**2.1 Sample homogenization**

266 Gel Electrophoresis – Advanced Techniques

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

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

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

functionally active enzymes.

TIMPs (Snoek-van Beurden and Von den Hoff, 2005).

to mass spectrometry.

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

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

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 degradation halos can be seen in Figure 2.

Applications of Zymography (Substrate-SDS-PAGE)

screening in an uncharacterized organism can be seen in Figure 4.

for Peptidase Screening in a Post-Genomic Era 269

of ions or reducing agents and finally assess the inhibition profile. A general flowchart for establishing such conditions is shown in Figure 3, and a general view of Gelatin-SDS-PAGE

Fig. 2. Degradation of different proteinaceous substrates co-polymerized to SDS-PAGE by a surface metallopeptidase from *Herpetomonas samuelpessoai*, an insect trypanosomatid. The following substrates were individually incorporated into SDS-PAGE to evidence the proteolytic activity: gelatin, bovine serum albumin, human serum albumin, casein, immunoglobulin G (IgG), hemoglobin, mucin and gut extract from *Aedes aegypti*. The gels were incubated for 20 h at 37oC in 50 mM sodium phosphate buffer pH 6.0 supplemented with DTT 2 mM (A). The degradation halos, which correlate with degradation capability, were densitometric measured and expressed as arbitrary units of proteolytic activity (B). For

**3. Practical examples of peptidases screening through SDS-PAGE-substrate** 

*Bodo* sp. is a free-living flagellate that belongs to the family Bodonidae, order Kinetoplastida. This bodonid isolate still has its taxonomic position unsolved, but it is phylogenetically related to *Bodo caudatus* and *Bodo curvifilus*, which are considered ancestral to the trypanosomatids. The Trypanosomatidae family comprises parasites that are of particular interest due to their medical importance, such as the etiologic agent of Chagas' disease (*Trypanosoma cruzi*), African trypanosomiasis (*Trypanosoma brucei* complex) and the various forms of leishmaniasis caused by *Leishmania* spp.. Due to their medical relevance, this family has been the focus of extensive research (Wallace, 1966; Vickerman, 1994). Peptidase characterization in *Bodo* sp. and comparison to peptidases from closely related pathogenic protozoa may help to understand peptidase function and evolution in general. The gold standard approach for such comparison would be a bioinformatic analysis of the *Bodo*

experimental details see Pereira et al. 2010b. Reprinted with permission of *Protist*.

**3.1 A first glance on** *Bodo* **sp. peptidases** 

Fig. 1. Extracellular and cell-associated proteolytic enzymes of *Crithidia deanei* cells, an insect trypanosomatid. Parasites were cultured in a complex medium (brain heart infusion) for 48 h at 28◦C. Then, cells were harvested by centrifugation, the culture supernatant was filtered in Millipore membrane 0.22 µm and concentrated 50-fold by dialysis (cut-off 9000 Da) against polyethylene glycol 4000 overnight at 4oC. The cells were lysed by: the addition of SDS, generating whole cellular extract, or by Triton X-114 to obtain the hydrophilic (cytoplasmatic and intravesicular fraction) and hydrophobic (membrane fraction) phases. The extracellular and cellular extracts were applied on gelatin-SDS-PAGE to evidence the proteolytic enzymes. The gels were incubated in 50 mM sodium phosphate buffer pH 5.5 supplemented with DTT 2 mM at 37oC for 24 h. MP, metallopeptidase and CP, cysteine peptidase. For experimental details see d'Avila-Levy et al. 2001, 2003.
