Rates of peptide proteolysis measured using liquid chromatography and continuous-flow fast atom bombardment mass spectrometry.
An immobilized digestive enzyme assay, which has been used to determine whether orally administered peptide drugs are hydrolyzed by the digestive system, was applied to the measurement of rates of proteolysis of biologically active peptides. In this study, the rates of hydrolysis by trypsin and chymotrypsin of the pressor agent angiotensin II, the peptide hormone [Arg8]vasopressin, and the peptide drug [deamino-Cys1,D-Arg8]vasopressin were measured. Enzyme immobilization prevented autolytic proteolysis and provided a stable enzyme preparation during the assays. For rate determinations, the disappearance of substrate was measured over time by using either flow injection continuous-flow fast atom bombardment (FAB) mass spectrometry with selected ion monitoring or reversed-phase high-performance liquid chromatography (HPLC) with UV absorbance detection. Compared to the HPLC method, continuous-flow FAB was faster, provided more confident identification of the analyte because molecular weight data was obtained, and could be used for all enzymatic reactions instead of only those in which complete chromatographic resolution of substrate from proteolytic fragments was obtained. The in vitro proteolytic rates measured for the vasopressins were compared to data from rat bioassays and confirmed that the limiting factor in the oral bioavailability of [Arg8]vasopressin was rapid hydrolysis by trypsin in the intestinal lumen. The more bioactive compound, [deamino-Cys1,D-Arg8]vasopressin, was more stable to chymotryptic digestion and completely resistant to trypsinization.
van Breemen
R B
RB
Department of Chemistry, North Carolina State University, Raleigh 27695-8204.
Davis
R G
RG
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment
D004800:Enzymes, Immobilized; D010455:Peptides