**2. Isoenzymes of lactate dehydrogenase and their electrophoretic separation**

LDH is a tetrameric enzyme that exists in three basic homotetrameric forms in vertebrates: H4 (LDH1 ), M4 (LDH5 ), and C<sup>4</sup> (LDH-X) [9–16]. Three structurally different polypeptide chains of homotetrameric LDH molecules are encoded by three different genes [13, 17–19]. Lactate dehydrogenase H4 and M4 forms, also called somatic LDH [20], are present in tissues of all studied vertebrates, while the tissue distribution of LDH-C<sup>4</sup> isoenzyme varies from organism to organism. In mammals and columbid birds, this isoenzyme is expressed only in mature testes [19, 21, 22]. Except H<sup>4</sup> and M4 homotetramers, somatic LDH also exists in three hybrid forms, thus being present in five structural entities in cells of most tissues. According to their different motility to the anode, somatic LDHs are denoted LDH<sup>1</sup> (H4 ), LDH2 (H3 M), LDH3 (H2 M2 ), LDH4 (HM3 ), and LDH5 (M4 ) with LDH1 having the highest and LDH5 the lowest migration rate to the anode. Three hybrid forms (LDH2 , LDH3 , and LDH4 ) are relatively equally spread between LDH<sup>1</sup> and LDH5 on the electrophoreogram. Protomers of the somatic LDH isoenzymes are, in general, designated H (heart) or B, and M (muscle) or A according to the domination of homotetrameric molecules in the cells of respective organs of adult vertebrates. Hybrid forms of LDH are present in various levels in the individual organs of an animal/human.

Although catalyzing the same overall reaction, LDH isoenzymes differ in their kinetic characteristics (Km, kcat) as documented in **Table 1** [4–6, 23–25].

As seen, H4 homotetramers have a lower value of Michaelis constant Km for pyruvate as substrate. Moreover, H4 is more sensitive to inhibition by high pyruvate concentration than M4 isoenzyme, which is relatively indifferent to substrate concentration [3].

Different structures of lactate dehydrogenase isoenzyme molecules predetermine their different net charge and, consequently, different migration rate in electric field. It is known that the charge of a protein and, therefore, its motility in the electric field vary with the pH of its environment. Maximum resolution is achieved when the proteins of interest have


**Table 1.** Kinetic constants determined for the catalytic action of H4 and M4 homotetramers of lactate dehydrogenase.

widely varying electrical charges. Conversely, no or poor separation of proteins is achieved when there are only small differences in the electrical charges of molecules at a given pH values. Changing the pH of the electrophoresis media may alter the charges on respective proteins, thus producing a better separation. Generally, a buffer system of pH 8.6 is chosen for the separation of the five lactate dehydrogenase isoenzymes. At pH 8.6, their electrophoretic migration depends on the two pure types, that is, the H4 and M4 forms [9]. The more widely the two homotetramers differ in charge (the case of mammalian lactate dehydrogenases), the more separable are the hybrids by electrophoresis. In the case of bird lactate dehydrogenases, the two pure types migrate relatively close together toward the negative pole at pH 8.6, and the observation of the hybrid forms under these conditions is very difficult [9]. To overcome this difficulty, we chose a gradient of pH 3–9 for the electrophoretic separation of bird lactate dehydrogenase isoenzymes (isoelectric focusing technique, IEF) [8]. By this technique, we achieved a good and clear resolution of all five forms of the enzyme in serum and tissues of chicken (adult as well as in embryonic), turkey, pheasant, and pigeon. We also compared patterns in bird tissues and sera with those of mammalian origin.
