Crystallization and preliminary X-ray diffraction studies on a trypsin/chymotrypsin double-headed inhibitor from horse gram

Abstract

The Bowman-Birk family of proteinase inhibitors from seeds of leguminous plants usually have a molecular mass of 8000 to 10,000 Da. Horse gram (Dolichos bifloros or Macrotyloma uniflorum) seeds contain an unusual Bowman-Birk inhibitor of molecular mass 15,500 Da active against both trypsin and chymotrypsin. In order to elucidate its three-dimensional structure, its evolutionary relationship with the more usual Bowman-Birk inhibitors and to study the structure-function properties, this inhibitor has been purified and crystallized. The purified protein crystallizes easily under a variety of conditions in different crystal forms. Crystals obtained by precipitating the protein (3 to 5 mg/ml in 50mM Tris·HCl (pH 8·0)) with 5% ammonium sulphate and 2 to 3% PEG 4000 appear to be suitable for structure determination by X-ray diffraction. The crystals belong to cubic space group P2₁3 (a=110·81 Å) and diffract X-rays to beyond 3·0 Å resolution.