Inhibition of catalase by 3,3''-diaminobenzidine.

3,3'-Diaminobenzidine strongly inhibits bovine liver catalase in two distinct ways. One of these was competitive with respect to H2O2, approached a limit of 100% inhibition and was rapidly reversed by dilution or by dialysis. The other was dependent upon H2O2, approached a limit of 60% and was not reversed by dilution or dialysis. Exposure to diaminobenzidine followed by dialysis did not modify the electrophoretic mobility of the enzyme, its Km for H2O2 or its optical spectrum, although Vmax. was halved. Diaminobenzidine protects catalase against the irreversible inactivation imposed by 3-amino-1,2,4-triazole plus H2O2. CN-protected catalase against the apparently irreversible inhibition by diaminobenzidine, and dialysis against CN- reversed this inhibition. Ethanol was similarly protective, and ethanol plus H2O2 reversed the inhibition by diaminobenzidine. Several other aromatic diamines inhibited in a fashion similar to that of diaminobenzidine, but were less effective. A scheme of reactions that accounts for these observations is proposed.     

Spectral studies of human erythrocyte catalase.

The optical absorption and circular dichroic spectra of human erythrocyte catalase (EC 1.11.1.6) and its cyanide, azide, and fluoride derivatives over the wavelength range of 210 to 700 nm are reported. Treatment with acid or alkaline solutions causes spectral changes which may be due to dissociation of the enzyme into subunits and removal of the heme group from the protein. The fractions of the protein structure present as alpha helix, beta pleated sheet, and unordered structure have been estimated from the CD spectrum in the far-ultraviolet region. The CD spectra also indicate that the protein conformation does not change appreciably after cyanide binding. The epr spectroscopy of the native enzyme and its cyanide complex are reported. The spectral results are compared with catalase obtained from other mammalian and bacterial sources.     

Biosynthesis of liver catalase in rats treated with allylisopropylacetylcarbamide. I. Immunochemical assay of catalase in liver cell fractions.

Rats were injected twice intraperitoneally with 20 mg of allylisopropylacetylcarbamide (Sedormid) per 100 g of body weight at an interval of 12 hr. The level of catalase [EC 1.11.1.6] in various liver cell fractions was determined both enzymatically and immunochemically 12 hr after the second injection. 1. The decrease in catalase protein assayed by the immunochemical method directly confirmed the inhibition of biosynthesis of the enzyme by this porphyrinogenic drug. 2. The occurrence of a considerable amount of catalase protein with no enzymatic activity was demonstrated both in the peroxisomes and in the supernatant fraction. 3. The amount of catalase-synthesizing polysomes in hepatic cell was reduced in Sedormid-treated rats by the extent comparable to the decrease in the concentration of liver catalase.     

The chemical modification of beef liver catalase. V. Ethoxyformylation of histidine and tyrosine residues of catalase with diethylpyrocarbonate.

In order to elucidate the possible roles of histidine and tyrosine residues of catalase [EC 1.11.1.6] in maintaining the quaternary structure and catalatic activity, diethylpyrocarbonate modification experiments were carried out. A method for the estimation of N-ethoxyformyl (EF)-His at pH 5--7 and of O-ethoxyformyl (EF)-Tyr in alkaline solution by measuring A 242 nm (ximM = 3.2) and A278 nm (ximM = 1.16), respectively, was developed. The formation of EF-His and EF-Tyr was an electrophilic reaction and was dependent on pH, exhibiting pK values of 6.8 and 9.9, respectively. The maximal yield of EF-His at pH 6.0 was 49% of the total histidine content, but no inactivation nor unfolding of the enzyme was observed. The formation of 12 EF-Tyr residues per mole of catalase at pH 8.1 did not cause any inactivation, but the formation of 8 more EF-Tyr residues at pH 8.9 resulted in both inactivation and unfolding. Nearly complete inactivation and partial splitting of catalase were observed when 43-46 EF-Tyr residues per mole were produced at pH 10.0. More EF-His residues were formed by the reaction of diethyl pyrocarbonate with cyanoethylated (CE)-catalase monomer (subunit) than with CE-catalase tetramer. The CE-catalase tetramer and monomer were extensively O-ethoxyformylated, reaching 100% EF-Tyr formation. These results indicate that a half of the histidine residues may lie outside the protein core and that three-quarters of the tyrosine residues are probably in the protein core of the enzyme. The production of 2--3 EF-Tyr residues per mole of the monomer by ethoxyformylation at pH 7.0 was accompanied by a decrease in the magnitude of the Soret peak. A possible interaction of those tyrosine residues with porphyrin of the heme group is discussed.     

