Ouchterlony Double Diffusion Test Research Articles

Identification of the species of origin of fresh, cooked and canned meat and meat products using antisera to thermostable muscle antigens by ouchterlony's double diffusion test

AbstractAntisera to thermostable muscle antigens (TMA) from 14 species of bovidae were raised in goats and/or sheep. To achieve species specificity the antisera were absorbed with serum from the other species. While the absorbed antisera to TMA of buffalo, impala, eland, waterbuck, wildebeest and oryx were rendered specific, the antiserum to cattle TMA cross‐reacted with buffalo fresh meat antigens (FMA) and cooked meat antigens (CMA) but not with buffalo thermostable muscle antigens. Fresh and cooked muscle antigens from these two species could be differentiated by the antiserum to buffalo TMA. A similar approach was used to differentiate the FMA, CMA, and TMA of kongoni, topi and wildebeest. Antiserum to cattle TMA proved useful in detecting the presence of beef meat in meat products that had undergone commercial sterilisation.

Ultrastructural and biochemical studies of two dynamically expressed cell surface determinants on Candida albicans.

Variability in the expression of two different cell surface carbohydrate determinants was examined with two agglutinating immunoglobulin M monoclonal antibodies (H9 and C6) and immunoelectron microscopy during growth of three strains of Candida albicans. A single strain of Candida parapsilosis did not express either antigen at any time during growth. Antigens were detected on the surface of C. albicans by agglutination tests with either H9 or C6 over a 48-h growth period. The difference in specificities of the monoclonal antibodies was demonstrated by Ouchterlony double-diffusion tests with solubilized antigens and by variabilities in the reactivity of the agglutinins among yeast strains. The antigenic determinants were isolated by specific immunoprecipitation and protease digestion and characterized by methods including high-pressure liquid chromatography, gas-liquid chromatography, and mass spectroscopy with both chemical and electron ionization. These determinants both contain mannose and glucose. In the case of antigen H9, an additional carbohydrate was detected with gas chromatography and mass spectroscopy. The location of antigens on individual cells was determined by indirect labeling of the determinants, first reacting cells with H9 or C6 followed by goat anti-mouse antibody conjugated with 20-nm colloidal gold particles. Transmission electron microscopy was used to examine cells. The antigens that were reactive with the monoclonal antibodies were associated with a flocculent surface layer. Expression of this layer and expression of the antigens is a dynamic process which is growth phase and strain dependent. The antigens were not expressed on very young cells and disappeared from the cell surface of most C. albicans strains with age. The use of monoclonal antibody to cell surface determinants may allow characterization of cell surface antigens of C. albicans and be helpful in establishing receptors which mediate adherence.

Ornithodoros moubata: Host immunoglobulin G in tick hemolymph

Hemolymph proteins of a soft tick, Ornithodoros moubata, were analyzed immunochemically and biochemically. The components of tick hemolymph proteins were shown to be totally different from the host (rabbit) serum proteins by polyacrylamide gel electrophoresis with sodium dodecyl sulfate and Coomassie blue or silver stain. However, in the hemolymph of ticks engorged from rabbits immunoglobulin G was detected by immunoblotting analysis with goat anti-rabbit immunoglobulin G. The concentration of rabbit Immunoglobulin G in tick hemolymph changed with the physiological stages after a blood meal. Immunoglobulin G was isolated from tick hemolymph by affinity chromatography on a Protein A-Sepharose 4B column. Analysis of the isolated immunoglobulin G from tick hemolymph with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony double diffusion test showed it to be composed of the same subunits as heavy and light chains of host (rabbit) immunoglobulin G. Tracer experiments showed that 125I-labeled heavy and light chains of immunoglobulin G were detected in an intact form in hemolymph from ticks that sucked 125I-labeled rabbit immunoglobulin G through an artificial membrane. These facts suggested that the host rabbit immunoglobulin G ingested in the tick midgut passed through the gut wall without digestion. By solid-phase enzyme immunoassay, immunoglobulin in the hemolymph was shown to retain its antibody activity.

Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary.

