These data are in agreement with earlier studies of recombinant proteins representing TI1 and TI2, where TI1 isoforms showed greater inhibition of chymotrypsin than did TI2, hypothesised to reflect the active site sequences , in agreement with tyrosine being a more effective amino acid at the P1�� position. As far as trypsin inhibition is concerned, there is some evidence to suggest that TI2 may also be less effective than TI1 , in agreement with the lower activity remaining in the C77Y mutant. It is also possible, however, that dimers of TI1 and TI2 contribute significantly to activity and that a loss of functional TI1 leads to a disproportionate loss of overall activity; this theory is not supported by analysis of the E109K mutant, where a reduction in the extent to which oligomers are formed did not diminish TIA or CIA significantly. The expression of TI1 and TI2 genes was approximately equal during seed development at stages when protein is being maximally synthesised; the slightly earlier expression of TI2 observed during seed development might reflect the genomic organisation of the two genes, which may be concluded not to influence their relative expression to any great degree. The E109K mutant showed marginal decreases in both TIA and CIA , consistent with the lack of direct participation of the carboxy-terminal domain in interactions with trypsin or chymotrypsin. Here, however, it was predicted that the charge variation might impact on interactions between monomers. The data obtained from size-exclusion chromatography indicated that the E109K mutation had a profound effect on the participation of TI1 in oligomeric forms of TI. The ARN-509 composition of eluted peaks from size-exclusion chromatography of the E109K mutant indicated that, whereas peak A from wild type consisted of unprocessed forms of TI1 and TI2, the TI1 unprocessed form was absent from peak A in the mutant E109K , where peak A was reduced in proportion overall. These data are supported by a suggested electrostatic interaction between the ��-amino group of K58 of one monomer and a negatively charged residue at the carboxy-terminal of the second monomer in the 1446712-19-1 format
