**2.3 Enzyme renaturation and proteolysis**

After electrophoresis, the enzymes are renaturated by replacement of the anionic detergent SDS by the non-ionic detergent Triton X-100, through gel washing. Then, gels are incubated under conditions ideal for detection of the desired peptidase. For instance, metallopeptidases are known to require neutral to basic pH for activity, while cysteine peptidases require an acidic pH and a reducing agent, usually DTT (Branquinha et al. 1996). However, it is common to screen biological samples, where there is no previous clue on what peptidase class shall be present, nor the best conditions for proteolysis. Therefore, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence

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

After electrophoresis, the enzymes are renaturated by replacement of the anionic detergent SDS by the non-ionic detergent Triton X-100, through gel washing. Then, gels are incubated under conditions ideal for detection of the desired peptidase. For instance, metallopeptidases are known to require neutral to basic pH for activity, while cysteine peptidases require an acidic pH and a reducing agent, usually DTT (Branquinha et al. 1996). However, it is common to screen biological samples, where there is no previous clue on what peptidase class shall be present, nor the best conditions for proteolysis. Therefore, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence

peptidase. For experimental details see d'Avila-Levy et al. 2001, 2003.

**2.3 Enzyme renaturation and proteolysis** 

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

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