Characterization of five new low-molecular-mass trypsin inhibitors from white mustard (Sinapis alba L.) seed

Five new low-molecular-mass trypsin inhibitors belonging to the RTI/MTI-2 f amily were identified from white mustard (Sinapis alba L.; MTI-2) seed. Pur ified MTI-2 consisted of a peptide mixture, displaying Ile or Arg at positi on 43, Trp or kynurenine (Kyn) at position 44, and C-terminal ragged ends. The occurrence of Ile or Arg at position 43 suggested that MTI-2 inhibitors originated from different genes. The presence of 5-oxo-proline (pyroglutam ic acid; 5-oxoPro1) and Kyn44 reflected post-translational processing of th e serine proteinase inhibitor. MTI-2 showed approximate to 70% amino-acid i dentity with low-molecular-mass trypsin inhibitors isolated from oil rape ( Brassica napus var. oleifera; RTI-III) seed and with serine proteinase inhi bitors mapped in Arabidopsis thaliana chromosome II (ATTI). Furthermore, MT I-2 was homologous to brazzein, the sweet-tasting protein from Pentadipland ra brazzeana Baillon fruit (approximate to 30% amino-acid identity). Althou gh snake-venom toxins showed a low amino-acid identity (< 20%) with MTI-2, RTI-III, and ATTI, some structurally relevant residues were conserved. The disulfide bridge pattern of MTI-2 (Cys5-Cys27, Cys18-Cys31, Cys42-Cys52, an d Cys54-Cys57) corresponded to that of RTI-III and of snake-venom toxins, b eing different from that of brazzein. Therefore, protein similarity might b e attributable to the three-dimensional arrangement rather than to the amin o-acid sequence. Values of K-a for MTI-2 binding to bovine beta-trypsin (tr ypsin) and bovine alpha-chymotrypsin were 6.3 x 10(9) M-1 and 2.0 x 10(6) M -1, respectively, at pH 8.0 and 21.0 degrees C. Moreover, values of k(on) f or MTI-2 binding to trypsin and of k(off) for the dissociation of the serin e proteinase:inhibitor complex were 5.6 x 10(5) M-1.s(-1) and 8.9 x 10(-5) M-1.s(-1), respectively, at pH 8.0 and 21.0 degrees C. Despite the heteroge neity of the purified inhibitor peptide mixture, the inhibition properties of the different MTI-2 inhibitors were indistinguishable.