Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the Brazilian tree Dipteryx alata (Leguminosae).

ABSTRACT

Dipteryx alata trypsin inhibitor (DATI) has been purified and completely sequenced. It showed homology to members of the Bowman-Birk family of inhibitors. The last step of DATI purification by RP-HPLC (narrow-borc C18 column) suggested the existence of some isoforms of the inhibitor due to the presence of a cluster of very close peaks in the chromatogram. By using electrospray ionization mass spectrometry (ESIMS) and laser desorption mass spectrometry (LDIMS), the identification of DATI isoforms was made possible. From the ESIMS data, the following molecular masses were found 6803.22 +/- 0.92 for isoform a; 6890.94 +/- 0.73 for b; 6977.58 +/- 0.39 for c; 7065.07 +/- 0.67 for d; 7151.42 +/- 0.86 for e; and 7291.70 +/- 0.43 for f. Similar masses were found when using LDIMS. Isoform b was the most abundant and its molecular mass matched the molecular mass of 6893 calculated from the sequence of DATI. The mass differences between a and b, b and c, c and d, and d and e were equal to 87, which corresponds to Ser. Isoform a might not have the N-terminal Ser present in isoform b, while the other additional Ser residues might comprise a row localized in the C- or N-terminal. The appearance of all these isoforms could result from posttranslational N- and C-terminal processing.