Calliandra selloi Macbride trypsin inhibitor: isolation, characterization, stability, spectroscopic analyses.
A trypsin inhibitor was purified from Calliandra selloi Macbride seeds (CSTI). SDS-PAGE under non-reducing conditions showed a single band of approximately 20,000 Da, while under reducing conditions two bands of 16,000 and 6000 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 of 4.0 was observed and one peak was obtained by reversed phase chromatography. Amino-terminal sequence analysis showed homology to Kunitz-type inhibitors. CSTI was able to inhibit trypsin (Ki 2.21 x 10(-7)M), alpha-chymotrypsin (Ki 4.95 x 10(-7)M) and kallikrein (Ki 4.20 x 10(-7)M) but had no effect on 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 beta-II protein. Thermal unfolding showed a two-state transition with a midpoint at 68 degrees 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 1mM DTT caused very slight changes in the far-UV CD spectrum, and only after carbamidomethylation was there was a marked loss observed in secondary structure.
Yoshizaki
Lucila
L
Instituto de Química y Fisicoquímica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Junín 956, 1113 Buenos Aires, Argentina.
Troncoso
María F
MF
Lopes
Jose L S
JL
Hellman
Ulf
U
Beltramini
Leila M
LM
Wolfenstein-Todel
Carlota
C
D000818:Animals; D002478:Cells, Cultured; D004912:Erythrocytes / Q000187:drug effects; D007887:Fabaceae; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011817:Rabbits; D012639:Seeds / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology
D014361:Trypsin Inhibitors