Capillary electrophoretic analysis of serine hydroxymethyltransferase in Escherichia coli fermentation broth.

Serine hydroxymethyltransferase (SHMT) expressed in Escherichia coli was analyzed in fermentation broth through the use of capillary electrophoresis (CE), a method which provided advantages over the traditional techniques of slab gel electrophoresis and chromatography. In addition, via CE the difficult resolution and quantitation of SHMT holoenzyme and apoenzyme were achieved. Using this method, a pyridoxal-5'-phosphate (PLP) cofactor/SHMT dimer molar ratio of 0.65 was estimated to be present in holoenzyme in the absence of excess PLP. This determination correlated well with results obtained by other techniques, including electrospray ionization mass spectrometry (ESI-MS). CE and ESI-MS analyses both provided evidence for significant differences between the folded conformations of SHMT holoenzyme and apoenzyme.