Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 degrees C and a pH range from 6 to 10, with optimum activity occurring at 90 degrees C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 degrees C and 3 h at 90 degrees C. The enzyme also demonstrated excellent stability at 70 degrees C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A K(m) of 35.5 mM and a V(max) of 20.3 mM/min.mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.     

Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6

Among the 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as strain QW6 showed high capacity in the removal of toxic oxyanions of tellurium in a wide range of culture medium factors including pH (5.5-10.5), temperature (25-45 degrees C), various salts including NaCl, KCl, and Na(2)SO(4) (0.5-4M), selenooxyanions (2-10mM), and at different concentrations of potassium tellurite (0.5-1mM) under aerobic condition. Phenotypic characterization and phylogenetic analyses based on 16S rDNA sequence comparisons indicated that this strain was a member of the genus Salinicoccus. The maximum tellurite removal was exhibited in 1.5M NaCl at 35 degrees C, while the activity reduced by 53% and 47% at 25 and 45 degrees C, respectively. The optimum pH for removal activity was shown to be 7.5, with 90% and 83% reduced removal capacities at the two extreme values of 5.5 and 10, respectively. The impact of different concentrations of selenooxyanions (2-10mM) on tellurite removal by strain QW6 was evaluated. The ability of strain QW6 in the removal of tellurite in the presence of 6mM selenite increased by 25%. The concentration of toxic potassium tellurite in the supernatant of the bacterial culture medium decreased by 99% (from 0.5 to 0.005mM) after 6 days and the color of the medium changed to black due to the formation of less toxic elemental tellurium.     

Phenol removal from aqueous solutions by peroxidase extracted from horseradish

Horseradish peroxidase (HRP) is one of the most recently used enzymes in the process of enzymatic phenol removal. It has a catalytic ability over a broad range of pH, temperature and contaminant concentrations. In this study we revealed the possibility of successful use the crude peroxidase obtained from horseradish roots for the phenol removal from aqueous solutions in the presence of the low molecular polyethylene glycol (PEG 300) at room temperature (20°C) and pH 7.2. Reaction was monitored by direct measuring of the absorbance changes in a samples taken at certain time intervals from the reaction mixture. At the first time PEG 300 was shown to be a more stabilizing effect on crude HRP and provided a higher phenol removal in comparison with PEG 3350. Crude HRP used in these study demonstrated a greater resistance on phenol and hydrogen peroxide inactivation that allowed a higher phenol removal. The highest phenol removal was achieved when the concentration of PEG 300, phenol and hydrogen peroxide were 300 mg/L, 2.0 and 2.5 mM, respectively.     

Spring cabbage peroxidases--potential tool in biocatalysis and bioelectrocatalysis

Two fractions of peroxidase activity, cationic Px-cat and anionic Px-ani, were isolated and partially purified (143.5- and 5.49-fold, respectively) from homogenate of spring cabbage heads. Optimum pH for both fractions is 6.0; however, Px-cat is almost equally active at neutral pH (7.0) while Px-ani reveals high activity in more acidic pHs (with 60% of maximum activity at pH 3.0). Optimal temperature for both fractions was 40 degrees C. Px-ani possessed much higher thermal stability at 40-50 degrees C (60% of remaining activity after 144h of incubation) than Px-cat. The peroxidases remained fully active during 4 weeks of storage at 4 degrees C. Kinetic studies revealed that Px-cat and Px-ani had lower apparent Km values for ABTS (0.0377 and 0.0625mM) and o-dianisidine (0.357 and 0.286mM) than for guaiacol (6.41 and 13.89mM). The best substrate for Px-cat was pyrogallol and for Px-ani-o-dianisidine. Px-cat immobilized on polyanionic PyBA-modified carbon electrode was found to produce linear repetitive signals upon consecutive additions of hydrogen peroxide during at least 1-week period and to work effectively under buffered and non-buffered conditions. These properties were comparable with those of commercially available horseradish peroxidase. Stability of the hybrid bioelectrocatalytic film and low costs of extraction and partial purification of Px-cat make it a highly promising enzyme for practical applications, including construction of bioelectrodes.     

