Catalase complexes

Further reduction of the catalase complex VI is shown in Figure 6.5 (peroxidase reaction) and Figure 6.6 (catalase reaction). These diagrams show that peroxidase and catalase reactions of catalase proceed by two-electron transfer mechanism in one stage and are practically equal. 

Any excess of free streptavidin is removed by gel-filtration using a Sephacryl S-200 HR column (60 mL). Collect the void volume peak (approx 22-25 mL fractions). This peak contains the SA/b-catalase complex. 

The purpose of the second step of the conjugation procedure is to conjugate the bimolecular complex 125I-labeled SA/b-catalase to b-Ab or control b-IgG utilizing residual biotin-binding sites of SA/b-catalase complex. 

Unexplained Effects. Some observations are difficult to attribute to any of the freezing mechanisms considered so far. Most amenable to conceptual accommodation are those cases where a particular reactive intermediate, or a particular conformer, appears to have been trapped. This is believed to occur at very low temperatures where mobility at the atomic level is severely limited partly because of little thermal motion and partly because of the physical constraint provided by the glassy or icy structures of the medium. Normally unstable free radical derivatives of proteins can be studied by trapping them at low temperatures (e.g. 79). This technique also can be used to stabilize what appear to be specific conformational isomers of normal structures. For example, this approach has enabled the detection of an ammonia-catalase complex (80), conformers of the iron proteins conalbumin and transferrin (81), and a conformer of the flavoprotein L-amino acid oxidase (82, 83). 

The inhibition of catalase by cyanide shows none of the characteristics of the azide or hydroxylamine inhibition as is to be expected if cyanide combines with the ferric form. At low peroxide concentrations about 10-3 M the equilibrium constant for the formation of the cyan-catalase complex (Ki) determined from kinetic data using the expression... 

Catalase was found to form an intermediate compound in the presence of hydrogen peroxide (Chance, 69). The spectrum was measured from 380-430 nqi and is slightly shifted toward the visible as compared with free catalase. The complex shows no similarities to cyan-catalase or the compound formed when peroxide is added to azide catalase. Its formation is very rapid, the bimolecular velocity constant having a value of about 3 X 107 M.-1 sec.-1. In the absence of added hydrogen donors, the complex decomposes slowly according to a first order reaction with a velocity constant of about 0.02 sec.-1. This catalase complex thus resembles the green primary hydrogen peroxide complex of peroxidase. 

About 300 individual compounds with antimutagenic properties are known. The frequency of mutations can be reduced by some amino acids (arginine, histidine, methionine, cysteamine etc ), vitamins and provitamins (a-tocopherol, ascorbic acid, retinol, p-carotene, phylloquinone, folic acid), enzymes (peroxidase, NADPH oxidase, glutathione peroxidase, catalase), complex compounds of plant and animal origin, chemical substances with antioxidant properties (derivatives of gallic acid, ionol, oxypyridines, selenium salts and others). 

This technique has been developed by Aizawa and coworkers. The goal is to build a convenient and specific detector using an enzymatic activity as signal. In the case of biotin determination, avidin coupled with catalase is bound to a membrane bearing covalently linked HABA residues. Addition of biotin destroys quantitatively the HABA-avidin-catalase complex. Washing the membrane and measurement of the remaining catalase activity afforded a sensitive (0.5 ng) and convenient titration of biotin (95). 

Three enzyme-substrate complexes with HjO are known for protohemin-containii peroxidases and catalases complex I, green complex II,... 

Even under these conditions, the value of k should increase linearly with the donor concentration and no maximum value of k exists as it does for the simpler mechanism of equations (3) and (4). Experimentally, maximal values of k are often quoted in various papers, but they may be attributed to insufficient substrate concentration (the inequality A)Xo kiOo is violated), or to enzyme inactivation due to the excess peroxide concentration. For example, catalase inactivation can be caused by the formation of the inactive catalase complex II. In these cases it is desirable to use a lower value of substrate concentration and a smaller enzyme turnover number in order to avoid the inactivation. 

The electrode has not been used as much as the ultraviolet spectro-photometric method for routine catalase assays, and does not cover a sufficiently wide range of peroxide concentration to be suitable for studying the activity-substrate concentration relationship. The technique is suitable for simultaneous measurements of catalase activity and the catalase complex as described below. 

Table V summarizes some of the classic measurements of magnetic susceptibility on heme compounds. The compounds fall into two classes high spin (H 0) and low spin (CN, Os), in agreement with the measurements on simpler complexes mentioned above in the discussion on ligand field theory. The hydroxide complex of heme 195) with hemoglobin has been shown to exist in at least two forms. The catalase complexes are peculiar in that an intermediate magnetic moment is foimd even for the cyanide complex. The ease of oxidation of the heme in this compound may account for part of this aberancy. Another possibility is the assumption of a peculiar (or absent) fifth ligand.

Fig. 10. The formation of hydrogen peroxide-catalase complex II in the hydroxylating mixture of Udenfriend. Catalase 5.4 x 10 mM 20 mM ascorbate 0.33 mM FeSO 6.66 mM versene 1.66 mM phosphate buffer pH 6.8. The second spectrum was recorded after 15 minutes of oxygenation. Spectrophotometer Beckman DK 1. From Zito and Kertesz (1962).

Fig. 12. Formation of the Soret band of the hydrogen peroxide-catalase complex II during the oxidation of ascorbic acid by the phenolase plus o-dihydroxyphenol system (compare with Fig. 4 of Chance, 1950) and its disappearance after the addition of a monohydroxyphenol. (1) Catalase 0.9 x 10 mAf 3.1 X 10 mM phenolase 5 x 10 mM catechol 20 vaM ascorbate 1.66 mM phosphate buffer pH 6.8 total volume 3 ml. (2) the same after 15 minutes of oxygenation and (3) immediately after the addition of 1 /tmole of phenol in 0.005 ml. The lower curves are the spectra of the controls without catalase. Spectrophotometer Beckman DK 1. From Zito and Kertesz (1961).

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