**Electrophoresis Application in Enzymology**

244 Gel Electrophoresis – Advanced Techniques

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**14** 

**Polyacrylamide Gel Electrophoresis** 

**an Important Tool for the Detection** 

**by Electrophoretic Zymograms** 

Carlos A. Leal-Morales, Alberto Flores Martínez, Patricia Ponce Noyola and Roberto Zazueta-Sandoval

 *University of Guanajuato* 

*México* 

**and Analysis of Enzymatic Activities** 

Reyna Lucero Camacho Morales, Vanesa Zazueta-Novoa,

Gel electrophoresis of enzymes is a very useful and powerful analytical method, which is at present widely used in many distinct fields of both biological and medical sciences and successfully applied in many different fields of human activity. The tremendous expansion of this methodology is mainly due to its simplicity and its high ability to separate both isoenzymes and alloenzymes, which have proven to be very useful genetic markers. The most important step of enzyme electrophoresis is the detection of native enzymes on electrophoretic gels; it means the procedure of obtaining electropherograms, or zymograms. Detection of enzymes on electrophoresis gels means the visualization of gel areas occupied by specific enzyme molecules after their electrophoretic separation. From this point of view, sometimes the testing of an enzyme-coding DNA sequence for expression of catalytically active enzyme is performed by zymograms, where the use of this technique for this purpose is very effective, cheap, and time saving. The number of applications of zymogram techniques for testing cloning enzyme-coding genes for their expression at the protein level is growing (Pfeiffeer-Guglielmi et al., 2000; Lim et al., 2001; Okwumabua et al., 2001). An absolute prerequisite for this is the specific and sensitive zymogram technique suitable for

detection of the enzyme inside the gel and the use of the appropriate substrates.

The zymograms has been used to detect a variety of oxidoreductases (Bergmeyer, 1983), including isoenzymes (Jeng & Wayman, 1987) and to classify various genera of yeast based upon the relative mobility of the activity bands produced by selected enzymes (Goto & Takami, 1986; Yamasaki & Komagata, 1983). Electrophorezed gels are placed in a staining solution containing a reduced substrate such as an alcohol, oxidized cofactor such as NAD+ or NADP+, a dye such as nitroblue tetrazolium, and an electron acceptor-donor such as phenazine methosulphate. At the location of the appropriate enzyme catalyzing oxidation of substrate and reduction of cofactor, a dark-purple band appears as a result of the precipitate

**1. Introduction** 

that forms upon reduction of the dye.
