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. 2013 Jun 24;8(6):e67705. doi: 10.1371/journal.pone.0067705

Figure 3. Iph1 and hIDE have similar protease activity.

Figure 3

A) SDS-PAGE of Iph1 elution fractions at 300 mM imidazole after Ni-NTA purification. 1: Iph1 wt, 2: Iph1-E71D. B) Activity of Iph1 WT, Iph1-E71D and hIDE towards Mca-RPPGFSAFK(Dnp). Error bars represent the standard deviation of 3 experiments. C) Titration of 1,10-phenanthroline inhibition of Mca-RPPGFSAFK(Dnp) digestion by Iph1 or hIDE. Activity is expressed as the percentage of activity of the enzyme in the absence of inhibitor. The error bars represent the standard deviation of duplicates. D) Reversed phase HPLC analysis of insulin digestions. Insulin was digested for 16 h at 37°C in the presence of 500 ng hIDE or 800 ng Iph1. E) Tricine SDS-PAGE analysis of insulin digestions. 185 ng insulin were digested for 16 h with Iph1 wt, hIDE or Iph1-E71D (0.7 or 3.5 or 14 ng). The left hand lanes show different dilutions of undigested recombinant insulin, corresponding to 100%, 50% and 25% of the starting amount used in digestions. One out of 3 experiments performed is shown. F) The percentage of digestion by the different enzymes was calculated by measuring insulin band intensity on Tricine SDS-PAGE gels.