A neutral endopeptidase (EC 3.4.24.5) that degrades alpha- and beta-crystallins occurs in mammalian lens. A procedure for purification of this enzyme from bovine lens is described. The enzyme appears to have a high molecular weight (Mr approximately equal to 700,000) and under denaturing conditions dissociates into at least eight polypeptide subunits with Mrs ranging from 24,000 to 32,000. A neutral proteinase in bovine pituitary has been reported previously to have similar structural characteristics. We have found that this enzyme purified from bovine pituitary is indistinguishable in molecular weight and in subunit composition from bovine lens endopeptidase. In addition, antiserum raised in rabbit against the purified lens enzyme crossreacts with bovine pituitary enzyme. When examined side by side in Ouchterlony double-diffusion tests, the two enzymes give a continuous precipitin line with no spurring. It is concluded that lens neutral endopeptidase and pituitary neutral proteinase are structurally closely similar, if not identical. This is a surprising result because it had been thought previously that the lens endopeptidase was unique to lens, where its crystallin substrates comprise a large proportion of the total tissue protein. In other tissues, crystallin is either absent or occurs, at most, in trace amounts.

Exposure to microorganisms, febrile and airway-obstructive symptoms, immune status and lung function of Swedish farmers.

A questionnaire was sent to 512 farmers, members of a local farmer's health association. Eighty were interviewed, and their serum precipitins and total serum immunoglobulin E (IgE) values were determined. Forty-five underwent extensive pulmonary function tests. On the basis of the clinical evaluation it was estimated that 19% of the farmers had experienced febrile reactions (fever and/or shivering) following exposure to organic dust, 50% of the 19% having been exposed within the last 2.5 years. Common causes were moldy grain, hay, and woodchips. The pulmonary function, gas exchange, and chest radiographs of those who had previously had febrile reactions were normal. Only 13% showed positive precipitin reactions in Ouchterlony double-diffusion tests. With more sensitive tests, positive precipitins were found in 59%, but they were negatively correlated with febrile episodes. Air samples collected during work with hay and grain on 21 farms contained between 10(7) to 2 X 10(9) microorganisms/m3. The highest values were associated with symptoms of alveolitis. Eleven percent of the farmers reported obstructive chest symptoms following exposure to organic dust. This group showed decreased pulmonary function and elevated serum IgE levels and included a high proportion of smokers and exsmokers. No correlation was found between febrile and obstructive reactions.

Lipid composition of Schistosoma mansoni and surface labeling of glycolipid components.

Schistosoma mansoni adult worms obtained from human patients and mice infected with cercariae were compared in lipid composition. Both worms possessed cholesterol ester, free fatty acid and cholesterol as neutral lipids, but the latter worms possessed triacylglyceride as another component. Phospholipid compositions of both worms were similar. Larger amounts of phosphatidyl choline diacylglyceride and smaller amounts of phosphatidyl ethanolamine diacylglyceride were detected. Adult worms from infected mice were surface-labeled by galactose oxidase treatment followed by sodium boro[3H]hydride reduction after or before trypsin treatment. Trypsin treatment was approximately 10 times more effective to label glycolipids than non-treatment. Mono-, di-, tri- and tetra-glycolyl components were labeled with trypsin treatment; however, mainly tri-glycolyl glycolipid was labeled without trypsin treatment. These results indicated that some glycolipid components are really exposed on the surface of the tegumental membrane. But no antigenicity was detected in the purified neutral and acidic glycolipid fractions by the Ouchterlony's double diffusion test using rabbit anti-S. mansoni sera or those obtained from patients with S. mansoni infection, though these sera, each gave two different precipitin lines against homogenate of the adult worms.

Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin.

Hemolysin of a non-O1 Vibrio cholerae strain was purified and characterized. The purified hemolysin gave a single protein band on conventional and sodium dodecyl sulfate-gel electrophoresis. Its molecular weight was estimated as 60,000 by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. It had a pI of 5.7. The purified hemolysin caused increased vascular permeability of rabbit skin and rapid death of mice on intravenous injection and also lysed erythrocytes of various animal species. An Ouchterlony double gel diffusion test using antiserum against the purified hemolysin indicated that the hemolysin from non-O1 V. cholerae was immunologically related, but not identical, to the hemolysin from El Tor V. cholerae. Antiserum against the purified hemolysin neutralized the hemolytic activity of the hemolysins from not only non-O1 but also El Tor V. cholerae.

Purification of histidine decarboxylase from the liver of fetal rats and its immunochemical and immunohistochemical characterization.

