meal or protein, were calculated. One trypsin inhibitor unit was defined as that which gives a purchase 146368-16-3 reduction in absorbance at 410 nm of 0.01, relative to trypsin control reactions, in 10 min in a defined assay volume of 10 mL. One chymotrypsin inhibitor unit was defined as that which gives a reduction in absorbance at 410 nm of 0.01, relative to chymotrypsin control reactions, in 16 min in a defined assay volume of 10 mL. Finely ground meal was added to 3 mL of 50 mM HCl and stirred for 2 h at 4. The extracts were centrifuged at 15,000 g for 15 min and supernatants were dialysed extensively against 25 mM sodium acetate buffer, pH 4.4, at 4. The protein extracts were fractionated on a MonoS 5/50 GL cation-exchange column. The elution was monitored at 280 nm and 0.5 mL fractions were collected. TIA measurements of eluted proteins were carried out in flat-bottom microtitre plates and assay products measured at OD405 nm, as previously Ganetespib described. CIA evaluation of fractionated proteins was carried out, using N-benzoyl-L-tyrosine ethyl ester , as previously described. The oligomerization pattern of pea TI was investigated using albumin proteins extracted from cv. Cameor, wild-type and E109K mutant seeds. Freeze-dried albumins were filtered through a 0.22 ��m filter and 2mL samples loaded onto a HiPrep 26/60 Sephacryl S-100 HR column in 50 mM Tris- HCl, pH7.5. Four replicates samples of each line were analysed. Size calibration of the column separation was based on three sets of standards, each in 2mL buffer. Fractions containing TIA were identified, corresponding to three peaks ; these were individually collected, dialysed against distilled water, freeze-dried and dissolved in 3 mL of 50 mMsodium acetate pH 4.4 for analysis by cation-exchange chromatography as above. Following cation-exchange chromatography of some lines, samples from chromatographic peaks having TIA were precipitated with acetone, dissolved in NuPAGE lithium dodecyl sulphate sample buffer , and the proteins separated by electrophoresis on Novex 12 Bis-Tris pre-cast gels as above. Bands were excised from Colloidal Blue -stained gels and subjected to in-gel trypsin digestion. Peptide fragments from di