Enzyme Assays Catalase Activity

The bacterium Lactobacillus plantarum and its closest allies are unusual in that they are aerobic organisms but do not produce a superoxide dismutase. This bacterium instead accumulates Mn(II) to an intramolecular level on the order of 25 mM (150-152). In vitro studies indicated that Mn(II) formed a complex with lactate which possessed significant superoxide activity (153). These bacteria are additionally unable to produce heme and, consequently, when grown in the absence of heme, produce a hemeless catalase, or pseudocatalase (154-158). Unlike heme-containing catalases, the enzyme is not inhibited by cyanide or azide, and the addition of either Mn or Fe into the growth medium increased the amount of the pseudocatalase present. However, neither of the metals could be detected in partially purified enzyme assays (157). 

Enzyme assays Intact chloroplasts were isolated and then osmotic-ally shocked. The lysates were used for determination of the stromal activities of ascorbate peroxidase (6), monodehydroascorbate reductase (7) and dehydroascorbate reductase (6). Superoxide dismutase (SOD) was assayed (8) in a protein extract derived from chloroplasts solubilized in the presence of 2.5% Triton-X-100. Catalase activity was determined (9) in leaf homogenates. 

Assay Methods. The conventional assay for catalase activity involves determination of the first-order rate constant under specified conditions 0.01 M H2O2 and 0.0067 M phosphate pH 6.3 at 0 C. One milliliter of enzyme is added to 50 ml. of reaction mixture and 5 ml. samples are titrated in H2SO4 with KMn04. The reaction constant is evaluated ... 

Suppression of peroxidase and catalase activity is of importance for the shelf life of heat-processed food. As long as the protein moiety has not been denatured, it is the lipoxygenase enzyme which is the most active for lipid peroxidation (cf. 3.7.2.2). After lipoxygenase activity is destroyed by heat denaturation, its role is replaced by the heme(in) proteins. As already suggested, an assay of heme(in) protein enzyme activity does not necessarily reflect its prooxidant activity. 

The detection and later the estimation of catalase activity during the first 40 years of research in this field was founded wholly on the appearance of 02 gas formed in the reaction. Thdnard (342), in 1818, estimated the H2O2 decomposing activity by collecting the oxygen gas evolved in graduated tubes placed under mercury. Jacobson (189) used a similar technique but replaced the mercury by water. Spitzer, in 1897 (326), published a quantitative assay method for this enzyme which was recognized as a separate entity 4 years later by Loew (233). 

Occasionally the problem of determining the activity of one hydroperoxidase in the presence of the other can arise. If physical separation of the enzymes is not possible, as in investigations involving whole cells, the catalase activity can be inhibited during the determination of the peroxidase activity, or the H20 can be replaced by MeOOH in the assay. On the other hand, a gasometric technique should permit the determination of the catalase activity free from interference by peroxidase. 

Galactose oxidase of P. circinatus was apparently inhibited by traces of BESOD. It can be inactivated by H Oj produced in the reaction unless catalase was added. There was an activation by traces of O J. In the absence of oxidants the reaction usually showed an induction period The enzyme, used at very low concentrations in the assays, was protected by proteins like serumalbumin. SOD did, however, not alter the reaction rate when added after 15 min This was interpreted by an inactivation of SOD by the H O accumulated in the reaction but it could just as well mean that SOD had no effect on the active enzyme, but that it did lower the activation in the induction period. Peroxidase activated galactose oxidase and suppressed the effect of SOD It did protect the enzyme against H O inactivation and could have been responsible for appreciable amounts of OJ, produced from O and from radicals formed in its action on a substrate. 

The autoxidation of ascorbate, a cosubstrate of dopamine P-monooxygenase, induces the degradation of most proteins including catalase and dopamine p-monooxygenase, but with the exception of (Cu,Zn)-SOD. Catalase protects dopamine P-monooxy-genase and is therefore generally added in the assay systems . The apparent activation or rather the stabilization of the enzyme (6.5 pg) by small amounts of catalase (3.1 pg) was enhanced by native but not by boiled SOD (100 pg) and also by similar amounts of serumalbumin (100 pg) or of boiled catalase (65 pg)... 

The [Eu(Tc)] assay for H202 can also be used for the determination of enzyme activities. This comprises all enzymes that produce (oxidases) or consume (catalase, peroxidases) H2O2. For example, the activity of glucose oxidase can be directly imaged after the addition of [Eu(Tc)] as indicator and glucose as substrate. Steady-state and time-resolved imaging schemes can be applied for the read-out of a microwell plate-based assay. The best results were obtained by the RLI method (Fig. 19) [115]. 

Table 11. Enzymic activity of different Cu2+ and/or Zn2+ apoerythrocuprein chelates. The reciprocal concentrations of these different metal apoprotein complexes were compared under equilibrium conditions ( Cyt credl X Cyt cox]-1 = 1)- For each metal protein 4 different assays were performed. Incubations were carried out at 25°. The assay mixture was composed of xanthine, 3.3 x 10 AM beef-heart cytochrome cox, 2.7 X 10 5M catalase, 1.6 x 10 SM xanthine oxidase, 2.1 x 10 1M HEPES buffer, 5 x 10 2M, pH 7.8 (78, 122)...

Enzyme Purification. Broccoli contained sufficient levels of peroxidase, lipase and cystine lyase to permit their isolation in the amounts needed. Only traces of lipoxygenase and catalase were present. Activities (units/g vegetable see assay methods below) were peroxidase, 220 lipase, 12 lyase, 0.26. Catalase was M) units/g in broccoli compared to 19 in English green peas lipoxygenase was 2 units/g in broccoli compared to 110 in English green peas. Peroxidase, lipase and cystine lyase were purified by... 

Alcohols Lower alcohols have been measured with alcohol oxidase (EC 1.1.3.13) from Candida boidinii or Pichia pastoris. The latter is available with a higher specific activity and has a somewhat different substrate specificity. Coimmobilization with catalase increases the stability of the enzyme column to several months with an operating range of 0.005-1 mmol 1 (0.5 ml samples) using 0.1 mol 1 sodium phosphate, pH 7.0, as the buffer. This assay is useful for the determination of ethanol in samples from beverages, blood, and for monitoring fermentation. 

Several established protocols have been adapted for 96-well plate readers including catalase, hyaluronidase, acetylcholinesterase, protein phosphatases and membrane-bound ATPases (22-26). In several instances these have involved novel protocols that are well suited to the ELISA format. For example, a sensitive, rapid microtitre-based assay for hyaluronidase activity was described by Frost and Stem (23). The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtitre plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtitre plate, artefacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples, with an interassay variation of less than 5%. 

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