Separation of canine pancreatic juice proteins by hydrophobic interaction chromatography preserves enzyme activity.
To measure the synthesis of pancreatic enzymes requires the separation of pancreatic juice proteins. The aim of the present study was to separate amylase, lipase, and trypsinogen present in dog pancreatic juice by using a hydrophobic interaction high-performance liquid chromatography (HPLC). During a 40-min, four-stage gradient of decreasing sodium sulfate concentration, dog pancreatic juice proteins were separated into 10 peaks based on hydrophobicity. Amylase, lipase, and trypsinogen were identified in HPLC fractions by measuring enzyme activity and molecular weight. Amylase and lipase were present in separate peaks. By sodium dodecyl sulfate (SDS) gel electrophoresis, peak 10, the only peak with amylase activity had a single protein, but peak 3, containing lipase, and peak 9, containing trypsinogen, had two or more proteins. Trypsinogen activity was also detected as a main protein in peak 5 and the molecular weight of this protein, 26 kDa corresponds to that of dog trypsinogen. Trypsinogen was not identified in peak 9 by SDS gel electrophoresis because other proteins were close to this location on the gel. In summary, the proteins and activities of amylase, lipase, and two trypsinogens secreted by the dog pancreas can be separated rapidly by hydrophobic interaction liquid chromatography. Also, this one step procedure recovers 87% of amylase from dog pancreatic juice as a single protein.
Yamadera
K
K
Division of Gastroenterology and Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905.
DiMagno
E P
EP
D000681:Amylases / Q000302:isolation & purification*; D000818:Animals; D002845:Chromatography / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D008049:Lipase / Q000302:isolation & purification*; D008970:Molecular Weight; D010189:Pancreatic Juice / Q000737:chemistry*; D014362:Trypsinogen / Q000302:isolation & purification*
D014362:Trypsinogen; D008049:Lipase; D000681:Amylases