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

Ano: 2010 | Volume: 22 | Número: 2

Autores: Leonardo A. Calderon, Humberto A. Almeida Filho, Rozeni C. L. Teles, Francisco J. Medrano, Carlos Bloch Jr, Marcelo M. Santoro, Sonia M. Freitas

Palavras-chave: inga cylindrica [vell] mart, leguminosae, mimosoideae, protease inhibitor, protein stability, trypsin inhibitor

Resumo Inglês:

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.