Cathepsin D: Rapid Isolation by Affinity Chromatography on Haemoglobin‐Agarose Resin

The intracellular proteinase, cathepsin D, has been isolated from bovine spleen and thymus, in times as short as several hours, by affinity chromatography of partially purified and unpurified tissue extracts on haemoglobin‐agarose resin. After subsequent separation from an inactive higher‐molecular‐weight protein by gel permeation chromatography, the enzyme from both tissues shows three dominant proteolytically active bands on gel electrophoresis at pH 4.3 and 9.5: this proteolytic activity is completely inhibited by the acid‐proteinase inhibitor, pepstatin. These enzyme electrophoretic patterns were approximately constant with variation in isolation time and with various preliminary purification procedures. The enzyme shows only traces of polypeptides other than that with an apparent molecular weight of 42000 on dodecylsulphate electrophoresis, in contrast to the enzyme prepared by conventional methods, which contains considerable amounts of smaller polypeptides. This difference in polypeptide composition is shown to be the result of degradation of the enzyme in vitro during isolation by the previously published methods.