Purification and structural stability of a trypsin inhibitor from Amazon Inga cylindrica [Vell.] Mart. seeds

Abstract

Inga cylindrica Trypsin Inhibitor (ICTI) was purified as a single polypeptide chain by one step anion-exchange chromatography from a crude extract of Inga cylindrica (Vell.) Mart. seeds. ICTI is a 19.5 kDa protein presenting a remarkable inhibitory activity against bovine trypsin (EC 3.4.21.4) (K i = 4.3 nM). Circular dichroism analysis revealed that this inhibitor is a β type protein (40.4% of β-strand; 24.6% of β-turn and 6.7% of α-helix) in accordance with properties displayed in Kunitz type inhibitors. ICTI is a thermal stable protein within a wide range of pH (1.6 to 10.0) exhibiting highest stability at pH 7.0 as indicated by T m of 70. ) indicate a reduced structural stability of the protein under these conditions. This is likely to result from pK a differences of the acid and basic side chains reflecting the changes in the non-covalent interactions in the folded state.

Exploiting the biological roles of the trypsin inhibitor from Inga vera seeds: A multifunctional Kunitz inhibitor

“…4B). These features have been observed in other trypsin inhibitors, such as I. laurina [39], I. cylindrica [46] and P. moniliformis [16]. Additionally, the inhibitory activity of IVTI was also examined after incubation with DTT.…”

“…However, most inhibitors studied belonging to the subfamily Mimosoideae are usually formed by two polypeptide chains linked by a double disulfide bridge, differentiating them from other Kunitz-type single-chain inhibitors from the Caesalpinioideae and Papilionoideae subfamilies [43]. The occurrence of a single-chain polypeptide in the Mimosoideae subfamily was also reported for the trypsin inhibitors from I. laurina [39], Inga cylindrical [46], Entada scandens [45] and E. acaciifolia [27]. Oliveira et al [27] suggested that the Inga and Entada genera appear to form a subgroup where the presence of single chain inhibitors is more commonly observed.…”

Thermal inactivation studies on toxic seeds from fruits of the Brazilian Central Plain

“…Other authors have described two groups of tripsin inhibitors with significantly different thermal stability in leguminous seeds such as kidney beans and soya beans (Carvalho et al, 2002;Khattab et al, 2009;Lajolo & Genovese, 2002;Shimelis & Rakshit, 2007). One of these fractions can be totally inhibited after 10 minutes heating at 70 °C (Calderon et al, 2010) while the other shows an activity reduction of 50% at 95 °C (Miura et al, 2005) or 70% at 100 °C (Kadam et al, 1986), both after 1 hour of heating. This fact together with the existence of high concentrations of antitrypsins, could explain the inactivation effect observed in Jatobá's toxicity at a higher temperature over a shorter time, suggesting the presence of the more stabile fraction of trypsin inhibitors.…”