Trypsin inhibitors from the garden four o’clock ( Mirabilis jalapa) and spinach ( Spinacia oleracea) seeds: Isolation, characterization and chemical synthesis

Abstract

Five serine proteinase inhibitors ( Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o’clock ( M. jalapa) and spinach ( S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5 M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP–HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants ( K a) with bovine β-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I–III) were found to be about 10 7 M −1. Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1–Cys4, Cys2–Cys5 and Cys3–Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.