Catalase hydrogen peroxide determination

By application of EMMA Regehr and Regnier developed several assays for enzymes that produce (galactose oxidase and glucose oxidase) or consume (catalase) hydrogen peroxide. Unlabeled enzymes were determined in the femto-mole mass range, while detection limits of less than 10,000 molecules were reported for catalase [101]. 

Campanella L, Roversi R, Sammartino MP et al (1998) Hydrogen peroxide determination in pharmaceutical formulations and cosmetics using a new catalase biostaisOT. Pharm Biomed Anal 18 105-116... 

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

Catalase was immobilized with gelatin by means of glutaraldehyde and fixed on a pretreated Teflon membrane served as enzyme electrode to determine hydrogen peroxide [248], The electrode response reached a maximum when 50mM phosphate buffer was used at pH 7.0 and at 35°C. Catalase enzyme electrode response depends linearly on hydrogen peroxide concentration between 1.0 X 10-5 and 3.0 X 10-3 M with response time 30 s. 

S. Akgol and E. Dinckaya, A novel biosensor for specific determination of hydrogen peroxide catalase enzyme electrode based on dissolved oxygen probe. Talanta 48, 363-367 (1999). 

Concerning the mode of formation of ES, we prefer the concept that the substrate in a monolayer is chemisorbed to the active center of the enzyme protein, just as the experimental evidence pertaining to surface catalysis by inorganic catalysts indicates that in these reactions chemisorbed, not physically adsorbed, reactants are involved. Such a concept is supported by the demonstration of spectroscopically defined unstable intermediate compounds between enzyme and substrate in the decomposition by catalase of ethyl hydroperoxide,11 and in the interaction between peroxidase and hydrogen peroxide.18 Recently Chance18 determined by direct photoelectric measurements the dissociation con-... 

FIGURE 4.19 Amino acid enantiomers are determined by reaction (A) with l- or D-amino-acid oxidase at pH 7-8.75 Added catalase decomposes the hydrogen peroxide (B), which would otherwise oxidize the a-oxoacid. Quantitation is achieved by measuring oxygen consumption, which is 0.5 mol/mol of substrate. 

In Investigation 6-B, you will write a detailed procedure to determine the rate law for the catalyzed decomposition of hydrogen peroxide. Instead of using catalase to catalyze the reaction, you will use an inorganic catalyst. 

Figure SJ Activity of the various states of the [NiFe] hydrogenase from A. vinosum as determined with a Pt electrode at 30°C.The reaction was performed in SOmM Tris/HCI (pH 8.0) in a volume of 2 ml. Oxygen was scavenged by adding glucose (90 mM) and glucose oxidase (2.5 mg/ml). Hydrogen peroxide was removed by catalase. When the system was anaerobic, an aliquot of H2-saturated water was added, and a little later enzyme (S-IOnM) was injected. Benzyl viologen (4.2mM) was used as electron acceptor.

Influence of catalase activity and electrical conductivity. Parallel to the determination of the oxygen activity. Beck and co-workers 38> investigated the activity for the decomposition of hydrogen peroxide by various chelates. In each case, 1 mg of freshly precipitated pigment was added to 1% hydrogen peroxide solution and the velocity of oxygen evolution was determined volumetri-cally. The results summarized in Table 5 show no correlation between electro-catalytic activity and catalase activity. 

New europium probes have been presented that can indicate changes in hydrogen peroxide concentration and are applicable for time-resolved imaging. As H2O2 is a product of the activity of almost all oxidases, quantitative assays for different substrates, e.g glucose, or the determination of the activity of the respective oxidases can be carried out. On the other hand, peroxidases and catalases consume H2O2, which can also be monitored via the europium tetracycline probe and can therefore be used for the imaging of ELIS As performed in microwell plates. 

Let us now consider the questions of the biological oxidation of various substrates with hydrogen peroxide in the presence of catalases and peroxidases. As Pratt notes [82], this group of enzymes is unique in that it is the only one in which intermediates are detected, and all stages of catalytic process are determined, identified and studied. Of special value is the characteristic that catalase consists of four subunits, whereas peroxidase possesses only one subunit. Using special technique, it is also shown that every iron atom (heme) binds one H202 molecule [83 ]. 

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. 

For the purpose of determining low hydrogen peroxide concentrations, the authors have designed the most cost-effective and simple to use potentiometric-biomimetic sensors based on immobilized catalase mimics. These sensors possess high hydrodynamic properties and the fastest speed of response. Figure 8.3 shows experimental data on catalase activity of biomimetic electrode in 0.03% aqueous H202. For the sake of comparison, catalase activities of aluminum electrode and aluminum electrode with applied adhesive are also shown. 

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. 

In studies of catalase, much effort has been directed toward a determination of whether or not hydrogen peroxide could be dissociated from the enzyme-substrate intermediates of catalases and peroxidases. It should be pointed out that catalase, as contrasted with cytochrome oxidase, has been studied only at room temperature, and if any lesson is to be learned from the study of cytochrome oxidase 150), it is that the complexes are most likely to be identified at low temperatures, as precursors of the compounds. In this sense, they are of first importance and not to be ignored in our understanding of the mechanism of enzymic reactions. 

The sensitivity of glucose determination by means of hydrogen peroxide detection may be decreased by the catalase that contaminates several GOD preparations. If no catalase-free GOD is available, this problem can be largely overcome by adding catalase inhibitors such as sodium azide or aminotriazole to the background solution. 

The activity of an enzyme is affected by environmental factors such as pH and temperature. Every enzyme has optimum conditions at which its reaction rate is fastest. In this ChemLab, you will study the decomposition of hydrogen peroxide as catalyzed by the catalase in carrot cells, and you will determine the optimum temperatures under which this enzyme works. 

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