Histidine decarboxylase was purified from fetal rat liver about 3,000-fold by the method described previously (Watanabe, T., Nakamura, H., Leu, Y.L., Yamatodani, A., and Wada, H. (1979) Biochem. Pharmacol. 28, 1149-1155) except that DEAE-cellulose column chromatography, Sephacryl S-300 gel filtration, and preparative polyacrylamide gel electrophoresis were added. The enzyme had a molecular weight of 110,000 in the native state and gave a single band of protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a position corresponding to a molecular weight of 54,000, indicating that it was a dimer. Its isoelectric point was pH 5.1. Antibody raised in rabbits against the enzyme gave single fused precipitation lines with the enzymes from fetal rat liver and adult rat brain and stomach in Ouchterlony's double diffusion test, and inhibited these three enzymes similarly and strongly. Using this antibody, histidine decarboxylase-like immunoreactive structures were located in fetal liver and peritoneal mast cells, stomach and brain of rats.

Variability in expression of a cell surface determinant on Candida albicans as evidenced by an agglutinating monoclonal antibody.

The expression of a surface immunodeterminant of Candida albicans was investigated with an agglutinating immunoglobulin M monoclonal antibody. The 96 strains of C. albicans tested, of which 76% were recent clinical isolates, were capable of expressing the antigen. The antigen was also produced by strains of Candida tropicalis and Torulopsis glabrata, but not by other yeast species. Expression of the surface immunodeterminant in C. albicans varied as a function of growth as indicated by agglutinin reactions and indirect immunofluorescence tests. When yeast cells were tested with the agglutinin, three patterns of reactivity were observed. In general, cells in the early logarithmic phase were less reactive than cells in the mid-logarithmic phase. Antigen expression, as determined by agglutinin reactivity, was also influenced by the nutritional composition of the growth medium, and in general, cells from broth cultures were usually more reactive than cells grown on solid media. The antigen was solubilized from the cell surface of C. albicans by hot 1 M NaCl. These water-soluble extracts were capable of binding antibody, and a single precipitin band formed when soluble antigen was reacted with the monoclonal antibody in an Ouchterlony double-diffusion test. Whole cell preparations and hot NaCl extracts from yeast strains which did not agglutinate when mixed with the antibody also did not absorb the agglutinin from solution. These data indicate that the expression of surface antigens on C. albicans is a dynamic process which may be influenced by a number of environmental factors. The use of monoclonal antibodies may allow characterization of surface antigens presented to the host during candidiasis.

Comparison of five chymotrypsin inhibitors from the silkworm haemolymph

1. 1. Using two silkworm strains selected from various electrophoretic variants, five haemolymph chymotrypsin inhibitors were partially purified and their properties were compared. 2. 2. Five inhibitor fractions separated on DEAE-Sephacel and Ultrogel AcA54 columns corresponded to the electrophoretic bands; these were classified into two groups, comparatively high, 39,000 (c and d) and low molecular weights, 5500–8500 (a, e and g). 3. 3. Inhibitor a was extremely heat stable, e and g were also stable considerably, but c and d were labile. 4. 4. Ouchterlony's double diffusion test showed that the inhibitors c and d are immunologically identical whereas no precipitate was observed between inhibitors a, e, g and anti-d serum.

Purification and Characterization of Four Forms of Cytochrome P-450 from Liver Microsomes of Phenobarbital-Treated and 3-Methylcholanthrene-Treated Rats1

Four forms of cytochrome P-450, tentatively designated PB-1, PB-2, MC-1, and MC-2, were purified from liver microsomes of rats treated with phenobarbital (PB-1 and PB-2) or 3-methylcholanthrene (MC-1 and MC-2). Each purified form showed a single protein-staining band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 (MC-1), 53,000 (PB-1), 53,000 (MC-2), or 49,000 (PB-2). PB-1 and MC-1 were the major cytochrome P-450 components inducible by phenobarbital (PB) and 3-methylcholanthrene (MC), respectively. Antibodies prepared against each form of purified cytochrome P-450 did not cross-react with heterologous antigens in Ouchterlony double diffusion tests, confirming the immunological distinctness of the four forms. The CO-compounds of reduced PB-1 and PB-2 had an absorption maximum at 450 nm, whereas those of MC-1 and MC-2 had a maximum at 447 nm. Judging from the oxidized absolute spectra, MC-2 was of high spin type and the others were of low spin type. Amino acid analysis revealed considerable differences among the purified four forms of cytochrome P-450, and the amino acid sequences of their NH2-terminal portions confirmed that the four forms were different proteins. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, PB-1 and PB-2 oxidized benzphetamine at high rates, but their oxidation of benzo(a)pyrene was much slower than that by MC-1, which catalyzed rapid hydroxylation of benzo(a)pyrene but had low activity with benzphetamine. The quantity of each form of cytochrome P-450 in microsomes was determined by quantitative immunoprecipitation, and selective induction of PB-1 and MC-1 by PB and MC, respectively, was confirmed. Some induction of PB-2 and MC-2 by the corresponding inducers was also noticed. PB group P-450's were not increased by MC treatment, nor were MC group P-450's by PB.

