**4. Separation of isozymes by isoelectric focusing**

The charge of proteins is determined by the ratio of acidous and alkaline molecule parts and the rate of their dissociation. The rate of dissociation is determined by the pH of the surroundings of the molecule. The isoelectric point (pI) of the protein is the pH at which the acidous and alkaline molecule parts equally dissociate, the protein's net charge being zero. In a surrounding where the pH is lower than the isoelectric point, the net charge of the protein will be positive, in turn when the pH is higher than the IP, the net charge will be negative (Hames, 1990).

Isoelectric focusing of proteins can be carried out in a gel, in which a pH gradient is generated. Under voltage the proteins migrate to the point of the gel, where their net charge is equal to zero (pI). For this method thin poliacrilamide or agarose gels are used (Fig. 3).

The advantages of isoelectric focusing in opposition of gel electrophoresis are that the isozyme variants can be identified based on their isoelectric points, which results more accurate determination of isoforms, than the identification based on Rf values. On the other hand, the used gels in isoelectric focusing are thinner, so the separation is faster (Patterson and Payne, 1989).

Previously isoelectric focusing had disadvantages, as it required practice and the staining of the gels, because of the wide pH gradient, was difficult (Patterson és Payne, 1989), but nowadays these cause no problem. With the use of ampholites, the preparation of pH gradient gels needs even less practice, and you can even purchase ones. Neither causes problems with the staining of this so-called immobilized pH gradient gels, because the pH gradient can be removed by the washing of the gel.

Fig. 2. Segregation of isozyme bands in PGM zone 4. Each column shows representative

The charge of proteins is determined by the ratio of acidous and alkaline molecule parts and the rate of their dissociation. The rate of dissociation is determined by the pH of the surroundings of the molecule. The isoelectric point (pI) of the protein is the pH at which the acidous and alkaline molecule parts equally dissociate, the protein's net charge being zero. In a surrounding where the pH is lower than the isoelectric point, the net charge of the protein will be positive, in turn when the pH is higher than the IP, the net charge will be

Isoelectric focusing of proteins can be carried out in a gel, in which a pH gradient is generated. Under voltage the proteins migrate to the point of the gel, where their net charge is equal to zero (pI). For this method thin poliacrilamide or agarose gels are used (Fig. 3).

The advantages of isoelectric focusing in opposition of gel electrophoresis are that the isozyme variants can be identified based on their isoelectric points, which results more accurate determination of isoforms, than the identification based on Rf values. On the other hand, the used gels in isoelectric focusing are thinner, so the separation is faster (Patterson

Previously isoelectric focusing had disadvantages, as it required practice and the staining of the gels, because of the wide pH gradient, was difficult (Patterson és Payne, 1989), but nowadays these cause no problem. With the use of ampholites, the preparation of pH gradient gels needs even less practice, and you can even purchase ones. Neither causes problems with the staining of this so-called immobilized pH gradient gels, because the pH

individuals of a selfed population of Cherry Bell-4. (Nomura et al., 1999.)

**4. Separation of isozymes by isoelectric focusing** 

gradient can be removed by the washing of the gel.

negative (Hames, 1990).

and Payne, 1989).

Fig. 3. Isoelectric focusing patterns of horseradish (HP), and grapevine (GP) peroxidases. IEP isoelectric points, P1-P7 names of isoenzyme bands, AP application zone, PK cathodic peroxidase. -a) anodic and cathodic pattern of peroxidases obtained from different organs of *Vitis riparia*. L Leaf, R root, C callus. The bands of horseradish peroxidases are distorted due to a margin effect on the slab. -b) Shematic diagram of the anodic part of peroxidase patterns. (Bachmann and Blaich, 1988)
