Two-step fast protein liquid chromatographic purification of the Serratia marcescens hemolysin and peptide mapping with mass spectrometry.

The pore forming toxin of Serratia marcescens (ShlA) is secreted and activated by an outer membrane protein (ShlB). Activation of inactive ShlA (termed ShlA*) by ShlB is dependent on phosphatidylethanolamine (PE). Activation may be a covalent modification of ShlA. To test this hypothesis, the responsible activation domain (in the N-terminal 255 amino acids of ShlA) was isolated from whole bacteria with 8 M urea in an inactive form (ShlA-255*) and from the culture supernatant in an active form (ShlA-255), followed by a two-step purification by anion-exchange chromatography and gel permeation chromatography. Comparison of a tryptic peptide map of both forms with subsequent electrospray mass spectrometry (ES-MS) and sequencing by tandem ES-MS revealed no modification. These data imply that ShlB presumably imposes a conformation on ShlA-255 that triggers activity.