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

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) (ki = 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 tm of 70.0 oc and δg25 of 48.5 ± 0.7 kj.mol-1. the values of δg25 at ph 1.6 (22.5 ± 1.2 kj.mol-1) and ph 10.0 (31.5 ± 1.0 kj.mol-1) indicate a reduced structural stability of the protein under these conditions. this is likely to result from pka differences of the acid and basic side chains reflecting the changes in the non-covalent interactions in the folded state.