以纸为碳源去除地下水硝酸盐的研究

研究了以纸为碳源和反应介质的生物反应器对水中硝酸盐的去除。结果表明,以纸为碳源的反应器启动快．反硝化反应受温度及水力停留时间影响大。25℃的反硝化速率是14℃的1．7倍。在室温25±1℃,进水硝酸盐氮浓度为45．2mg·L^-1、水力停留时间8．6h时,反应器对硝酸盐氮的去除率在99．6％以上,当水力停留时间为7．2h,氮去除率只有50％。反硝化反应受pH值和溶解氧的影响小,反应进行过程中,纸表面形成了生物膜,纸也被消耗了．采用反应器出水再经活性炭吸附的工艺流程处理高硝酸盐氮地下水,<33．9mg·L^-1的硝酸盐氮完全去除,没有出现NC2-N,最终出水水质DOC<11mg·L^-1。     

The use of extracellular enzymes from Streptomyces albus ATCC 3005 for the bleaching of eucalyptus kraft pulp

The suitability of culture supernatant from Streptomyces albus ATCC 3005 for use in the biobleaching of eucalyptus kraft pulp was investigated. S. albus was found to grow on a minimal salts medium containing oat spelts xylan and yeast extract as the main carbon and nitrogen sources, respectively. Maximal extracellular xylanase and peroxidase production was detected after 120 h (11.97 U ml(-1)) and 72 h (0.58 U ml(-1)), respectively. Importantly, no cellulase activity could be detected. When the effect of pH on enzyme activity was examined, maximal xylanase and peroxidase activity was obtained at pH 6.5 and pH 9.9, respectively. The optimum hydrogen peroxide (H2O2) concentration for peroxidase activity was found to occur at 20 mM, with peroxidase remaining active at 100 mM H2O2 after 1 h incubation at 53 degrees C; the half-life of the enzyme at that temperature was estimated to be 33 min. Short-term (1 h) biobleaching of eucalyptus kraft pulp with culture supernatant from S. albus in the presence of H2O2 resulted in a significant reduction of kappa number (2.85 units) with no change in viscosity. These results suggest a potential application of cellulase-free culture supernatants from S. albus in biobleaching.     

Polyethylene glycol improves phenol removal by immobilized turnip peroxidase

Purified peroxidase from turnip (Brassica napus L. var. esculenta D.C.) was immobilized by entrapment in spheres of calcium alginate and by covalent binding to Affi-Gel 10. Both immobilized Turnip peroxidase (TP) preparations were assayed for the detoxification of a synthetic phenolic solution and a real wastewater effluent from a local paints factory. The effectiveness of phenolic compounds (PC's) removal by oxidative polymerization was evaluated using batch and recycling processes, and in the presence and in the absence of polyethylene glycol (PEG). The presence of PEG enhances the operative TP stability. In addition, reaction times were reduced from 3h to 10min, and more effective phenol removals were achieved when PEG was added. TP was able to perform 15 reaction cycles with a real industrial effluent showing PC's removals >90% PC's during the first 10 reaction cycles. High PC's removal efficiencies (>95%) were obtained using both immobilized preparations at PC's concentrations <1.2mM. Higher PC's concentrations decreased the removal efficiency to 90% with both preparations after the first reaction cycle, probably due to substrate inhibition. On the other hand, immobilized TP showed increased thermal stability when compared with free TP. A large-scale enzymatic process for industrial effluent treatment is expected to be developed with immobilized TP that could be stable enough to make the process economically feasible.     

