The synthesis, kinetic characterization and application of biotinylated aminoacylchloromethanes for the detection of chymotrypsin and trypsin-like serine proteinases

Abstract

The synthesis of two biotinylated affinity labels for chymotrypsin and trypsin-like serine proteinases is described, along with their kinetic characterization and application to the detection of these proteinases after PAGE and Western blotting. Thus the chloromethane analogues biotinylphenylalanylchloromethane (Bio-Phe-CH2Cl; reagent 1) and biotinylarginylchloromethane (Bio-Arg-CH2Cl, reagent 2), have been shown to be potent active-site-directed inactivators of chymotrypsin and trypsin respectively. The apparent overall second-order rate constants (kobs./[I]) for the inactivation of chymotrypsin and trypsin by reagent 1 (-4.9 x 103 M-1 min-') and reagent 2 (-1.0 x 105 M-1 min-') respectively are comparable with those obtained by other workers with simple urethane-protected analogues and demonstrates that the presence of the bulky biotinyl moiety is compatable with inhibitor effectiveness. Samples of chymotrypsin and trypsin that have been inactivated by reagents 1 and 2 respectively and which have been subjected to SDS/PAGE and Western blotting can be revealed with a streptavidin/alkaline phosphatase label. We can presently detect down to 20 ng of inactivated proteinase by using this system. The utility of the arginine derivative for the detection of the plasma trypsin-like proteinases plasmin and thrombin has also been demonstrated, thus holding out the possibility that this reagent may find general application as an active-site-directed label for this class of proteinase.

Protease-Activated Receptor-2 Involvement in Hypotension in Normal and Endotoxemic Rats In Vivo

“…We also used a trypsin inhibitor, biotin-SKGR-chloromethylketone, a peptide that specifically blocks the proteolytic activity of trypsin. 13 The finding that trypsin-induced hypotension was inhibited by biotin-SKGR-chloromethylketone indicates that proteolysis is required for the induction of hypotension. Unexpectedly, and strikingly, none of these inhibitors affected the hypotension caused by PAR-2AP.…”

“…After L-NAME, the hypotension was 29.3Ϯ10.4 mm Hg (nϭ4; PϽ0.02), 28.7Ϯ5 mm Hg (nϭ4; PϽ0.02), and 25.7Ϯ5.6 mm Hg (nϭ4; PϽ0.02) after 20, 30, and 40 minutes after administration, respectively ( Figure 3C). Biotin-SKGR-chloromethylketone (0.1 mg/kg IV), a potent irreversible inhibitor of trypsin proteolysis, 13 given 30 minutes before trypsin (2.4 mg/kg) reduced the hypotensive response significantly, from 41.5Ϯ6.4 to 21Ϯ6.9 mm Hg (nϭ5; PϽ0.01). Strikingly, none of the drugs tested, including the trypsin inhibitor, caused a significant inhibition of the PAR-2AP-induced hypotension.…”

“…RNA purity was estimated by measurement of optical density at 260/280 nm. Total RNA (5 g) was subjected to first-strand cDNA synthesis in a 10-L reaction containing 250 mmol/L Tris-HCl (pH 8.3 at 20°C), 375 mmol/L KCl, 15 mmol/L MgCl 2 , 1 mmol/L DTT, 1 mmol/L of each dNTP, and 20 U RNase inhibitor, in the presence of 1.5 g oligo dT (12)(13)(14)(15)(16)(17)(18) primer and 200 U Superscriptase (all from Life Technologies). After completion of first-strand cDNA synthesis, the reaction was stopped by heat inactivation (5 minutes, 95°C) and diluted to 50 ng/L RNA equivalents with water.…”

Utilization of biotinylated diphenyl phosphonates for disclosure of serine proteases

“…Detection of biotinylated proteases by SDS-PAGE and Western blotting was carried out as described in [3].…”

“…a-Aminoalkyl diphenyl phosphonate esters have been shown to be superb inactivators of serine proteases with no activity against cysteine proteases, are nontoxic in vivo, and are stable under physiological conditions [1,2]. Biotinylated aminoacyl chloromethanes and isocoumarins have found use as active-site-directed affinity labels for the disclosure of serine proteases from a variety of biological milieu and using techniques ranging from SDS-PAGE and Western blotting to immunocytochemistry and affinity purification [3][4][5][6]. We have previously reported on the synthesis of N-protected diphenyl phosphonate analogues of ornithine and lysine [7] and in this paper we detail the synthesis of the biotinylated analogues and report on their kinetic characterization and utilization for the detection of trypsin-like serine proteases using SDS-PAGE and Western blotting.…”