Characterization of three forms of cytochrome P-450 isolated from liver microsomes of rats treated with 3-methylcholanthrene.

Three forms of cytochrome P-450, designated as P-450MC-I, P-450MC-II, and P-450MC-III, were isolated from liver microsomes of rats treated with 3-methylcholanthrene (MC) by using a high performance liquid chromatography (HPLC) technique. The major MC-inducible forms, P-450MC-I and P-450MC-II showed a single protein band on SDS-polyacrylamide gel electrophoresis giving a minimum molecular weight of 56,000 daltons. The oxidized absolute spectra of both cytochromes P-450 were of low spin type, having a Soret absorption peak at 417 nm. The CO-reduced difference spectra of these two cytochromes P-450 showed a peak at 447 nm. In a reconstituted system, both cytochromes P-450 exhibited similar high levels of catalytic activity for benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation. Anti-P-450MC-I IG and anti-P-450MC-II IG, which were produced against the corresponding cytochromes P-450, each formed a single continuous precipitin line with both P-450MC-I and P-450MC-II in Ouchterlony double diffusion tests. Amino acid sequence analysis revealed that the sequence of the NH2-terminal 18 amino acids of both enzymes was the same. Therefore, the major MC-inducible forms, P-450MC-I and P-450MC-II, were highly homologous, being indistinguishable from each other in terms of apparent molecular weight, spectral properties, substrate specificity and the NH2-terminal 18 amino acid residues, but clearly separable by HPLC. The characteristics of both P-450 forms appear to correspond to those of the previously reported P-450c (1). On the other hand, a minor form, P-450MC-III was different from P-450MC-I and P-450MC-II in chromatographic properties, apparent molecular weight, substrate specificity and immunochemical properties, and did not correspond to any P-450 species previously purified from MC-treated rat liver microsomes.

Fibronectin promotes epithelial migration of cultured rabbit cornea in situ.

We investigated the effect of fibronectin on epithelial migration onto the stroma in cultured rabbit cornea. Rabbit plasma fibronectin was purified by affinity chromatography using gelatin-Sepharose 4B, and its purity was confirmed by SDS polyacrylamide slab gel electrophoresis. Antibody against rabbit plasma fibronectin raised in guinea pigs formed a single precipitin line against rabbit plasma and purified rabbit plasma fibronectin by Ouchterlony double diffusion test. When rabbit cornea was cut into small blocks and cultured in TCM-199 medium alone, corneal epithelial cells began to migrate on the cut edge of the corneal stroma. The addition of purified rabbit plasma fibronectin to the culture medium significantly enhanced epithelial migration. The degree of enhancement depended on the amount of fibronectin added. When guinea pig IgG anti-rabbit plasma fibronectin was added, epithelial migration was significantly inhibited when compared with that in control cultured corneal blocks. The results demonstrate that fibronectin promotes epithelial migration in the cornea and thus plays an important role in corneal wound healing.

Immunochemical evidence for participation of NADPH-cytochrome c reductase in microsomal fatty acyl-CoA desaturation of Tetrahymena cells lacking in cytochrome P-450

Rabbit antibody was prepared against NADPH-cytochrome c reductase of Tetrahymena microsomes. When examined by the Ouchterlony double diffusion test, anti-NADPH-cytochrome c reductase immunoglobulin formed a single precipitation line with Tetrahymena reductase but not rat liver one. The antibody inhibited the NADPH-cytochrome c reductase activity of Tetrahymena microsomes, but it did not affect either NADH-ferricyanide or NADH-cytochrome c reductase activity of Tetrahymena microsomes. The NADPH-dependent desaturation of stearoyl-CoA in Tetrahymena microsomes was inhibited by anti-reductase immunoglobuline, while the NADH-dependent desaturation was affected by neither anti-reductase nor control immunoglobuline. It was suggested that the temperature associated-alteration of NADPH-cytochrome c reductase activities would be important for regulation of microsomal NADPH-dependent desaturase activities in Tetrahymena which contains no cytochrome P-450.

Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168.

Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis.

Characterization of periplasmic hydrogenase from Desulfovibrio vulgaris Miyazaki K.