Effect of reaction conditions on phenol removal by polymerization and precipitation using Coprinus cinereus peroxidase

The quantitative relationships between removal efficiency of phenol and reaction conditions were investigated using Coprinus cinereus peroxidase. The most effective ratio of hydrogen peroxide to phenol was nearly 1/1 (mol/mol) at an adequate enzyme dose. 12.2 U of the enzyme was needed to remove 1 mg of phenol when our peroxidase preparation was used. At an insufficient peroxidase dose, the optimum pH value was 9.0, and lowering the reaction temperature led to the improvement of removal efficiency. At an excess peroxidase dose, almost 100% removal of phenol was obtained over a wide range of pH (5-9) and temperature (0-60 degrees C). Despite the presence of culture medium components, it was shown that Coprinus cinereus peroxidase had the same phenol polymerization performance as horseradish peroxidase or Arthromyces ramosus peroxidase.     

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Addition of veratryl alcohol oxidase activity to manganese peroxidase by site-directed mutagenesis

Manganese peroxidase and lignin peroxidase are ligninolytic heme-containing enzymes secreted by the white-rot fungus Phanerochaete chrysosporium. Despite structural similarity, these peroxidases oxidize different substrates. Veratryl alcohol is a typical substrate for lignin peroxidase, while manganese peroxidase oxidizes chelated Mn2+. By a single mutation, S168W, we have added veratryl alcohol oxidase activity to recombinant manganese peroxidase expressed in Escherichia coli. The kcat for veratryl alcohol oxidation was 11 s-1, Km for veratryl alcohol approximately 0.49 mM, and Km for hydrogen peroxide approximately 25 microM at pH 2.3. The Km for veratryl alcohol was higher and Km for hydrogen peroxide was lower for this manganese peroxidase mutant compared to two recombinant lignin peroxidase isoenzymes. The mutant retained full manganese peroxidase activity and the kcat was approximately 2.6 x 10(2) s-1 at pH 4.3. Consistent with relative activities with respect to these substrates, Mn2+ strongly inhibited veratryl alcohol oxidation. The single productive mutation in manganese peroxidase suggested that this surface tryptophan residue (W171) in lignin peroxidase is involved in catalysis.     

Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.     

Suicide-peroxide inactivation of horseradish peroxidase in the presence of sodium n-dodecyl sulphate: a study of the enzyme deactivation kinetics

In the presence of the anionic surfactant sodium n-dodecyl sulphate (SDS), horseradish peroxidase (HRP) undergoes a deactivation process. Suicide inactivation of horseradish peroxidase by hydrogen peroxide(3 mM) was monitored by the absorbance change in product formation in the catalytic reaction cycle. The progress curve of the catalytic reaction cycle was obtained at 27degrees C and phosphate buffer 2.5 mM (pH = 7.0). The corresponding kinetic parameters i.e., intact enzyme activity (alpha i); the apparent rate constant of suicide inactivation by peroxide (ki); and the apparent rate constants of enzyme deactivation by surfactant (kd) were evaluated from the obtained kinetic equations. The experimental data are accounted for by the equations used in this investigation. Addition of SDS to the reaction mixture intensified the inactivation process. The deactivation ability of denaturant could be resolved from the observed inactivation effect of the suicide substrate by applying the proposed model. The results indicate that the deactivation and the inactivation processes are independent of each other.     

A novel thermo-alkali stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis: purification and characterization

