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

Paolo Ascenzi, Margherita Ruoppolo, Angela Amoresano, Piero Pucci, Roberto Consonni, Lucia Zetta, Stefano Pascarella, Fabrizio Bortolotti, Enea Menegatti

Research output: Contribution to journalArticlepeer-review


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 P(n)...P1 and P1'...P(n)', 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 x 109 M-1 and 2.4 x 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.

Original languageEnglish
Pages (from-to)275-284
Number of pages10
JournalEuropean Journal of Biochemistry
Issue number1
Publication statusPublished - Apr 1 1999


  • H-NMR investigation
  • Amino acid sequence determination
  • Brassica napus var. oleifera
  • Disulphide bridge location
  • Inhibitory properties
  • Oil-rape seed
  • Trypsin inhibitors

ASJC Scopus subject areas

  • Biochemistry


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