Affinity chromatography studies with the pyruvate dehydrogenase complex of wild-type Escherichia coli.
1. The lifetime of thiamine pyrophosphate-Sepharose 2B affinity matrices synthesized according to Matsuura et al. (Matsuura, A., Iwashina, A. and Nose, Y. (1973) Biochem. Biophys. Res. Commun. 51, 241-246) has been improved. The matrix interacts with bacterial pyruvate dehydrogenase complexes. 2. The synthesis of a stable thiochrome-Sepharose 2B matrix is described. 3. Both matrices bind the pyruvate dehydrogenase complex of Escherichia coli in a 50 mM phosphate buffer, pH 7.0. Elution is possibly by an increase in ionic strength but not by the cofactor or metal-cofactor complexes. 4. The presence of Mg2+, reduces the capacity of the affinity matrices but leads to higher specificity for the multienzyme complex. 5. The pyruvate dehydrogenase complex of E. coli has been successfully purified by combining a classical purification step with these affinity chromatography systems. The method is less suitable for large scale operation.
D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium; D008970:Molecular Weight; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*
D011768:Pyruvate Dehydrogenase Complex; D008274:Magnesium