Isoelectric focusing of basic proteases in immobilized pH gradients

By exploiting a new, alkaline immobilized pH gradient spanning the pH 10-11 interval, it has been possible to focus and to detect, by in situ zymogramming with cellulose acetate foils impregnated with fluorogenic substrates, 2 alkaline proteases, namely elastase and trypsin. Elastase gave a sharp array of 3 bands, with the following pIs (at 10 degrees C): 10.60 (major component), 10.53 (intermediate species) and 10.45 (minor isoform). Trypsin was resolved into 2, about equally abundant, species having pIs of 10.70 and 10.53. However, the latter enzyme gave smears in between these 2 forms and also anodic to the lower pI species. As hydrophobic interaction with the Immobiline matrix was excluded, it is suggested that these smears represent product of auto-digestion due to the very alkaline pH during the focusing process.