FIGURE 4.

Purification of two types of ASP. A, the crude sample was loaded onto a hydroxyapatite column equilibrated with 1 mm phosphate buffer (pH 7.2). After washing the column, the adsorbed material was eluted first with a linear gradient of 1–120 mm phosphate buffer and then with a gradient of 120 to 600 mm phosphate buffer. B, fractions presenting proteolytic activity are indicated by the horizontal bar in A. The active fractions from A were collected and concentrated 20×, after which the sample was diluted 3-fold with water and loaded onto a hydroxyapatite column in a HPLC system. After washing, the adsorbed material was eluted with a linear gradient of 1–100 mm phosphate buffer. Two peaks, designated fractions X and Y, expressed proteolytic activity.