Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column.
An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochome b1 oxidoreductase, EC 1.2.2.2), a peripheral membrane flavo-enzyme, which is much more reproducible and requires considerably less time than the original purification scheme. The key element in this protocol is a new Sepharose-based affinity resin designed for the isolation of thiamine pyrophosphate-requiring enzymes. The synthesis, partial characterization, and use of two such affinity resins is described. Pyruvate oxidase is a pure, homogenous protein as it is eluted from the affinity resin. The enzyme is a tetramer with a subunit molecular weight of approx. 60 000. The subunits appear to be identical. The isoelectric point of pyruvate oxidase is 5.6.
D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D004983:Ethanolamines; D008970:Molecular Weight; D011771:Pyruvate Oxidase / Q000302:isolation & purification* / Q000378:metabolism; D013835:Thiamine Pyrophosphate
D004983:Ethanolamines; D011771:Pyruvate Oxidase; D013835:Thiamine Pyrophosphate