Article

Journal of Protein Chemistry

, Volume 20, Issue 8, pp 625-632

Purification and Characterization of a New Trypsin Inhibitor from Dimorphandra mollis Seeds

  • Gláucia C. MelloAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP)
  • , Maria Luiza V. OlivaAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP)
  • , Joana T. SumikawaAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP)
  • , Olga L. T. MachadoAffiliated withCentro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense
  • , Sérgio MarangoniAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP)
  • , José C. NovelloAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP)
  • , Maria Lígia R. MacedoAffiliated withDepartamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP) Email author 

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Abstract

A second trypsin inhibitor (DMTI-II) was purified from the seed of Dimorphandra mollis (Leguminosae-Mimosoideae) by ammonium sulfate precipitation (30–60%), gel filtration, and ion-exchange and affinity chromatography. A molecular weight of 23 kDa was estimated by gel filtration on a Superdex 75 column SDS-PAGE under reduced conditions showed that DMTI-II consisted of a single polypeptide chain, although isoelectric focusing revealed the presence of three isoforms. The dissociation constant of 1.7 × 10−9 M with bovine trypsin indicated a high affinity between the inhibitor and this enzyme. The inhibitory activity was stable over a wide pH range and in the presence of DTT. The N-terminal sequence of DMTI-II showed a high degree of homology with other Kunitz-type inhibitors.

Dimorphandra mollis Mimosoideae trypsin inhibitor N-terminal sequence Kunitz family