High-performance liquid chromatographic assay for tryptase based on the hydrolysis of dansyl-vasoactive intestinal peptide.
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 the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.
Delaria
K
K
Biotechnology Division, Bayer Corporation, West Haven, Connecticut, 06516-4175, USA.
Muller
D
D
D001331:Automation; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D003619:Dansyl Compounds; D006801:Humans; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008407:Mast Cells / Q000201:enzymology; D012697:Serine Endopeptidases / Q000032:analysis*; D015842:Serine Proteinase Inhibitors / Q000032:analysis; D053802:Tryptases; D014660:Vasoactive Intestinal Peptide / Q000031:analogs & derivatives / Q000378:metabolism
D003619:Dansyl Compounds; D015842:Serine Proteinase Inhibitors; D014660:Vasoactive Intestinal Peptide; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; D053802:Tryptases