High-Performance Liquid Chromatographic Assay for Tryptase Based on the Hydrolysis of Dansyl-Vasoactive Intestinal Peptide

• Published in: Analytical biochemistry 1996-04, Vol.236 (1), p.74-81
• Main Authors: Delaria, Katherine, Muller, Daniel
• Format: Article
• Language: English
• Subjects: Automation; Chromatography, High Pressure Liquid; Chymases; Dansyl Compounds; Humans; Kinetics; Lung - enzymology; Mast Cells - enzymology; Serine Endopeptidases - analysis; Serine Proteinase Inhibitors - analysis; Tryptases; Vasoactive Intestinal Peptide - analogs & derivatives; Vasoactive Intestinal Peptide - metabolism
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1996.0133

A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While theKmvalue was unaffected, thekcatvalue decreased, yielding akcat/Kmratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in thekcat/Kmratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.