Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses
Lucila Yoshizaki1, María F Troncoso, Jose L S Lopes
1Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
Phytochemistry
|July 27, 2007
Summary
A novel trypsin inhibitor from Calliandra selloi seeds (CSTI) comprises two polypeptide chains and exhibits Kunitz-type homology. This inhibitor effectively targets trypsin, alpha-chymotrypsin, and kallikrein, demonstrating stability across various conditions.
Area of Science:
- Plant biochemistry
- Protein chemistry
- Enzyme inhibitors
Background:
- Calliandra selloi Macbride seeds are a potential source of bioactive compounds.
- Plant-derived protease inhibitors play crucial roles in regulating enzymatic activity.
- Understanding the structural and functional properties of these inhibitors is vital for biotechnological applications.
Purpose of the Study:
- To purify and characterize a novel trypsin inhibitor from Calliandra selloi seeds, designated CSTI.
- To elucidate the structural features, inhibitory activity, and stability of CSTI.
- To investigate the secondary structure and conformational stability of CSTI using biophysical techniques.
Main Methods:
- Purification of CSTI using standard biochemical techniques.
- SDS-PAGE and mass spectrometry for molecular weight and subunit analysis.
- Enzyme inhibition assays to determine inhibitory activity against various proteases.
- Circular dichroism spectroscopy (Far-UV and Near-UV) for secondary structure and conformational analysis.
- Thermal denaturation studies to assess protein stability.
Main Results:
- CSTI was purified as a single band on SDS-PAGE under non-reducing conditions, dissociating into two chains (16 and 6 kDa) under reducing conditions.
- Mass spectrometry confirmed molecular masses consistent with a two-chain structure.
- CSTI demonstrated potent inhibition of trypsin, alpha-chymotrypsin, and kallikrein, with Ki values in the 10(-7) M range.
- The inhibitor exhibited remarkable stability over a wide pH and temperature range.
- Far-UV CD spectra indicated CSTI is a beta-II protein, with thermal unfolding showing a two-state transition at 68°C.
- DTT treatment followed by carbamidomethylation led to a significant loss of secondary structure.
Conclusions:
- CSTI is a novel Kunitz-type trypsin inhibitor from Calliandra selloi seeds with a distinct two-polypeptide chain structure.
- Its potent inhibitory activity and stability suggest potential applications in various fields.
- The structural analysis reveals a predominantly beta-sheet secondary structure, with susceptibility to reductive cleavage impacting its integrity.
