Stability and spectroscopic analyses of a trypsin inhibitor isolated from Calliandra selloi seeds

YOSHIZAKI L; TRONCOSO MF; LOPES JL; BELTRAMINI LM; WOLFENSTEIN-TODEL C

Rosario, Santa Fé

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

A novel trypsin inhibitor was purified from Calliandra selloi seeds (CSTI). SDS-PAGE under non reducing conditions showed a single band of approximately 20,000 Da while in reducing conditions two bands of 16,000 and 6,000 Da were observed, indicating that CSTI consists of two polypeptide chains. Molecular masses of 20,078 and 20,279 were obtained by mass spectrometry, although only one pI corresponding to 4.0 was observed. Amino-terminal sequence showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21x10 -7 M), chymotrypsin (Ki 4.95x10 -7 M) and kallikrein (Ki 4.2x10-7 M) but had no inhibitory activity towards elastase. Trypsin inhibitory activity was stable over a wide range of pH and temperature. CSTI was particularly susceptible to DTT treatment, followed by addition of iodoacetamide. Far-UV circular dichroism measurements revealed that CSTI is a â-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68°C. Far-UV CD spectra of CSTI at pH extremes showed little changes, while more pronounced differences in near-UV CD spectra were detected.  Remarkably, treatment with 1 mM DTT caused slight changes in the far-UV CD spectrum, and only after carbamidomethylation there was a marked loss of secondary structure.