Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography.
Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).
Michel
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Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps Universität, Marburg, Germany.
Buckel
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Linder
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D000595:Amino Acid Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003013:Clostridium / Q000201:enzymology*; D003038:Cobamides / Q000302:isolation & purification*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D019751:Intramolecular Transferases; D007535:Isomerases / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D013056:Spectrophotometry, Ultraviolet
D002793:Cholic Acids; D003038:Cobamides; D003902:Detergents; D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; C061157:3-methylitaconate delta-isomerase; D019751:Intramolecular Transferases; C047997:2-methyleneglutarate mutase; C000913:cobamamide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate