Preparation of Escherichia coli pyruvate oxidase utilizing a thiamine pyrophosphate affinity column

Thomas A. O'Brien, Herbert L. Schrock, Patricia Russell, Robert Blake, Robert B. Gennis

Research output: Contribution to journalArticlepeer-review

Abstract

An improved procedure is reported for the purification of Escherichia coli pyruvate oxidase (pyruvate:ferricytochrome 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, homogeneous 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.

Original languageEnglish (US)
Pages (from-to)13-29
Number of pages17
JournalBBA - Enzymology
Volume452
Issue number1
DOIs
StatePublished - Nov 8 1976

ASJC Scopus subject areas

  • Medicine(all)

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