Catalase , activity units

Table 3.13. Effects of aeration on catalase activity (units/mg protein) in propionic acid bacteria...

Application and Principle This procedure is used to determine the catalase activity, expressed as Baker Units, of preparations derived from Aspergillus niger var., bovine liver, or Micrococcus lysodeikticus. The assay is an exhaustion method based on the breakdown of hydrogen peroxide by catalase and the simultaneous breakdown of the catalase by the peroxide under controlled conditions. 

For blood heparinized venous blood is centrifuged and the upper layer is removed. Wash the erythrocyte sediment three times with 0.9% (w/v) NaCl solution. Haemolyse the washed red cells by adding 4 parts (v/v) of distilled water per volume of packed cells to give a stock haemolysate solution (approx. 5% (w/v)). For assay, dilute the stock solution 1 500 with the phosphate buffer immediately before the assay is to be carried out and determine the haemoglobin content of the solution by the method of Drabkin. The catalase activity is expressed per unit of haemoglobin. 

From the linear part of the curve calculate catalase activity as units per milligram of protein (1 U decomposes 1 pM of H202/min). 

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... 

Catalase (CAT) [EC 1.11.1.6] was measured by determining the reduction of UV absorbancy in the presence of H2O2 at 230 nm using, UV spectrophotometer (Chance, 1954 Aebi, 1984). The specific activity unit was defined as the enzyme equivalent reducing 1 mM H202/min per mg protein. 

All tubes contained the following components (in /nmoles unless stated otherwise) potassium phosphate, pH 6.5, 100 ascorbate, 6.0 fumarate, 50 tyramine-/3/3 -3H, 2.0, specific activity 1.15 X 10 CPM//nmole catalase, 300 units, dopamine /3-hydroxylase (4), Final volume, 0.68 ml. at 25°C. Octopamine determined by a minor modification of a published procedure (17). A 0.05 ml. sample of water was obtained by lyophilization and dissolved in 10 ml. of Bray s scintillation mixture. Radioactivity determined in a Packard liquid scintillation spectrometer total counts collected were sufficient to yield a 5% coefficient of variation. The expected tritium release was calculated from the octopamine formed, assuming that the amount of tritium was the same in both /3 positions of the tyramine. The results for the amount of tritium released have been corrected for the amount of exchangeable tritium initially present in the tyramine. 

CAT - catalase activity in units/min/gm foliage 2 POD - peroxidase activity in OD470/min/gm foliage RGR - relative larval growth rate (mg/day/mg larva). 

We also checked the effect of catalase on Me since a recent report of Baker et al, (1976) has shown that this enzyme enhances the stearyl-CoA desaturation. For this reason the catalase activity of Sp, Cytosol and other fractions tested in the reactivation of Me were measured. Sp and the cytosol have catalase activity but the last one was relatively more active than the first one. Fig. 3 shows the comparative reactivation capacity of Sp, cytosol, DEAE Cellulose fraction and pure catalase on A6 desaturation and the content of units of catalase of each fraction. Results demonstrate that there is no correlation between the Sp reactivation capacity of a6 desaturation reaction and their catalase activity content. 

The protein factor contains catalase activity and pure catalase reactivates the desaturation. However there is no correlation between the protein factor reactivation capacity on a6 desaturation and catalase activity content. Pure catalase has less reactivation capacity than Sp although it contains many more units of catalase activity. 

That these mechanisms are valid has been concluavely shown by Chance (1-4), who studied the kinetics intensively by means of rapid optical methods that allow him to measure the rates of the separate reaction steps. He could show (4,101) that k[, the reaction constant for over-all catalase activity, is related to ki and k i of equations (4) and (5). But ki and 4 cannot be determined individually from the over-all reaction kinetics unless the ratio of the steady state concentrations of the enzyme-substrate compound to the free enzyme is known. In the case of peroxidatic activities where the substrate and donor are different molecules, their relative concentrations determine whether h or h is measured by the over-all activity usually an ill-de6ned mixture of the two reaction velocity constants is measured. The conditions appropriate for the evaluation of ki and kt from the over-all activity are discussed in a recent paper (4) by Chance, and the calculation of ki from the kinetics of peroxidase reactions and the standard peroxidase activity unit PZ (see p. 389) has also been carried out. Any sound procedure for activity determination should depend to as great an extent as possible upon the measurement of only one reaction velocity constant of the enzymic mechanism. 

