|Fontanini, Debora - UNIVERSITY OF WISCONSIN|
|Jarvinen, Marita - VTT, FINLAND|
|Pekkarinen, Anja - VTT, FINLAND|
Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 10, 1998
Publication Date: N/A
Interpretive Summary: Endoproteinases are enzymes that degrade proteins by breaking the bonds between their amino acids. They are essential enzymes for many normal cellular processes occurring in plants, animals, and microorganisms. They are also important in industrial processes such as the malting of barley. It thus often becomes necessary to measure the activities of these enzymes that are present in various systems. In the past, there has not been a good, simple way to measure their activities. In this paper, we report an improved and simple method that we have developed for measuring the activities of these endoproteinases. The method consists of preparing solutions of the proteins gelatin and/or azogelatin, allowing the enzymes to degrade these proteins, removing the undegraded protein with either trichloroacetic acid (azogelatin) or isopropanol (gelatin) precipitation, and measuring the amount of degraded protein that is left in solution. The method is shown to work with enzymes from papayas, barley and from the fungus Fusarium. In general, the assay using azogelatin is preferable to that with gelatin, because it is more sensitive and less affected by contaminants. This method will allow researchers and industry personnel to more easily and accurately measure the various endoproteinases present in a variety of systems.
Technical Abstract: Endoproteinase assays with gels containing incorporated gelatin have shown that this substrate is exceptional 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, a wavelength 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.