Properties of a Trypsin Inhibitor from Job’s-tears (Coix lacryma-jobiL. var.Ma-yuenStapf)

The amino acid sequence of a cereal Bowman‐Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma‐jobi L.)

“…The inhibitor also behaved anomalously on SDS-PAGE giving a single narrow band of apparent molecular mass 11 kDa in the absence of reducing agent, and broader more diffuse bands (17-20 kDa) after treatment with 2-mercaptoethanol. The higher values of molecular mass observed after treatment with reducing agents probably resulted from reoxidation to form inter-chain disulphide bonds and confirm the observations of Ohtsubo et al [24] that the Coix inhibitor had a similar mobility to cytochrome c (12.5 kDa) on SDS-PAGE, but eluted after this protein during gel filtration on Sephadex G-100. The complete amino acid sequence of the Coix trypsin inhibitor is shown in fig.2 together with details of the overlapping peptides from which it was deduced.…”

“…The sequence contained 64 amino acid residues, corresponding to 7262Da, and was in good agreement with the amino acid composition of the protein. The composition calculated from the sequence is also very similar to that reported for the Coix 12 kDa trypsin inhibitor by Ohtsubo et al [24] when the latter is recalculated for a protein of 7 kDa. Fig.3 shows the clear sequence homology which exists between the Coix trypsin inhibitor, rice bran inhibitor [23], wheat germ inhibitors [22] and Bowman-Birk proteinase inhibitors from a number of legumes [31][32][33][34][35][36][37].…”

“…Application of the preliminary steps of the purification method of Ohtsubo et al [24] followed by preparative reverse-phase HPLC yielded two peaks of trypsin inhibitory activity, one major and one minor, together with a number of other protein peaks ( fig.l). The protein in the major peak (TI 1) was eluted at an acetonitrile concentration of 24°70 which is very similar to the value reported for the wheat germ trypsin inhibitors of the 7 kDa type II [22].…”

“…The rice bran inhibitor on the other hand appears to ex-ist only in the inhibitor I (14.5 kDa duplicated) form [23]. Other workers have recently reported the isolation of a 12 kDa trypsin inhibitor from seeds of the cereal crop Jobs' tears (Coix lachryma-jobO [24], which they suggest might be similar to the inhibitor I forms in wheat germ and rice bran.…”

“…The preliminary steps of the purification of the trypsin inhibitor from defatted flour of seeds of Jobs' tears (extraction with 50 mM NaH2PO4-K2HPO4 buffer, pH 7.5, containing 0.1 M NaCI; heat treatment; precipitation with (NH4)2SO4 (40-95070); ion-exchange chromatography on DEAE-Sepharose CL6B; and gel-filtration on Sephadex G-75) were carried out as described by Ohtsubo et al [24].…”