SIMPLIFIED ENDOPROTEINASE ASSAYS USING GELATIN OR AZOGELATIN

Endoproteinase assays with gels containing incorporated gelatin have s hown that gelatin is an exceptional substrate Tor studying enzymes fro m 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 hydro lysis reactions after the substrate has been precipitated with trichlo roacetic acid). In this paper we demonstrate that gelatin can be used for such analyses by using isopropanol as precipitating reagent. Alter natively, azogelatin, which is precipitated by trichloroacetic acid, c an be used. Azogelatin also serves as a very good substrate. One probl em with using gelatin (or any nonderivatized protein) as substrate for measuring the activity of crude enzyme preparations is that protein c ontaminants in the enzyme preparation are hydrolyzed, The resulting pe ptides are impossible to differentiate from those released from the ge latin substrate. This problem is obviated when azogelatin is used, sin ce its peptides are detected at 440 nm, where nonderivatized peptides do not absorb. Unlike some azo-derivatized proteins, azogelatin is sol uble from pH 3.0 to 9.0. This, together with the fact that it is hydro lyzed by many different endoproteinases, makes it suitable for many ap plications.