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Detection of Amoebal Proteases

The so called Zymogram technique has been used by many workers to identify proteases from amoeba (mainly Acanthamoeba where the extra cellular protease is implicated in corneal degradation).We have modified this method for use in the "Mini-Protean" gel system by BioRad.  From the methods of Heussen & Dowdle, 1980, Kleiner & Stetler-Stevenson 1994, Mitra et al (1994).  It is convenient to use Fish Skin Gelatin (Sigma G-7765).

1).  It is important that b-ME and DTT be kept out of any sample buffer for which protease activity is to be detected.  Samples 
must therefore be made from non-reducing sample buffer:-

                        Working Concentration            Stock                           100mls
                        20% Glycerol                                   -                                   20g
                          5% SDS                                        -                                    5g
                        0.4M Tris pH6.8                              0.5M                            80mls

                        (Bromephenol Blue added to give colour)

2).  Make up gel mix including the gelatin at a suitable concentration (0.1%).  The following is for
 
the BioRad ôminiproteanö system and is enough for two gels:-

            Stock solutions                        8%                   10%                 12%                 14%     
            Distilled Water                          4.64mls            4.31mls            3.31mls            2.64mls
            1.5M Tris pH8.8                        2.5mls              2.5mls              2.5mls              2.5mls
            10% SDS                                 100Ál                100Ál                100Ál                100Ál
            45% Fish skin gelatin                  22Ál                  22Ál                  22Ál                  22Ál
            30% Acrylamide mix                2.67mls            3.0mls              4.0mls              4.67mls
            10% APS                                 50Ál                  50Ál                  50Ál                  50Ál
            TEMED                                   20Ál                  20Ál                  20Ál                  20Ál      

            Total                                        10mls               10mls               10mls               10mls

3).  Run gel at 200v as usual, and then incubate the gel with 2.5% Triton X-100 ; 50mM Tris-HCl pH 7.0  
for 1 hour and then overnight at in 50mM Tris-HCl pH 7.0, 2mM CaCl2 at room temperature. 

Coomassie blue Staining 

After overnight incubation, stain with Coomassie as normal:-


            0.1%                Coomassie R-250
            40%                 Methanol
            10%                 Acetic acid


Rock the gel in the stain for about one hour, then destain by briefly washing the gel in tap water and then rocking in destain solution:-


            10%                 Acetic acid
            5%                   Methanol

Figure 1. Equal quantities of material from culture supernatants of various Acanthamoeba were added to the wells of a 10% gel containing 0.1% fish skin gelatin. The gel was incubated in triton buffer for 1 hour to remove SDS. Staining was by usual Coomassie blue method.
Another method that is useful for certain applications utilizes the fact that unprocessed film has a gelatinous backing whose digestion can be assayed (Cheung et al, 1991).  This method is especially useful for detecting pH optima (Buckwold et al, 1999) and inhibitors.
Figure 2. This method is simply to take a strip of untreated film and to pipet on equal volumes of solution containing the protease and to incubate at a suitable temperature in a moist environment (so that the drop does not evaporate.  After a suitable time (try an hour at first?) the film is gently washed under flowing water and the gelatin coat very gently but equally rubbed to reveal activity.
  Back to Amoeba methods   Acanthamoeba proteases & pathogenesis
References:-

Alfieri, S.C., Correia, C.E.B., Motegi, S.A., & Pral, E.M.F. (2000). "Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites." J.Parasitol. 86, 220-227.

Buckwold, V. E., Alvarado, M., Carraso, R. M. & Amils, R. (1999) A method for the determination of the pH optima of proteases using unexposed photographic film., Anal.Biochem. 267, 420-421.

Cao, Z., Jefferson, D. M. & Panjwani, N. (1998) Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba, J.Biol.Chem. 273, 15838-15845.

Cheung, A. L., Ying, P. & Fischetti, V. A. (1991) A method to detect proteinase activity using unprocessed X-ray films., Anal.Biochem. 193, 20-23.

He, Y.-G., Niederkorn, J. Y., McCulley, J. P., Stewart, G. L., Meyer, D. R., Silvany, R. & Dougherty, J. (1990) In vivo and in vitro collagenolytic activity of Acanthamoeba castellanii, In.Oph.Vis.Sci. 31, 2235-2240.

Heussen, C. & Dowdle, E.B. (1980) Anal.Biochem. 102;196-202.

Hong, C.-Y., Kong, H.-H., Ock, M.-S., Kim, I.-S., & Chung, D.-I. (2001). "Isolation and characterization of a cDNA encoding a subtilisin-like serine proteinase (AhSUB) from Acanthamoeba healyi". Mol.Biochem.Parasitol. 111, 441-446.

Kleiner, D.E. & Stetler-Stevenson, W.G. (1994) Anal.Biochem. 218; 325-329.

Mitro, K., Bhagavathiammai, A., Zhou, O.-M., Bobbett, G., McKerrow, J. H., Chokshi, R., Chokshi, B. & James, E. R. (1994) Partial characterization of the proteolytic secretions of Acanthamoeba polyphaga, Exp.Parsitol. 78, 377-385.

Na, B.-K., Kim, J.-C. & Song, C.-Y. (2001) Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii., Microbial Pathogenesis. 30, 39-48.

 
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