Improved Purification Process of β- and α-Trypsin Isoforms by Ion-Exchange Chromatography

The purpose of this work was to improve the separation and yield of pure βand α-trypsin isoforms by ionexchange chromatography and to characterize some physical–chemical properties of these isoforms. Purification of trypsin isoforms was performed by ion-exchange chromatography in 0.1 mol/L tris-HC buffer, pH 7.10 at 4C. The sample loading, salt concentration, flow rate and pH of mobile phase were varied to determine their effects on the resolution of the separation. The resolution was optimized mainly between βand α-trypsin. Pure isoforms were obtained by chromatographying 100 mg of commercial trypsin during seven days, yielding 51 mg of high purity βtrypsin and 13 mg of α-trypsin partially pure, with small amounts of contaminating of ψ-trypsin. Thus, time and resolution of purification were optimized yielding large amounts of pure active enzymes that are useful for several research areas and biotechnology.