Catalase , activity units determination

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. 

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. 

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. 

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. 

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 principal advantages of the Clark oxygen electrode are that proteins and other materials which can foul solid eletrode do not contact the platinum and reference dectrodes. The entire sensor assembly can be immersed in the test solution as a single unit without an external reference electrode. Hence the oxygen electrode is well suited to construct a flow-through enzyme immunosensor with such a porous antibody-bound membrane using GOD or catalase as a label. Since the enzyme immunosensor has an excellent response time, the enzyme activity on the sandwich structure membrane can be determined with flow injection mode, which makes the detecting procedure simple and convenient. 

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