The nature and stability of trypsin inhibitor isoforms in New Zealand grown pea (Pisum sativum L.) cultivars

Seventeen New Zealand grown, spring and winter pea cultivars were assayed for trypsin inhibitor activity, tannin and protein content. Trypsin isoinhibitors for ten of the cultivars were extracted using hydrochloric acid and purified by size exclusion chromatography and anion exchange chromatography. Isoelectric points and molecular masses were determined by isoelectric focusing and SDS-PAGE, respectively. Trypsin inhibitor activity (TIA) in New Zealand grown pea cultivars ranged from 0.33 to 0.75 TIU/mg DM; much lower than reported for most European cultivars. Phenotypic characteristics in relation to trypsin inhibitor activity and condensed tannin content were observed. In general, marrowfat peas contained the highest levels of trypsin inhibitor activity and soluble protein whereas maple peas had low levels of soluble protein and trypsin inhibitor activity. Winter-sown and smooth-seeded peas exhibited greater levels of TIA than their spring-sown or wrinkled-seeded counterparts. Condensed tannins were most abundant in maple peas (purple-flowered varieties) which had brown coloured hulls. This indicates that condensed tannin levels may be responsible for darker coloured hulls. However, increased tannin content was also observed in the wrinkled pea, Bolero. Total trypsin inhibition in New Zealand pea cultivars was found to be negatively correlated to tannin content. Thus it appears that the presence of tannins does not contribute to trypsin inhibition. Both maple and wrinkled varieties exhibited decreased levels of TIA and increased tannin levels, indicating that increased levels of one anti nutritional factor may result in decreased levels of the other. During the separation of trypsin inhibitors by size exclusion chromatography, trypsin-like activity was also observed, indicating the possible presence of an endogenous trypsin or similar protease in the pea seed. Between 6 and 10 trypsin isoinhibitors were observed in each cultivar; with twelve different isoinhibitors in total. Isoelectric points ranged from 4.6-7.6. Cultivars with similar gene pools were found to exhibit the same number and/or type of isoinhibitors. The isoelectric points for the individual pea seed trypsin isoinhibitors of the marrow fat cultivar Primo were pI 5.9 & pI 6.3, pI 4.9 and pI 6.8, according to elution from the anion exchange column. Molecular masses of the proteins were observed at ~6.5, ~14 and ~21 kDa. These observations confirm previous reports on the similarity between pea protease inhibitors and the Bowman-Birk family of inhibitors. These protein bands may represent the inhibitors in single as well as multiple forms (eg, dimers, trimers and tetramers). After soaking and cooking, pea cultivars exhibited an average reduction in trypsin inhibitor activity of 77.5%. It appears that the residual TIA after heat treatment is negatively correlated to the amount of TIA in the raw seed; ie, the greater the TIA level in the raw pea seed, the greater the proportional reduction or inactivation of TIA after heat treatment. Isoinhibitors responded differently to heat treatment. In all ten cultivars, only three of the original isoinhibitors, with isoelectric points of 5.1, 5.9 and 7.6, remained indicating increased heat stability over the other isoforms. Therefore, these three isoforms would be undesirable to select for in breeding programmes.