**1. Introduction**

264 Gel Electrophoresis – Advanced Techniques

Nikolova, P. & Ward, O. P. (1991). Production of L-phenylacetyl Carbinol by Biotransformation:

Penning, T. M., Burczynski, M. E., Hung, C. F., McCoull, K. D. Palackal, N. T. & Tsuruda, L.

Pfeiffeer-Guglielmi, B., Broer, S., Broer, A. & Hamprecht, B. (2000). Isoenzyme pattern of

*Research*, Vol. 25, No. 11 (November 2000), pp. 1485-1491. ISSN 0364-3190. Silva, J. H., Zazueta-Novoa, V., Durón, C. A., Rodríguez, R. C., Leal-Morales, C. A. & Zazueta-

*Leeuwenhoewk*. Vol. 96, No. 4 (November 2009), pp. .527-535. ISSN 0003-6072. Smith, R. V. & Rosazza, J. P. (1974). Microbial Models of Mammalian Metabolism. Aromatic

Sutherland, J. B., Fu, P. P., Yang, S. K., Von Tungeln, L. S., Casillas, R. P., Crow, S. A. &

Torres-Martínez, S., Garre, V., Corrochano, L., Eslava, A. P., Baker, S.E. & others. (2009).

Van Herwijnen, R., Springael, D., Slot, P., Govers A. J. H. & Parsons, R. J. (2003).

Yamazaki, M. & Komagata, K. (1983). An Electrophoretic Comparison of Enzymes of

Zazueta-Sandoval, R., Durón, C. A. & Silva, J. H. (2008). Peroxidases in YR-1 strain of *Mucor* 

*Microbiology*. Vol. 58, No. 3 (September 2008), pp. 421-426. ISSN 1590-4261. Zazueta-Sandoval, R., Zazueta-Novoa, V., Silva-Jiménez, H. & Ortíz, R. C. (2003). A

*Biotechnology*. Vol. 108, No. 1-3 (March 2003), pp. 725-736. ISSN 0273-2289.

59, No. 7 (July 1993), pp. 2145-2149. ISSN 0099-2240.

Project CBS277.49, v1.0 http//genome.jgi-PSF.org.

(February 1983), pp. 115-143. ISSN 0022-1260.

Vol. 69, No. 1 (January 2003), pp. 186-190. ISSN 0099-2240.

*Toxicology.* Vol. 12, No. 1 (December 1998), pp. 1-18. ISSN 0893-228X. Pereira, E. F., Aracava, Y., Aronstam, R. S., Barreiro, E. J. & Alburquerque, E. S. (1992).

331-340. ISSN 0022-365.

pp. 551-558. ISSN 003-9861

*Bioengineering*. Vol. 38, No. 5 (August 1991), pp. 493-498. ISSN 1097-0290. Okwumabua, O., Persaud, J. S., & Reddy, P. G. (2001). Cloning and characterization of the gene

204, No. 1, (February 1992), pp. 113-120. ISSN 0014-2956.

Chain Alcohol Dehydrogenase Superfamily. *European Journal of Biochemistry*. Vol.

Product and By-Product Formation and Activities of the Key Enzymes in Wild Type and ADH Isoenzyme Mutants of *Saccharomyces cerevisiae. Biotechnology and* 

encoding the glutamato dehydrogenase of *Streptococcus suis* serotype 2. *Clinical and Diagnostic Laboratory Immunology.* Vol. 8, No. 2 (March 2001), pp. 251-257. ISSN 556-6811.

S. (1999). Dihydrodiol dehydrogenases and polycyclic aromatic hydricarbon activation: Generation of reactive and redox *o*-quinones. *Chemical Research in* 

Pyrazole, an Alcohol Dehydrogenase Inhibitor, Has a Dual Effects on N-methyl-Dasp. *Journal of Pharmacology and Experimental Theory.* Vol. 261, No.1 (April 1991), pp.

glycogen phosphorylase in the rat nervous system and rat astroglia-rich primary cultures: electrophoretic and polymerase chain reaction studies. *Neurochemical* 

Sandoval, R. (2009). Intracellular Distribution of Fatty Alcohol Oxidase Activity in *Mucor circinelloides* YR-1 Isolated from Petroleum Contaminated Soils. *Antonie Van* 

Hydroxylation. *Archives of Biochemistry and Biophysics*. Vol. 161, No. 2 (April 1974),

Cerniglia, C. E. (1993). Enantiomeric Composition of the *Trans*-Dihydrodiols Produced by Phenanthrene by Fungi. *Applied and Environmental Microbiology.* Vol.

