Chymotrypsin Inhibitor I from Potatoes: LARGE SCALE PREPARATION AND CHARACTERIZATION OF ITS SUBUNIT COMPONENTS

Abstract

Abstract A method for the large scale preparation of chymotrypsin Inhibitor I from potato tubers is described. The method utilizes the inhibitor's stability to acid at elevated temperatures and its dissociation in 4 m guanidine hydrochloride into subunits (protomers) to produce the pure protein with a 47% recovery and a 40-fold purification. Ion exchange chromatography on sulfoethylcellulose in the presence of 0.1 m formic acid in 8 m urea resolves Inhibitor I protomers into two major and two minor types. After reassociation to the tetramer form by dilution, one of the two major rechromatographed protomers was shown to be a powerful inhibitor of both chymotrypsin and trypsin, whereas the other strongly inhibits chymotrypsin but only weakly inhibits trypsin. All four purified protomers resolved from Inhibitor I can be reassociated either individually or hybridized with each other to form tetrameric isoinhibitors. Tetrameric Inhibitor I species prepared from each of the four protomeric types all have an NH2-terminal glutamic acid. However, they differ quantitatively from each other in amino acid composition, reactivity with chymotrypsin and trypsin, digestibility by pepsin, and electrophoretic mobility. Tetrameric Inhibitor I in potatoes is therefore a heterogeneous mixture of isoinhibitors whose properties reflect those of its individual protomers. The molecular weights of tetrameric Inhibitor I and its complex with chymotrypsin were re-evaluated. The molecular weight of Inhibitor I was found to be 39,000 ± 2,000, and that of its complex saturated with chymotrypsin, 140,000 ± 4,600.