Submitted to: Analytical Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2004
Publication Date: 1/31/2005
Citation: Vinokurov, K.S., Oppert, B.S., Elpidina, E.N. 2005. An overlay technique for postelectrophoretic analysis of proteinase spectra in complex mixtures using p-nitroanilide substrates. Analytical Biochemistry 337: 164-166. Interpretive Summary: Current methods of proteinase detection have some inherent problems, such as low sensitivity, retention of activity, and lack of response to inhibitors. We improved these methods by developing a new overlay technique, which involved impregnation of a membrane with substrate to detect proteinase activity. In addition, we determined that addition of inhibitors after electrophoresis provided superior analysis of inhibitor activity. These improvements will lead to enhanced analysis of proteinase activity in complex mixtures.
Technical Abstract: A simple method with improved sensitivity is described for the detection of different groups of proteinases in complex mixtures separated by polyacrylamide gel electrophoresis and overlaid with a nitrocellulose membrane impregnated with specific p-nitroanilide (pNA) substrates. A drawback of the direct testing of proteinase activity in a polyacrylamide gel with chromogenic peptide substrates is the high degree of diffusion of low molecular mass reaction products. Others have proposed the transfer of proteinases to a nitrocellulose membrane after electrophoresis in a 1% agarose gel with subsequent detection of activity after incubation with pNA substrate solutions. Later this technique was transferred to SDS-PAGE by means of electrotransfer and was actively used for the characterization of digestive proteinases in insects. However, conservation of proteinase activity during electrotransfer is time and temperature dependent, and enzyme activity can be lost after 1 h of electrotransfer, even for relatively stable serine proteinases. In this report, analysis by an overlay of nitrocellulose impregnated with pNA substrates on a polyacrylamide gel was more effective than electrotransfer for the preservation of proteinase activity in complex mixtures. Instead of SDS-PAGE, native PAGE at neutral pH in Hepes-imidazol buffer was used, which can be performed in two directions, for anionic and cationic proteins. This electrophoretic method retained proteolytic activity, especially for cysteine proteinases, which may be reduced or even irreversibly lost in denaturing conditions and/or alkaline pH in SDS-PAGE. Specific inhibitors used for characterization of proteinases are added to proteinase mixtures typically before or after PAGE. We demonstrate that the addition of inhibitors after PAGE was more reliable and excluded the possible breakdown of proteinase-inhibitor complexes during electrophoresis. The methods were developed with preparations of digestive proteinases from the yellow mealworm (Tenebrio molitor) larval anterior midgut (AM), where the pH of contents is 5.6 and cysteine proteinases are predominant, and the posterior midgut (PM) where the pH of contents is 7.9 and serine proteinases are predominant.