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Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of
the native Brazilian tree Copaifera langsdorffii (Caesalpinoideae, Leguminosae).
The purification procedure involved ammonium sulfate fractionation, ion-exchange
chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose,
and reversed-phase (RP) HPLC. RP-HPLC yielded two forms (TDI-I and TDI-II), as
confirmed by isoelectric focusing, with pI values between 7.0 and 8.1. The
molecular mass of the TDI forms was 24 kDa based on FPLC gel filtration on
Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses
of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Ki values were 1.1 and
1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on
chymotrypsin. Amino acid analysis revealed high levels of aspartic acid,
glutamic acid, serine, glycine, proline, and lysine but low levels of methionine
and aromatic amino acids in both inhibitors; the calculated molecular masses
were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal
sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin
inhibitors, whereas TDI-II showed no homology to any other protein. This
observation suggests that TDI-II belongs to a new inhibitor subclass of
low-molecular mass proteins in the subfamily Caesalpinoideae.
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