Periplasmic hydrogenase [hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1] from Desulfovibrio vulgaris Miyazaki K (MK) was purified to homogeneity. Its chemical and immunological properties were examined and compared with those of other Desulfovibrio hydrogenases. The pure enzyme showed a specific activity of 1,000 mumol H2 evolution min-1 (mg protein)-1. The enzyme had a molecular weight of 50,000 as estimated by gel filtration and consisted of a single polypeptide chain. The absorption spectrum of the enzyme was characteristic of an iron-sulfur protein and the extinction coefficients at 400 and 280 nm were 34 and 104 mM-1. cm-1, respectively. It contained 9.4 mol iron and 6.9 mol of acid-labile sulfide per mol. The amino acid composition of the preparation was very similar to the value reported for D. desulfuricans NRC 49001 hydrogenase. Rabbit antisera were prepared against the enzyme of D. vulgaris MK. Ouchterlony double diffusion and immunotitration tests of crude extracts from several strains of Desulfovibrio revealed that the enzyme from MK cells was immunologically identical with those from D. vulgaris Hildenborough and D. desulfuricans NRC 49001, but different from those from D. vulgaris Miyazaki F (MF) and Miyazaki Y, and D. desulfuricans Essex 6 strains. It is concluded that among Desulfovibrio hydrogenases, those from D. vulgaris MK, D. vulgaris Hildenborough and D. desulfuricans NRC 49001 form one group in terms of both subunit structure and antigenicity.

Purification and some properties of a non-o1 Vibrio cholerae enterotoxin that is identical to cholera enterotoxin.

Cholera-like enterotoxin was isolated and purified from the culture supernatant of a non-O1 strain of Vibrio cholerae, E8498, isolated from the environment. Enterotoxin was purified by aluminum hydroxide absorption and elution and successive gel filtrations on Sephadex G-100, Bio-Gel A-5m, and Sephadex G-75. Purified enterotoxin gave a single stained band on polyacrylamide gel disc electrophoresis, and the mobility was the same as that of cholera enterotoxin. The specific biological activity of the purified enterotoxin was almost the same as that of cholera enterotoxin in the Chinese hamster ovary cell assay, fluid accumulation in mouse ligated intestine, increase in vascular permeability in rabbit skin, and passive immune hemolysis. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed that the purified enterotoxin consisted of subunits A and B, identical to those of cholera enterotoxin, and Ouchterlony double gel diffusion tests indicated that the two toxins were immunologically identical. Enterotoxins prepared from several non-O1 strains isolated from human patients were also immunologically identical to cholera enterotoxin.

Physiological and pathological characteristics of Erwinia chrysanthemi isolates from potato tubers

Nine isolates of Erwinia chrysanthemi from rotting potato tubers were compared with six type or reference strains of this species. Phenotypic properties of the potato isolates closely agreed with those of Erw. chrysanthemi pv. zeae and with the characteristics proposed for Dickey's infrasubspecific subdivision IV (1979) and Samson & Nassan‐Agha's biovar 3 (1978), where Zea mays was among the most common host species. Pathogenicity tests on 20 ornamental and agricultural species showed only Cyclamen sp. and Z. mays to be susceptible. In Ouchterlony double diffusion tests, antisera to whole live cells of one potato strain reacted with four of the six pathovars of Erw. chrysanthemi. Tuber isolates did not produce blackleg symptoms in inoculated stems. The rationale of intensive pathogenicity testing is discussed.

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450 C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450 C21 and guinea pig adrenal microsomes against anti-cytochrome P-450 C21 IgG. Anti-cytochrome P-450 C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450 C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450 C21 IgG did not inhibit the activities of 17α-hydroxylase and C 17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450 C21 catalyzes the 17α-hydroxylation and C 17,20 bond cleavage. The stimulation of 17α-hydroxylation and C 17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.

A novel non-heme iron-containing dioxygenase. Chloridazon-catechol dioxygenase from Phenylobacterium immobilis DSM 1986.

Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double-diffusion tests to consist of a single protein species. No cross-reaction was observed with other meta-cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (epsilon = 310 mM -1 cm -1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (epsilon = 1.14 mM -1 cm -1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the pl of 4.5. Surprisingly the enzyme per se is completely inactive, although it contains one non-dialysable iron atom per submit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non-activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta-cleaving enzymes, this enzyme seems to represent a new type of a non-heme iron dioxygenase. We tentatively propose the name Chloridazon-catechol dioxygenase for this enzyme.