Figure 8.

RP-HPLC fractionation. Cation exchange-concentrated ileostomy fluid, of control patient (A) and Crohn’s patients (B and D), was applied to C18 RP-HPLC column, and fractions immunoreactive (by dot blot) for HD-5 were collected (arrowed on traces). A: Control patient (formerly with ulcerative colitis). Only a single peak (arrows), at 50% acetonitrile, was immunoreactive for HD-5 and had N-terminal sequence ATCYCRT, identical to predicted mature HD-5 peptide. B: Crohn’s patient CD21. HD-5 eluted at 50% acetonitrile (small arrow) and 59% acetonitrile (large arrow). After application to PVDF membrane, N-terminal sequencing was performed on protein eluting at 50% acetonitrile, which revealed the sequence ATCYCRT, which matches that for the mature form of HD-5. The HD-5-positive 59% acetonitrile fraction was subjected to AU-PAGE Western blot analysis (see Figure 9A), SDS-PAGE Western blot analysis (see Figure 9B) and ES-QToF MS (see Figure 9, C and D). C: Predicted amino acid sequence (from cDNA) of trypsin-1 (cationic trypsin). Underlined are amino acid sequences from one CD (CD1) ileostomy fluid fraction eluting at 59% acetonitrile. D: Crohn’s patient CD11. Three fractions were immunoreactive for HD-5 (39%, 50%, and 59% acetonitrile, respectively, arrowed). The large arrowed peak (59% acetonitrile) was run on AU-PAGE, and N-terminal sequences of the immunoreactive band obtained were IVNGE (chymotrypsin chain B) and CGVPA (chymotrypsinogen).