Senter (313) studied the kinetics of catalase action by plotting the data obtained at 5, 10, 20, and 30 minutes of reaction time. In 1924 Hennichs (175) based the Kat.f. unit (see p. 362) on measurements at 5, 10, 15, 20, and 25 minutes. Von Euler and Josephson (140) changed this to 3, 6, 9, and 12 minutes. Their test was to become the standard assay for catalase activity. The choice of such long reaction times was quite unfortunate, because the rate of the catalytic reaction decreases considerably even before the first measurement is made (71). Extrapolation of the data to zero time, suggested by Sumner (338), solved the problem only to a small extent. By plotting experimental data obtained in the first minute of the reaction, Bonnichsen et al. (71) illustrated the drawbacks of extrapolating the results obtained from long reaction times. 

Critically analyzing the mechanism (6.8)-(6.12), one may note the unsuitability of the currently presented interaction between complexes E-Fe3+—OH and E-Fe3+ OOH and substrates (H202 and H2D), because it is unclear how the substrate is activated. Moreover, intensification of the catalase reaction induces a non-classical peroxidase activity increase in ethanol and formic acid oxidation reactions. This indicates the existence of a unit common to these two processes [82, 83], The alternative action of catalase (catalase of peroxidase reaction) in the biosystem with solidarity of elementary stage mechanisms should be noted [88, 89], Peroxidase action of catalase requires a continuous supply of H202 for ethanol and formic acid oxidation, which can be explained by oxidation according to conjugated mechanism [90],... 

The change of electrode potential (E) of the catalase reaction with time was measured by a voltmeter. pH and E values for aqueous hydrogen peroxide were determined simultaneously for possible correlations between pH metric and potentiometric results of enzymatic activity of catalase-biomimetic sensors. The electrochemical unit was also equipped with a magnetic mixer. 

FIGURE 35.1. Representative profiles of the activity of the AChE molecular forms in soleus, EDL, and hemidiaphragm muscles. Profiles at the top of each column are from untreated muscles followed by profiles of activity of AChE molecular forms of muscles 24 h and 7 days, respectively, after receiving an acute dose of soman (100 pg/kg, s.c.). The AChE activity scale is in arbitrary units based on the pmole substrate hydrolyzed/min by the enzyme activity in each fraction. The sedimentation values of the AChE molecular forms are given in the profiles of untreated muscles above the associated peaks. Sedimentation values were determined by the location of the added sedimentation standards, P-galactosidase (16.0 S), catalase (11.1 S), and alkaline phosphatase (6.1 S), following velocity sedimentation of the gradients. 

Isoniazid interferes with mycolic acid synthesis by inhibiting an enoyl reductase (InhA) which forms part of the fatty acid synthase system in mycobacteria. Mycolic acids are produced by a diversion of the normal fatty acid synthetic pathway in which short-chain (16 carbon) and long-chain (24 carbon) fatty acids are produced by addition of 7 or 11 malonate extension units from malonyl coenzyme A to acetyl coenzyme A. InhA inserts a double bond into the extending fatty acid chain at the 24 carbon stage. The long-chain fatty acids are further extended and condensed to produce the 60-90 carbon (3-hydroxymycolic acids which are important components of the mycobacterial cell wall. Isoniazid is converted inside the mycobacteria to a free radical species by a catalase peroxidase enzyme, KatG. The active free radicals then attack and inhibit the enoyl reductase, InhA, by covalent attachment to the active site. 

In addition to Km and ymax, the turnover number (molar activity) and the specific activity are important parameters for the characterization of enzyme reactions. Both are determined under substrate saturation. With highly purified enzymes the turnover number reflects the number of substrate molecules converted in unit time by a single enzyme molecule (or a single active center). Catalase, one of the most potent enzymes, has a turnover number of 2-105/s. 

The dinuclear units are closely related to the dinuclear Mn units believed to be present at the active site of manganese (pseudo)catalases. 7 This view is supported by the previously reported biomimetic (catalytic) activity of 1 (see also below). 

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