Protein chips for screening of protease-protease inhibitor interactions

Two Kunitz-type inhibitors (KPI) of class A, five of class B, and six of class C, in mixed or high pure fraction, in glycerol 40%, were spotted in NaHCO3 in triplicate on glass slides at increasing concentration 0.1, 0.2, 0.4 ?g/?l. KPIs binding to proteases, and in vitro inhibition was detected. Presence of full-length PI proteins and correlation with total protein concentration was checked by western blot with anti-KPI B10 polyclonal antibodies. Physical interaction with trypsin and chymotrypsin was detected, differentially characterising three KPIs. Inhibition assays show PI protease activity at each concentration. On the chip, protein-protein interaction data provide additional information and screening of PIs: chips were useful to show that cathepsin B, a cysteine protease, was actively bound by PIs, information that allowed to test the in vitro inhibition. Kunitz-PIs may be evaluated on the chip with anti-KPI B10 antibodies, for homogeneous presentation of PI proteins and estimation of amount of protein bound to the surface.