title: Effects of denaturing and stabilizing agents on the inhibitory activity and conformational stability of Schizolobium parahyba chymotrypsin inhibitor.
abstract: The conformational stability of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) was investigated based on conformational changes and inhibitory activity in the presence of chaotropic and stabilizing agents. At 90 degrees C, the half-lifetime of SPCI was 154 min, while in the presence of 1 M KCl and 20% PEG 20,000, it was drastically reduced to 6 and 3 min, respectively. In contrast, at 90 degrees C, the SPCI structure remained unaltered with the addition of 1 mM DTT and 56% glycerol. The reduction of the two disulfide bonds caused conformational changes in the SPCI without altering the inhibitory activity, suggesting that disulfide bonds are irrelevant to the maintenance of SPCI conformation. Unfolded structures were formed in the presence of 6 M GdnHCl, while in the presence of 8 M urea, destabilization was due to peptide bond rupture. These results suggest that the thermal inactivation of SPCI involves conformational changes and that hydrophobic and electrostatic interactions play a significant role, while the disulfide bonds are of secondary importance in maintaining the high thermal stability of SPCI.
authors.affiliation: Department of Cellular Biology, Universidade de Brasilia, Brazil.
authors.forename: E M
authors.initials: EM
authors.lastname: de Souza
authors.forename: R C
authors.initials: RC
authors.lastname: Teles
authors.forename: E M
authors.initials: EM
authors.lastname: de A Siqueira
authors.forename: S M
authors.initials: SM
authors.lastname: de Freitas
mesh_terms: D002918:Chymotrypsin / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010455:Peptides; D010940:Plant Proteins; D011487:Protein Conformation; D011489:Protein Denaturation; D019685:Rosales / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D002918:Chymotrypsin