Proteolytic artifacts in SDS-PAGE analysis of selected periodontal pathogens

The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic (Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola) and non-proteolytic (Fusobacterium nucleatum) bacteria. Conditions to limit or prevent proteolysis were also investigated. Bacterial cells were incubated in solubilizing buffer (SDS+ beta mercaptoethanol) at room temperature for various periods of time before boiling. A control assay consisted of trichloroacetic acid-treated bacterial cells. Cellular proteins were separated by electrophoresis and stained with Coomassie blue. Proteolysis occurred very rapidly in the case of P. gingivalis (< 30 s), whereas a longer incubation time (> 1 h) was required to observe similar effects in P. nigrescens and T. denticola. No proteolysis was observed for F. nucleatum. In all cases, heat (100 degrees C) and low pH (< 4) treatments of bacterial cells could avoid production of proteolytic artifacts. Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis. More particularly, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P. gingivalis, P. nigrescens and T. denticola, respectively. When outer membranes of P. gingivalis were prepared in the presence of TLCK, numerous additional protein brands, not seen in the absence of TLCK, were detected. The present study suggests that specific protease inhibitors, effective in preventing proteolysis, should be identified and added during cell fractionation and protein purification procedures.