Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor.
beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.
Schaschke
Norbert
N
Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany. schachk@biochem.mpg.de
Gabrijelcic-Geiger
Dusica
D
Dominik
Andreas
A
Sommerhoff
Christian P
CP
D002846:Chromatography, Affinity / Q000379:methods*; D004151:Dipeptides / Q000138:chemical synthesis / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000378:metabolism; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D053802:Tryptases
D004151:Dipeptides; D004791:Enzyme Inhibitors; D008024:Ligands; D010456:Peptides, Cyclic; C104330:cyclo(aspartyl-aspartyl); D010649:Phenylalanine; D012697:Serine Endopeptidases; D053802:Tryptases