**3.2 Pectinase**

Method I – After conducting a PAGE 4.5 or 8.9 (depending on the enzyme pI), the gel containing the enzyme must be incubated with the substrate – a solution containing citric pectin or sodium polypectate 1% in the suitable buffer of the enzyme under study. In Fig. 2B there was the use of 1% of sodium polypectate in a sodium acetate buffer 100mM, pH 4.0 and incubation at 50ºC (enzyme optimum temperature), for 2 hours for the dying with 0.02% Ruthenium red [(Ru3O2(NH3) 14)Cl6.4H2O)], a dye capable of interacting with the pectic substates (Sterling, 1970). Thus, in the region where the protein migrated to and hydrolyzed the substrate, there is a halo with a whitened coloration that contrasts against the rest of the red-colored gel.

Method II – The citric pectin must be dissolved in gel buffer with the aid of a magnetic agitator, followed by the addition of acrylamide, bis-acrylamide and TEMED solutions. Crystals of ammonium persulfate are added immediately before the plate gel is overflown. After the run, incubate the gel for 1-2 hours with 100 mL of malic acid 0.1M, at 4ºC, in order to cause a gradual change to pH 3.0. Such period allows the enzyme to interact with the pectin polymerized in the acrylamide gel in its suitable pH range. Wash with distilled water and color in Ruthenium red 0.02%, during 30 to 120 min. Wash with distilled water.

Result: against a redish gel, it is possible to notice the polygalacturonase activity due to the formation of clear, opaque or colorless areas.
