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

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Abstract
A new low‐molecular‐mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil‐rape (Brassica napus var. oleifera) seed, designated 5‐oxoPro1‐Gly62‐RTI‐III. The 5‐oxoPro1‐Gly62‐RTI‐III isoinhibitor is longer than 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 P1‐P1′ reactive site bond (where residues forming the reactive site have been identified as Pn…P1 and P1′…Pn′, where P1‐P1′ is the inhibitor scissile bond) has been identified at position Arg21‐Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5‐Cys27, Cys18‐Cys31, Cys42‐Cys52 and Cys54‐Cys57. The disulphide bridge arrangement observed in the RTI‐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 found in epidermal growth factor domains. Preliminary 1H‐NMR data indicates the presence of ααNOEs and 3JαNH coupling constants, typical of the β‐structure(s). These data suggest that the three‐dimensional structure of the RTI‐III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three‐finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI‐III isoinhibitors binding to bovine β‐trypsin and bovine α‐chymotrypsin are 3.3 × 109 m−1 and 2.4 × 106 m−1, respectively, at pH 8.0 and 21.0 °C. The serine proteinase : inhibitor complex formation is a pH‐dependent entropy‐driven process. RTI‐III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low 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.