at the substrate concentrations indicated are plotted in A, B, C, and D. For A, B, and D, the initial product (CIT and COASH) concentrations are zero. In C, flux was measured with COASH added in various concentrations to investigate the kinetics of product inhibition. E and F show fits to kinetic data on the reverse operation of kidney enzyme, with product concentrations indicated in the figure. All data were obtained at pH = 8.1 at 28 °C. Model fits in all cases are plotted as solid lines.

Figure4.11 Fits to kinetic data from [63] on the forward operation of liver enzyme. Measured flux in arbitrary units was obtained from Figures 1,2, 5, 6,13, and 14 of [63], For all cases the product (CIT and COASH) concentrations are zero and total substrate and inhibitor concentrations are indicated in the figure. Data obtained with no inhibitors present are plotted in A and B. In C the relative activity (normalized to its maximum) of the enzyme is plotted as functions of [ATP], [ADP], and [AMP] measured at [ACCOA] = 11 TM and [OAA] = 1.9 uM. D. The measured flux is plotted as a function of [ACCOA] at [OAA] = 34 qM with ATP, ADP, and AMP present as indicated in the figure. In E the relative activity of the enzyme is plotted as functions of [ATP] at [Mg2+] = 0 mM (shaded circles), 0.5 mM (shaded triangles), 1.0 mM (shaded squares), 2.0 mM (open circles), and 4.0 mM (diamonds). In F relative activity is plotted as a function of pH. Substrate concentrations are [ACCOA] = 21 qM and [OAA] = 8.6 qM. All data were obtained at 25 °C. pH is fixed a 7.4 for A. Model fits are plotted as solid lines.

The routine unit of enzyme activity has been the international unit (I.U.), namely xmoles P formed (or S consumed) per minute. The specific activity of an enzyme preparation is the number of xmoles P formed (or S consumed) per minute per milligram of protein (clearly this will be very low in a crude cell extract and have a maximal value for a pure preparation of the enzyme). If the molecular mass is known, the specific activity of a pure enzyme measured in saturating (Fmax conditions) can be used to calculate the turnover number (or molecular activity ) of an enzyme, namely the number of P molecules formed (or S molecules transformed) per molecule of enzyme per second (units sec- ). If we recall that the maximal velocity (Fmax) equals k+2 (sec " ) [ET], we can see that the molecular activity equals k+2 (sec -1), that is, fal (sec-1). The katal is the S.I. unit of enzyme activity (moles substrate transformed sec -I) from whence come the corresponding units for specific activity (katal kilogram-1) and molar activity (katal per mole of enzyme). 

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