Kunitz-type trypsin inhibitors from legume seeds have been characterized structurally. The presence of Cys-Cys in single or double chains shows a new pattern of proteins structurally not so closely related to STI. Therefore, briefly, with regard to cysteine content, plant Kunitz proteinase inhibitors may be classified into four groups: no Cys-Cys at all, one, two and more than two Cys residues. Functional properties and diversity of these proteins are also briefly discussed.

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

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Araucaria angustifolia seeds are characterised by a relatively high content of starch and protein. This study aimed to verify the presence of α-amylase inhibitors in the seeds and to characterise a trypsin inhibitor found in the embryo tissues. Inhibitor purification was carried out by the saline extraction of proteins, acetone precipitation and affinity chromatography. Two protein bands of molecular weight estimated by SDS-PAGE at about 35 kDa were further examined by high-performance liquid chromatography coupled to a mass spectrometer and were shown to be 36.955 Da (AaTI-1) and 35.450 Da (AaTI-2). The sequence of the N-terminal region shows that AaTI-1 and AaTI-2 are structurally similar to plant inhibitors of the serpin family. A mixture of AaTI-1 and AaTI-2, identified as AaTI, shows selectivity for the inhibition of trypsin (K 85 nM) and plasmin (K 7.0 μM), but it does not interfere with the chymotrypsin, human plasma kallikrein, porcine kallikrein or other coagulation enzymes activity.

Protease inhibitors are involved in the regulation of endogenous cysteine proteases during seed development and play a defensive role because of their ability to inhibit exogenous proteases such as those present in the digestive tracts of insects. Araucaria angustifolia seeds, which can be used in human and animal feed, were investigated for their potential for the development of agricultural biotechnology and in the field of human health. In the pine nuts extract, which blocked the activities of cysteine proteases, it was detected potent insecticidal activity against termites (Nasutitermes corniger) belonging to the most abundant termite genus in tropical regions. The cysteine inhibitor (AaCI-2S) was purified by ion-exchange, size exclusion, and reversed-phase chromatography. Its functional and structural stability was confirmed by spectroscopic and circular dichroism studies, and by detection of inhibitory activity at different temperatures and pH values. Besides having activity on cysteine proteases from C. maculatus digestive tract, AaCI-2S inhibited papain, bromelain, ficin, and cathepsin L and impaired cell proliferation in gastric and prostate cancer cell lines. These properties qualify A. angustifolia seeds as a protein source with value properties of natural insecticide and to contain a protease inhibitor with the potential to be a bioactive molecule on different cancer cells.