Purification and physicochemical characterization of a new rat plasma proteinase inhibitor, alpha 1-inhibitor III.

A three-stage procedure was used to isolate an additional proteinase inhibitor in rat plasma tentatively called alpha 1-inhibitor III. A 20% yield was obtained after two successive gel filtrations on Ultrogel Ac-A. 33-4 followed by an ion-exchange chromatography on DEAE-cellulose. This method was chosen since it permits further study of the enzyme binding properties of the isolated molecule. The purified material was first controlled to retain an inhibiting capacity towards serine proteinases using bovine chymotrypsin. The isolated molecule has an apparent molecular weight of 215 000, a pI of 4.65, an E1%1cm, 280 nm of 7.50 and a sedimentation coefficient of 8.6 S. It contains approx. 15% carbohydrates and is made up of a single peptidic chain. Study of the periodic structure by circular dichroism has demonstrated a low alpha-helix content (4--5%) whereas the beta-sheet conformation accounts for approx. 30% of the peptidic moiety. Tryptophan residues have been shown to be mainly responsible for the molecular fluorescence most of them being non-accessible to the solvent since only 25% of the tryptophanyl fluorescence was quenched in presence of I.