Purification and characterization of a cysteine proteinase inhibitor (Cystatin) from seeds of foxtail millet, Setaria italica

A cysteine proteinase inhibitor was purified from the extract of seeds of f oxtail millet, Setaria italica, to an electrophoretically homogeneous prote in by heat treatment, salting-out, ion-exchange chromatography on DEAE-Seph arose CL-6 B, gel filtration on Sephadex G-50, and chromatofocusing on PBE 94. The inhibitor (FMCPI) was a single polypeptide with a molecular weight of 12 000 and an isoelectric point of 5.2. It possessed the amino acid comp osition characterized by fairly high contents of aspartic acid, glutamic ac id, and alanine and by no half-cystine, FMCPI was relatively thermostable s ince it maintained more than 50% of the original activity after treatment o f 100 degrees C for 20 min at pH 2 or 7. However, the inhibitor almost lost its whole activity by the above treatment at pH 10. FMCPI inhibited papain in a 1 : 1 protein molar ratio : the K-i value was 2.4 x 10(-11) M. Comple x formation between FMCPI and papain derivatives demonstrated that the inhi bitor was capable of combining with even a papain molecule without the cata lytic ability.