Degradation of Anthracene by *Mycobacterium* sp. Strain LB501T Proceeds Via a Novel Pathway Throught o-Phtalic Acid. *Applied and Environmental Microbiology*.

Ballistosporogenous Yeasts. *The Journal of General Applied Microbiolgy*. Vol. 29, No. 2

*circinelloides* a Potential Bioremediator of Petroleum-Contaminated Soils. *Annals of* 

Different Method of Measuring and Detecting Mono and Di-Oxygenase Activities: Key Enzymes in Hidrocarbon Biodegradation. *Applied Biochememistry and*  Peptidases are enzymes that catalyze the hydrolysis of peptide bonds in proteins or peptides. The hydrolysis can be specific or unspecific, leading to highly regulated cleavage of specific peptide bonds, or to complete degradation of proteins to oligopeptides and/or amino acids. Peptidases can be classified as endo- or exopeptidases, the latter only act near the ends of the polypeptide chain. Endopeptidases are divided into six major families by virtue of the specific chemistry of their active site: aspartic, serine, metallo-, cysteine, glutamic and threonine peptidases (Rawlings et al. 2010).

Zymography is an electrophoretic technique, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a substrate (e.g. gelatin, casein, albumin, hemoglobin, etc.) co-polymerized with the polyacrylamide matrix. Proteins are prepared by the standard SDS-PAGE buffer under non-reducing conditions (no boiling and no reducing agent), and are separated by molecular mass in the standard denaturing SDS-PAGE co-polymerized with a protein substrate. After electrophoresis, peptidases are renatured by the removal of the denaturing SDS by a non-ionic detergent, such as Triton X-100, followed by incubation in conditions specific for each peptidase activity (time, temperature, ions, ionic strength), when the enzymes hydrolyze the embedded substrate, then proteolytic activity can be visualized as cleared bands on a Coomassie stained background (Heussen and Dowdle, 1980). Therefore, only endopeptidases can be detected by substrate-SDS-PAGE, which requires a considerable degradation of the substrate for visualization of the degradation haloes. Alternatively, an overlay with specific chromogenic or fluorogenic peptide substrate can be done after SDS-PAGE separation of the proteins and renaturation with Triton X-100, which allows the detection of specific peptidases in complex biological samples.

This technique has many benets: (1) it is relatively inexpensive, requires short assaying times, and peptidases with distinct molecular masses can be detected on a single gel; (2)

Applications of Zymography (Substrate-SDS-PAGE)

indicating possible variations.

**2.1 Sample homogenization** 

**substrates** 

for Peptidase Screening in a Post-Genomic Era 267

Several research groups perform substrate-SDS-PAGE to assess, screen and characterize peptidases in complex or purified biological preparations. After the original publication from Heussen and Dowdle (1980), several adaptations have been implemented to improve the detection of a specific peptidase class. Below we will present a generalized protocol

The preparation of the biological sample is critical for the success of the zymography, all the procedure must be performed at 4oC, the addition of detergents such as Triton X-100, SDS or CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) for the solubilization and recovery of hydrophobic enzymes is necessary, if one is interested in such enzymes, also addition of proteolytic inhibitors to undesired peptidase classes is also an interesting strategy. Alternatively, the separation of hydrophobic from hydrophilic proteins can be achieved during phase partition in solutions of Triton X-114, which occurs at 37oC preserving enzyme integrity (Figure 1) (Bouvier et al. 1987). After sample preparation, SDS-PAGE sample buffer is added to the biological sample (62.5 mM Tris HCl, pH 6.8, 2% SDS, 10% (v/v) glycerol, and 0.002% bromophenol blue). A concentrated sample buffer can be used to avoid sample dilution, which is critical for the detection of low abundant enzymes. The proteins are not denatured since sample is kept at 4oC, there is no sample boiling, nor the addition of reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol, as usual in sample preparation for standard SDS-PAGE analysis. The sample must maintain its

**2. Comments on peptidase screening through substrate-SDS-PAGE** 

native form due to the the further step of substrate degradation.

degradation halos can be seen in Figure 2.

