Reduction in size of ASP-2 upon incubation at acidic pH proposes autocatalytic pro-peptide cleavagePanel A: Pure ASP-2 samples were dialysed at ph 5 and pH 8.5 immediately following purification and subsequently incubated for 2 hrs at room temperature as described in the methods. The initial non-dialysed sample was also incubated before all the samples were all analysed by SDS-PAGE and western blotting. A reduction of the size of ASP-2, ph 5 was observed compared to the initial eluate and the pH 8.5 samples. Note the presence of higher Mw bands ~140-k which also displayed increased mobility at ph 5. Panel B: The same ASP-2 samples dialysed at different pH were treated with PNGase for better resolution of the protein bands. The samples dialysed at pH 5 were either not incubated (first two lanes) or pre-incubated before PNGAse treatment. These samples needed to be titrated to pH 8.5 before deglycosylation. Similarly samples at pH 8.5 were treated or not with PNGase as indicated on the figure. Compared to the respective sample at pH 8.5, the ph 5 deglycosylated ASP-2 reveals the lowest ASP-2 band visible on this blot at ~50-k, possibly mature protein without the pro-peptide and sugars, suggesting maturation of ASP-2. Pre-incubation of the sample at 37°C increased the intensity of this band compared to the samples that were not pre-incubated implying that its appearance is time-dependent on incubation of the protein at a certain pH. For both panels sizes of molecular weight standard proteins are indicated on the left. Both panels were probed with anti-ASP-2 IgG and developed with ECL.
Sidera et al. BMC Biochemistry 2002 3:25 doi:10.1186/1471-2091-3-25