Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape(Brassica napus var. oleifera) seed

A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has bee n isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5- oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer t han the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P-1-P-1' reactive site bond (where residues forming the reactive site have been ide ntified as P-n...P-1 and P-1'...P-n', where P-1-P-1' is the inhibitor sciss ile bond) has been identified at position Arg21-Ile22. The inhibitor disulp hide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42- Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RT T-III isoinhibitors is reminiscent of that found in a number of toxins (e.g . erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that foun d in epidermal growth factor domains. Preliminary H-1-NMR data indicates th e presence of alpha alpha NOEs and 3J alpha NH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional str ucture of the RTI-III isoinhibitors may be reminiscent of that of toxins an d epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equ ilibrium constant for RTI-III isoinhibitors binding to bovine P-trypsin and bovine a-chymotrypsin are 3.3 x 10(9) M-1 and 2.4 x 10(6) M-1, respectivel y, at pH 8.0 and 21.0 degrees C. The serine proteinase: inhibitor complex f ormation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the l ow molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.