Purification and characterization of catalase-1 from Bacillus subtilis

1987 ◽  
Vol 65 (11) ◽  
pp. 939-947 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Hydrogen Peroxide ◽  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Catalase Activity ◽  
Purification And Characterization ◽  
Ph Range ◽  
Sulfhydryl Compounds ◽  
Native Molecular Weight

The catalase activity produced in vegetative Bacillus subtilis, catalase-1, has been purified to homogeneity. The apparent native molecular weight was determined to be 395 000. Only one subunit type with a molecular weight of 65 000 was present, suggesting a hexamer structure for the enzyme. In other respects, catalase-1 was a typical catalase. Protoheme IX was identified as the heme component on the basis of the spectra of the enzyme and of the isolated hemochromogen. The ratio of protoheme/subunit was 1. The enzyme remained active over a broad pH range of 5–11 and was only slowly inactivated at 65 °C. It was inhibited by cyanide, azide, and various sulfhydryl compounds. The apparent Km for hydrogen peroxide was 40.1 mM. The amino acid composition was typical of other catalases in having relatively low amounts of tryptophan and cysteine.

Purification and characterization of spore-specific catalase-2 from Bacillus subtilis

1988 ◽  
Vol 66 (7) ◽  
pp. 707-714 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Molar Absorptivity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Extreme Stability ◽  
Native Molecular Weight ◽  
Soret Absorption

Catalase-2, the catalase found in spores of Bacillus subtilis, has been purified to homogeneity from a nonsporulating strain. The apparent native molecular weight is 504 000. The enzyme appears to be composed of six identical protomers with a molecular weight of 81 000 each. The amino acid composition is similar to the composition of other catalases. Like most catalases, catalase-2 exhibits a broad pH optimum from pH 4 to pH 12 and is sensitive to cyanide, azide, thiol reagents, and amino triazole. The apparent Km for H2O2 is 78 mM. The enzyme exhibits extreme stability, losing activity only slowly at 93 °C and remaining active in 1% SDS – 7 M urea. The green-colored enzyme exhibits a spectrum like heme d with a Soret absorption at 403 nm and a molar absorptivity consistent with one heme per subunit. The heme cannot be extracted with acetone–HCl or ether, suggesting that it is covalently bound to the protein.

Purification and characterization of catalase HPII from Escherichia coli K12

1986 ◽  
Vol 64 (7) ◽  
pp. 638-646 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl ◽  
Purification And Characterization ◽  
Unusual Structure ◽  
Escherichia Coli K12 ◽  
Molecular Weights ◽  
Ph Range ◽  
Heme Cofactor ◽  
Native Molecular Weight

Catalase (hydroperoxidase II or HPII) of Escherichia coli K12 has been purified using a protocol that also allows the purification of the second catalase HPI in large amounts. The purified HPII was found to have equal amounts of two subunits with molecular weights of 90 000 and 92 000. Only a single 92 000 subunit was present in the immunoprecipitate created when HPII antiserum was added directly to a crude extract, suggesting that proteolysis was responsible for the smaller subunit. The apparent native molecular weight was determined to be 532 000, suggesting a hexamer structure for the enzyme, an unusual structure for a catalase. HPII was very stable, remaining maximally active over the pH range 4–11 and retaining activity even in a solution of 0.1% sodium dodecyl sulfate and 7 M urea. The heme cofactor associated with HPII was also unusual for a catalase, in resembling heme d (a2) both spectrally and in terms of solubility. On the basis of heme-associated iron, six heme groups were associated with each molecule of enzyme or one per subunit.

Purification and characterization of a post-proline dipeptidyl aminopeptidase fromStreptococcus cremorisAM2

1990 ◽  
Vol 57 (1) ◽  
pp. 89-99 ◽  
Author(s):  
Mary Booth ◽  
Ide Ni Fhaoláin ◽  
P. Vincent Jennings ◽  
Gerard O'Cuinn
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Cheese Ripening ◽  
Streptococcus Cremoris ◽  
Scissile Bond ◽  
Dipeptidyl Aminopeptidase

SummaryThe present study describes the purification of a post-proline dipeptidyl aminopeptidase from the cytoplasm ofStreptococcus cremorisAM2. On the basis of its elution from a calibrated Sephadex G200 column, the enzyme had a molecular weight of 117000 and exhibited a broad pH optimum activity between 6·0 and 9·0. The activity was most comprehensively inhibited by phenylmethylsulphonylfluoride and more modestly inhibited by 1,10-phenanthroline and 8-hydroxyquinoline but not by EDTA. A range of peptides containing either proline or alanine as the penultimate amino acid residue could act as substrates. The presence of proline on the carboxy side of the scissile bond prevented hydrolysis. However the enzyme could release Pro-Pro from Pro-Pro-Gly-Phe-Ser-Pro. The significance of this substrate specificity is considered in the context of removal of either single proline residues or prolylproline sequences from oligopeptides during cheese ripening.

Purification and Characterization of a Novel Rhamnose/Fucose-Specific Lectin from the Hemolymph of Oak Tasar (Antheraea proylei J.) Silkworm

2020 ◽  
Vol 27 (7) ◽  
pp. 649-657
Author(s):  
Hijam Kiranbala Devi ◽  
Sanjenbam Kunjeshwori Devi ◽  
Huidrom Rully ◽  
Sorokhaibam Jibankumar Singh ◽  
Wayenbam Sobhachandra Singh ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Thermal Stability ◽  
Molecular Weight ◽  
Tandem Mass Spectrometry ◽  
Metal Ion ◽  
Tandem Mass ◽  
Sequence Matching ◽  
Purification And Characterization ◽  
Native Molecular Weight

Background: Lectins are proteins or glycoproteins of non-immune origin which bind specifically but reversibly to carbohydrates or glycoconjugates. They play a crucial role in various biological processes including host defense mechanism, inflammation and metastasis. Therefore, there is an expanding scientific emphasis on purification and characterization of novel lectins possessing different useful biological properties. Objective: The present investigation is concerned with purification and characterization of a novel lectin from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. Methods: The lectin was purified from the hemolymph by a procedure involving successive steps of hemocyte-free hemolymph preparation, ammonium sulfate (0-40%) fractionation and affinity chromatography on a column of Sephadex G-50 covalently coupled with L-rhamnose. It was then characterized by various physico-chemical methods including SDS-PAGE, gel filtration, hemagglutination assay, hemagglutination inhibition assay and tandem mass spectrometry (LCMS/ MS) coupled with Mascot sequence matching software (Matrix Science). Results: The lectin was purified to electrophoretic homogeneity from the silkworm hemolymph and was found to be a monomeric protein with a native molecular weight of 39.5 kDa. It was specifically inhibited by L-rhamnose and D-fucose, the former being sixteen times more inhibitory than the latter. The hemagglutinating activity was further characterized by independency of metal ion, optimum at pH 7-7.5 and thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not purified and characterized earlier. Conclusion: A novel rhamnose/fucose-specific lectin was purified to electrophoretic homogeneity from the hemolymph of oak tasar (Antheraea proylei J.) silkworm. The lectin was found to be a monomeric protein with a native molecular weight of 39.5 kDa. Its activity was found to be independent of metal ion, optimum at pH 7-7.5 and characterized by thermal stability with t1/2 of 60°C. Analysis with tandem mass spectrometry coupled with Mascot sequence matching software confirmed the purified lectin to be a protein not characterized earlier.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

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