Enzyme-Activity Measurement

Detection of free radicals can be performed using light absorption, luminescence, oxygen consumption, electrical conductivity, and enzyme activity measurements. A number of examples of relatively common systems are given in Table 1. 

Rather thorough studies have now been made of the properties of the PM of tomatoes,20 21 44 62 63 66 orange peel,21 tobacco3 61 64 and alfalfa.49 The activity of PM is profoundly affected by the pH of the reaction mixture, the salt concentration and the cation component of the salt. In salt-free solutions, the activity of the PM of higher plants is nearly zero at pH 4.25 and increases linearly with a steep slope as the pH is increased to 8. Above 8 the pectinic acids are also demethylated by the action of alkali and the enzyme activity measurements therefore become unreliable. The relationship between activity and pH is also dependent on the salt content of the reaction mixture. 

Enzyme activity measured, in vitro, in extracts of the muscle (for details of assay technique, see Chapter 3). 

Enzyme activity measurements are greatly affected by buffer systems used in analysis. In measuring alkaline phosphatase activity under optimal conditions, ethylaminoethanol buffer yielded activity 3.8 times... 

Enzymatic hydrolysis, of p-glucan, 753 Enzyme activity measurements conditions, activity units, and reaction rate, 331 -334... 

In studies with alkaline phosphatase it has been found that the enzymic activity measured by the release of p-nitrophenol from p-nitrophenyl phosphate increases with the concentration of tris buffer much faster than it increases with the ionic strength of other salts such as NaCl and Mg2SO< (4, 50). This behavior of tris was shown by Dayan and Wilson (122, 123) to result from a transphosphorylation reaction, where 0.5 M tris reacts with phosphoryl enzyme to form tris phosphate at the same rate as does 55 M water to form orthophosphate. 

Various methods are used for evaluating the quality, ie, physical strength and enzyme dust formation, of the granulate. In the elutriation process, a sample of product is fluidized in a glass tube with a perforated bottom plate for 40 minutes. Dust from the sample is collected on a filter and the enzyme activity measured. An acceptable dust level is when less than 5—10 ppm of the activity of the sample has been collected. In the so-called Heubach method, 20 g of granulate is elutriated. During the elutriation, four steel balls are rotated in the bed in order to evaluate the impact of attrition on the dust release of the enzyme. The dust is collected on a filter and measured. The acceptable dust level is very low. 

Following work by the same group addressed some of the major problems arising when electrochemical biosensors are in contact with food matrices pH effect and particle effect. Both problems were solved treating the biosensor surface with a Tween20 /phosphate buffer solution (pH 7.5) after the incubation with pesticide. The treatment was successful in removing the particulate, the correct pH for enzyme activity measurement was attained and the pesticide enzyme inhibition... 

Table 4 Interlaboratory comparison of enzyme activity measurements. Source Austrian Proficiency Testing Programme...

Cellulase activity of the samples was determined as filter paper activity (FPA) expressed in filter paper units (FPU) using Mandels procedure (15), and (3-glucosidase activity was assayed using 4-nitrophenyl-(3-D-glucopyranoside substrate according to Berghem and Petterson s (16) method. All samples were analyzed in triplicate and the mean values were calculated. The relative standard deviation of enzyme activity measurements was always below 5%. 

The activity of the cellulytic enzymes was measured as filter paper activity units. A1 x 6 cm strip of Whatman No. 1 filter paper was added to a total volume of 1.5 mL of enzyme solution containing 0.05 M citrate buffer, pH 4.8. All enzyme activity measurements were carried out in triplicate. The samples were incubated for 1 h at 50°C. Reducing sugars were determined after stopping the hydrolysis by adding 3 mL of dinitrosalicylic acid solution followed by boiling for 5 min. After cooling, 20.0 mL of distilled water was added and the absorbance was read at 540 nm (21). 

Fig. 5.13 Phosphatidylserine (PS) dependence of Ca2+-stimulated PKC activity as a function of palmitoyl- (Po), oleoylphosphatidylserine POPS concentration in the POPS/PE LUVs. Enzyme activity measured relative to 100% POPS. Total phospholipid concentration in the incubation...

Prepare one sample tube for the enzyme activity measurements. Add 1.0 mL of reagent cocktail and 0.7 mL of distilled water. Next, add 0.2 mL NADP+ solutions. Be careful to pipet exactly 0.2 mL NADP+. Mix the contents of the sample tube. Readjust the spectrophotometer with the Blank (prepared in step no. 3) to read 0.00 absorbance or 100.00% transmission. Remove the Blank and save it for future readjustments. 

Preconditioning, Amino Acid Concentrations and Enzyme Activity Measurement... 

The in situ method for enzyme activity measurement is the most efficient. Enzyme activity can also be measured from cell homogenates or microsomal preparations. Besides activity, one can also measure mRNA and protein levels. 

Use the quantity of PNP liberated per unit of time to calculate the enzyme activity. Measure the PNP liberated against a quantity of a standard preparation of PNP by measuring the absorbance of the solutions at 400 nm after adjusting the pH of the reaction mixture to pH 8.0. 

When the substrate and the enzyme are electrophoretically mixed, most of the enzyme will appear in the enzyme-substrate complex. Returning to the example used by Bao and Regnier, the net charge of the enzyme-substrate complex will be 10 - 2 = 8. Since enzyme activity measurements are performed under substrate-saturating conditions, all the enzyme will be bound to substrate, and the mobility of the enzyme should be the mobility of the ES complex. 

Garcia-Vargas, G.G., Hemandez-Zavala, A. (1996). Urinary porphyrins and heme biosynthetic enzyme activities measured by HPLC in arsenic toxicity. Biomed. Chromatogr. 10 278-84. 

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