ISOLATION, CHARACTERIZATION, AND PROPERTIES OF A TRYPSIN-CHYMOTRYPSININHIBITOR FROM AMARANTH SEEDS

A trypsin-chymotrypsin inhibitor was isolated from the seeds of amaran th-a highly nutritious protein source. The purification of the inhibit or (AmI) was carried out by affinity chromatography on trypsin-Sepharo se and by HPLC. AmI is a single-chain protein of 8 kD, as determined b y electrophoresis on SDS-polyacrylamide gels and by gel exclusion on S ephadex G-50 column. It is stable at neutral and alkaline pH and is re latively thermostable. AmI inhibits trypsin and chymotrypsin from the digestive system of insects such as Tribolium castaneum and Locusta mi gratoria, supporting the hypothesis that inhibitors may have evolved a s defense mechanisms of seeds against insects. AmI lost its inhibitory activities when submitted to limited proteolysis with trypsin, while limited proteolysis with chymotrypsin had almost no effect. The partia l amino acid sequence of 45 amino acids from the amino terminus of AmI differs significantly from the known sequences of legume-seed and cer eal-grain protease inhibitor families. Differences in the chemistry at the inhibitory site(s) and in the amino acid sequence of AmI in compa rison to that of other cereal and legume inhibitors suggest that AmI i s a member of a new family of serine protease inhibitors. AmI was foun d to inhibit the anchorage-independent growth of MCF-7 breast cancer c ells, suggesting that AmI may have anticarcinogenic activity.