Isolation and Amino Acid Sequence of a Serine Proteinase Inhibitor from Common Flax (Linum usitatissimum) Seeds

LUTI (Linum usitatissimum trypsin inhibitor), a member of the potato inhibitor I family, has been isolated from seeds of flax by ethanol fractionation, ion exchange chromatography on CM‐Sephadex C‐25, affinity purification on immobilized methylchymotrypsin (α‐chymotrypsin in which His 57 has been converted to 3‐methylhistidine) in the presence of 5 M NaCl, and finally by reversed‐phase HPLC. The 7655 Da inhibitor consists of a single polypeptide chain of 69 residues with one disulfide bridge. The molecule is acetylated at the N terminus. Its primary structure has been determined after limited proteolysis of the native molecule with trypsin at the reactive site, cleavage with cyanogen bromide or arginyl endopeptidase (Arg‐gingipain), and alcoholytic deacetylation of the N‐terminally blocked serine. The association constants (Ka) of LUTI with bovine β‐trypsin and α‐chymotrypsin are 3.58×1010 M−1 and 5.02×105 M−1, respectively. High NaCl concentration (3 M) increased the association constant of LUTI with α‐chymotrypsin to 6.64×107 M−1. To our knowledge, LUTI is the first serine‐proteinase‐type inhibitor isolated from a plant of the Linaceae family. Affinity chromatography on immobilized methylchymotrypsin in the presence of 5 M NaCl was a key step in the purification of LUTI (Linum usitatissimum trypsin inhibitor), a member of the potato inhibitor I family of serine proteinases. The two protein peaks obtained upon elution with water and 0.1 n HCl (I and II, see picture) were shown to have identical properties. The primary structure of LUTI (69 amino acids, Mr=7655 Da) and its association constants (Ka) with some serine proteinases were determined.