Identification and characterisation of serine protease inhibitors from Araucaria angustifolia seeds

Abstract

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.

Biotechnological Potential of Araucaria angustifolia Pine Nuts Extract and the Cysteine Protease Inhibitor AaCI-2S

“…This was also confirmed by our investigation since, in the present study, the saline extract of A. angustifolia seeds did not inhibit trypsin or other serine proteases. A trypsin inhibition has been detected in the embryo tissues only after sample concentration with acetone precipitation [24]. In contrast, the saline extract inhibited two cysteine proteases, papain, and the enzyme cruzain (a recombinant form of the cysteine protease cruzipain from Trypanosoma cruzi) [25].…”

Plant-Derived Compounds and Extracts as Modulators of Plasmin Activity—A Review

“…Studies on peptide-based plant inhibitors of serine proteases evidently indicate their higher efficiency when compared to (poly)phenolic compounds, for example, AVPI-12, a protease inhibitor from Aloe vera L. leaves with a unique RDWAEPNDGY motif starting its N-terminal region inhibited the plasmin-mediated hydrolysis of human fibrin(ogen) in vitro with efficiency comparable to α2-macroglobulin [ 56 ]. Both the Kunitz and other types of peptide inhibitors of different plant origin act mostly at nanomolar and even sub-nanomolar concentrations [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ] ( Table 4 ). In contrast, the most active plant polyphenolics are able to inhibit serine proteases or other enzymes at micromolar concentrations ( Table 3 ).…”

“…Kuntze seeds. The inhibitor did not influence activities of chymotrypsin, human plasma kallikrein, or other coagulation enzymes; however, AaTI was more active towards trypsin (K i = 85 nM) than plasmin (K i = 7.0 μM) [ 59 ].…”