Simplified endoproteinase assays using gelatin and azogelatin

Endoproteinase assays with gels containing incorporated gelatin have shown that gelatin is an exceptional substrate for studying enzymes from different sources. However, due to its solubility in trichloroacetic acid, gelatin is not suited to "in-solution" assays carried out in the classic manner (by reading the absorbance of supernatants of hydrolysis reactions after the substrate has been precipitated with trichloroacetic acid). In this paper we demonstrate that gelatin can be used for such analyses by using isopropanol as precipitating reagent. Alternatively, azogelatin, which is precipitated by trichloroacetic acid, can be used. Azogelatin also serves as a very good substrate. One problem with using gelatin (or any nonderivatized protein) as substrate for measuring the activity of crude enzyme preparations is that protein contaminants in the enzyme preparation are hydrolyzed. The resulting peptides are impossible to differentiate from those released from the gelatin substrate. This problem is obviated when azogelatin is used, since its peptides are detected at 440 nm, where nonderivatized peptides do not absorb. Unlike some azo-derivatized proteins, azogelatin is soluble from pH 3.0 to 9.0. This, together with the fact that it is hydrolyzed by many different endoproteinases, makes it suitable for many applications.