The catalase activity of Nalpha-acetyl-microperoxidase-8.

Nalpha-Acetylated microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for peroxidases. We report here that Ac-MP-8 catalyzes catalase-type reaction in addition to peroxidase-type and cytochrome P450-type reactions. The catalase activity of Ac-MP-8 was determined by the Clark oxygen electrode, which measures the production of oxygen in solution. The Km and kcat of the decomposition of hydrogen peroxide (H2O2) catalyzed by Ac-MP-8 are 40.9 mm and 4.1 per s, respectively. The specificity constant (kcat/Km) of Ac-MP-8 in catalase-type reaction of H2O2 is 100.2,/m/s, which is 5- to 12- and 50- to 100-fold less than those of MPs in cytochrome P450-type reaction of aniline/H2O2 and peroxidase-type reaction of o-methoxyphenol/H2O2, respectively. These results indicate that Ac-MP-8 can catalyze three different types of reactions, and the relative catalytic specificities of Ac-MP-8 with a histidyl ligand exhibit the following orders: peroxidase-type > cytochrome P450-type > catalase-type reactions. Comparisons of the enzyme activities of Ac-MP-8 suggest that the fifth ligands of hemoproteins influence the ratio of the three types of reactions.     

Isolation from Micrococcus sp. n. of a homogeneous heme-containing catalase and a crystalline protein with catalase activity

A method for isolation and purification of catalases from the culture of Micrococcus sp. n. grown under aeration conditions is described. Heme-containing catalase (I) and the protein possessing a catalase activity (II) were separated by fractionation with ammonium sulfate. The specific activity of the highly purified protein causing degradation of H2O2 is 200 times less than that of the heme-containing enzyme. The molecular weights of catalases I and II as determined by electrophoresis in polyacrylamide gel gradient 4/30% are 240000 and 130000, respectively. The method described is designed at rapid isolation of preparative amounts of catalases from Micrococcus sp. n.     

Resuscitation effects of catalase on airborne bacteria.

Catalase incorporation into enumeration media caused a significant increase (greater than 63%) in the colony-forming abilities of airborne bacteria. Incubation for 30 to 60 min of airborne bacteria in collection fluid containing catalase caused a greater than 95% increase in colony-forming ability. However, catalase did not have any effects on enumeration at high relative humidities (80 to 90%).     

Regulation of catalase synthesis in Salmonella typhimurium.

The specific activity of catalase in Salmonella typhimurium and other enteric bacteria decreased during the logarithmic phase of growth and increased at the onset and during the stationary phase. The increase in catalase synthesis at the end of the exponential phase in S. typhimurium cells coincided with the lowest pH value reached by the culture. Maintenance of the pH at a constant neutral value did not alter the typical pattern of synthesis in contradiction of the results previously reported (McCarthy and Hinshelwood. 1959). A sudden decrease in the pH value of an S. typhimurium culture during exponential growth by addition of HC1 did not cause an alteration in the catalase synthesis pattern. Addition of hydrogen peroxide to S. typhimurium cultures within the range 1 muM TO 2MM during the exponential growth phase stimulated catalase synthesis. The extent of catalase synthesis depended on the concentration of hydrogen peroxide; the maximum stimulation was observed at 80 muM. Increased catalase synthesis was not detected for 10 to 15 min after hydrogen peroxide addition. Hydrogen peroxide was produced by S. typhimurium cultures during the exponential and stationary growth phases. However, no direct relationship between hydrogen peroxide accumulation and synthesis of catalase was observed.