**2.2 Polyacrylamide gels containing sodium dodecyl sulfate and co-polymerized** 

Here, to the standard Laemmli protocol (Laemmli, 1970), a substrate can be co-polymerized to the gel (Heussen and Dowdle, 1980). Alternatively, an overlay with fluorogenic or chromogenic peptide substrates can be done (Cadavid-Restrepo et al. 2011). The acrylamide concentration in gels varies more commonly from 7 to 15%, which impact on protein separation; low molecular mass proteins usually require higher acrylamide concentration for better protein resolution. The co-polymerized substrate can be virtually any protein. Gelatin is commonly used as a protein substrate because it is easily hydrolyzed by several peptidases and does not tend to migrate out of the resolving gel in electrophoretic tests performed at 4°C, and is inexpensive (Michaud et al. 1996). In addition to gelatin, several other proteins have been used, such as: casein, bovine serum albumin, human serum albumin, hemoglobin, mucin, immunoglobulin, and collagen (d'Avila-Levy et al. 2005; Pereira et al. 2010a). Also, complex mixtures of proteins can be used, which may reflect a functional role of the enzyme. For instance, our research group employed gut proteins from an insect to co-polymerize in acrylamide gels. Then, extracts from a protozoan believed to interact with the insect gut were assayed, revealing the peptidases capable of degrading the insect gut proteins (Pereira et al. 2010a). An example of zymographies performed with a set of eight distinct proteinaceous substrates, as well as, a densitometric measure of the

separation of proteins by molecular mass through non-reducing electrophoretic migration allows a presumptive correlation with known peptidases; (3) incubation with proteolytic inhibitors provides powerful information about enzyme classification; (4) pH and temperature changes help to assess peptidase characteristics; (5) several substrates can be co-polymerized to assess peptidase degradation capacity; (6) densitometry can be used for quantitative analysis. Ultimately, in organisms with complete genome sequences, bioinformatic analysis provides rich information on putative peptidases, such as: peptidase classification, approximated molecular mass, possible cellular localization through classical motifs, evolutionary and functional relationships, and so on. However, it cannot be ascertained if the described ORFs are indeed expressed and active. Therefore, a zymographic assay coupled with bioinformatic analysis may allow the detection of functionally active enzymes.

The advantages of this technique are exemplified by its application nowadays to unveil peptidases in biological systems, which possesses genome information, but still zymography is the method of choice for peptidase screening, identification and characterization. Wilder and colleagues, for instance, report that zymography can selectively distinguish cathepsins K, L, S and V in cells and tissues by its electrophoretic mobility and by simply manipulating substrate and pH. The sequence homology among these cathepsins leads to a substrate promiscuity, which precludes desired specificity for in solution assays with specific chromogenic or fluorogenic peptide substrate (Wilder et al. 2011). Zymography allows the detection of a 37 kDa (cathepsin K), 35 kDa (cathepsin V), 25 kDa (cathepsin S) and 20 kDa (cathepsin L). Cathepsin K activity disappeared and V remained when incubated at pH 4.0 instead of 6.0, allowing the visualization of each enzyme (Wilder et al. 2011). Kupai and colleagues also highlighted that substrate zymography is the method of choice, among several analyzed, to detect the activity of the different matrix metallopeptidase (MMP) isoenzymes from a wide range of biological samples (Kupai et al. 2010). Also, it allows high throughput screening of specific MMP inhibitors, especially because the nature of the residues in the enzyme's active site is highly conserved among the different MMPs, therefore, once again, in solution enzymatic assays are not applicable (Devel et al. 2006, Kupai et al. 2010). Also, for the screening of tissue inhibitors of metallopeptidases (TIMPs), reverse zymography is a powerful approach. This technique is based on the ability of the inhibitors to block gelatinase activity of a MMP, usually MMP-2. A calibrated solution of gelatinase-A (MMP-2) is co-polymerized with gelatin in the polyacrylamide gel. The samples possibly containing TIMPs are then separated by electrophoresis, SDS is removed and the gel is incubated in a buffer that allows the gelatinase to digest the gelatin, except where it is inhibited by TIMP proteins. After staining with Coomassie blue, the result is a gel with a pale blue background (where gelatin was degraded by the gelatinase) with blue bands showing the positions and relative amounts of TIMPs (Snoek-van Beurden and Von den Hoff, 2005).

In view of this, below we will present comments on peptidase screening through zymography discussing possible protocol variations and its implications, and then we present and discuss practical examples of the application of zymography to generate critical data in organisms that still do not possess genome information. Finally, we will discuss the possibility of direct peptidase identification through two-dimensional zymography coupled to mass spectrometry.