A novel thermo-alkali-stable catalase–peroxidase from Oceanobacillus oncorhynchi subsp. incaldaniensis subsp. nov., strain 20AG, was purified and characterized. The protein purified from the cells resulted in 110-fold purification with a specific activity of 35,000 U/mg. The enzyme consisted of four identical subunits of 72 kDa as determined by SDS-PAGE and the total molecular mass measured by gel filtration was 280 kDa. The heme content was determined to be 1 heme per homodimer. The enzyme showed a Soret peak at 406 nm in the oxidized form and was easily reduced by dithionite. The enzyme showed an appreciable peroxidase activity in addition to high catalase activity. The behaviour of this heme-enzyme was typical of the class of prokaryotic catalase–peroxidases, which are sensitive to cyanide and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme was active over a temperature range from 30 to 60°C and a pH range from 5 to 10, with an optimum pH about 9.0 and an optimum temperature of 40°C. The enzyme was stable in the pH range of 5.0 to 10.0 after 1 h of treatment at 40°C. The enzyme was stable for 24 h at 40°C with a half-life of 4 h 60°C. The enzyme had a K _m of 24 mM for hydrogen peroxide. The amino terminal amino acid sequence of the catalase–peroxidase from strain 20AG was SEKRKMTTAFGA and it showed no homology with other catalases.     

Induction of an anionic peroxidase in cowpea leaves by exogenous salicylic acid

Two isoperoxidases were detected in cowpea (Vigna unguiculata) leaves. Treatment of the primary leaves with 10mM salicylic acid increased the total peroxidase activity contributed by the anionic isoform. To isolate both the anionic and cationic peroxidases the leaf crude extract was loaded on a Superose 12 HR 10/30 column followed by chromatography on Mono-Q HR 5/5. Both enzymes were stable in a pH range from 5 to 7. The optimum-temperatures for the cationic and anionic peroxidase isoforms were, respectively, 20-30 degrees C and 30 degrees C. The dependence of guaiacol oxidation rate varying its concentration at constant H(2)O(2) concentration showed, for both enzymes, Michaelis-Menten-type kinetic. Apparent K(m)(s) were 0.8 and 4.8 microM for the cationic and anionic isoperoxidases, respectively.     

A microcolorimetric assay of inorganic pyrophosphatase

A procedure is described for the assay of inorganic pyrophosphatase in tissues by a microcolorimetric procedure, taking advantage of the marked color intensification of phosphomolybdate by malachite green. Conditions are described for optimum enzyme activity, color stability, and sensitivity. With 1-cm cuvettes the AM660 is 100,000, allowing accurate measurement of Pi in the 1-nmol range. Reaction is conducted at 25 degrees C for 10 min in 0.5 ml of a 50 mM histidine buffer, pH 7.2, containing 0.2 mM inorganic pyrophosphate and 4 mM Mg2+, terminated by addition of 0.05 ml 2.4 M HClO4, cooled in ice, and 0.45 ml of color reagent is added. After standing 10 min at 0 degrees C, the contents are transferred to 1-cm cuvettes and the absorbance is read at 660 nm. Blanks are low, nonenzymatic hydrolysis of PPi is negligible, and color is stable without addition of detergents. The high sensitivity makes this procedure well-adapted to measurement of optimal activities in crude tissue preparations.     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.     

High-valent intermediates in the reaction of N alpha-acetyl microperoxidase-8 with hydrogen peroxide: models for compounds 0, I and II of horseradish peroxidase.

N-acetyl microperoxidase-8 (Ac-MP-8) is a water soluble, ferric heme model for the peroxidases. The reaction of Ac-MP-8 with H2O2 in 10 mM potassium phosphate over the pH range of 7-11 gives rise sequentially to relatively stable green and red species with properties that closely mimic those of HRP compounds I and II, respectively. Low-temperature stopped-flow studies of this reaction carried out in 50% v/v methanol/10 mM potassium phosphate, pH* 9.1 at -25.8 degrees C indicate that the pseudo-first-order rate constant, kobs, that describes the formation of the green intermediate exhibits saturation kinetics as a function of [H2O2] with kmaxobs = 95 s-1 and KM = 87 mM. Rapid-scan studies carried out with [H2O2] = 200 mM at -38.0 degrees C show that a compound 0 species with a characteristic band near 340 nm is formed whose conversion to the green species is rate limiting. Thus, Ac-MP-8 has high-valent forms that are models for all three known intermediates in the peroxidase cycle of horseradish peroxidase.