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

• Autor(a) principal: Calderon, Leonardo A
• Data de Publicação: 2010
• Outros Autores: Almeida Filho, Humberto A, Teles, Rozeni C. L, Medrano, Francisco J, Bloch Jr, Carlos, Santoro, Marcelo M, Freitas, Sonia M
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UnB
• Texto Completo: http://repositorio.unb.br/handle/10482/27891; https://dx.doi.org/10.1590/S1677-04202010000200001
• topic: Inga cylindrica [Vell.] Mart; Leguminosae; Mimosoideae; protease inhibitor; protein stability; trypsin inhibitor

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.