**3. Preparation of the samples for electrophoresis**

as isoenzymes. They catalyze the same biochemical reaction but differ from each other in their kinetic characteristics (Km, kcat), physicochemical properties (different net charge), response to the inhibition by substrate (pyruvate) [4–6], and immunological response. Despite a profound understanding of LDHs, there was a lack of data concerning their relative distribution and pattern in bird tissues and sera. This gap in our knowledge of these enzymes was probably due to the fact that electrophoretic techniques routinely used in separating mammalian LDH isoenzymes did not produce a satisfactory separation of all bird isoenzymes [7, 8]. Thus, their application in research as well as in diagnostic practice remained in birds limited to the determination of the total LDH activity without the possibility for the correct interpretation of data in relation to their quantitative patterns in respective tissues/organs. The aim of our work was to separate lactate dehydrogenase isoenzymes of bird origin and to characterize the pattern of their relative distribution in tissues and sera using a suitable electrophoretic technique.

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

LDH is a tetrameric enzyme that exists in three basic homotetrameric forms in vertebrates: H4

of homotetrameric LDH molecules are encoded by three different genes [13, 17–19]. Lactate

to organism. In mammals and columbid birds, this isoenzyme is expressed only in mature

forms, thus being present in five structural entities in cells of most tissues. According to their

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

Although catalyzing the same overall reaction, LDH isoenzymes differ in their kinetic charac-

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

homotetramers have a lower value of Michaelis constant Km for pyruvate as sub-

is more sensitive to inhibition by high pyruvate concentration than M4

) with LDH1

(LDH-X) [9–16]. Three structurally different polypeptide chains

forms, also called somatic LDH [20], are present in tissues of all

homotetramers, somatic LDH also exists in three hybrid

having the highest and LDH5

on the electrophoreogram. Protomers of the somatic

, LDH3

(H4

, and LDH4

isoenzyme varies from organism

), LDH2

(H3

M), LDH3

the lowest

) are relatively

**separation**

), M4

dehydrogenase H4

), LDH4

animal/human.

As seen, H4

strate. Moreover, H4

(LDH5

82 Electrophoresis - Life Sciences Practical Applications

testes [19, 21, 22]. Except H<sup>4</sup>

(HM3

equally spread between LDH<sup>1</sup>

), and C<sup>4</sup>

and M4

studied vertebrates, while the tissue distribution of LDH-C<sup>4</sup>

), and LDH5

migration rate to the anode. Three hybrid forms (LDH2

teristics (Km, kcat) as documented in **Table 1** [4–6, 23–25].

and M4

different motility to the anode, somatic LDHs are denoted LDH<sup>1</sup>

(M4

and LDH5

isoenzyme, which is relatively indifferent to substrate concentration [3].

(LDH1

(H2 M2

> Sera from various animal species were obtained from rested and clinically healthy animals. The blood collection for the serum samples was performed by standard procedures, and no hemolyzed sera were used for the examination. The determination of total lactate dehydrogenase activity as well as separation of the isoenzymes was performed on the same day as the blood collection.

> Tissue samples were taken from birds after decapitation and immediately put into ice-cold buffer saline. The tissues were cut into small pieces and washed in buffered saline to remove excess blood and connective tissue. Two grams of tissue were homogenized in 10 volumes of cold buffer (0.05 mol/L Tris-HCl buffer, pH 7.3 with 0.01% EDTA). The homogenate was centrifuged at 19,000 × *g* for 60 min at 4°C, and the supernatant was used for enzyme assay and electrophoretic separation.

> Blood for erythrocytes LDH pattern was collected in test tubes containing sodium heparin and centrifuged at 450 × *g* for 10 min at 4°C. Plasma was discarded and erythrocytes washed

thoroughly three times in twice their volume, and the supernatant discarded. The cells were then lysed with the addition of three volumes of cold distilled water and centrifuged at 19,000 × *g* for 60 min at 4°C.

up to 30 ml with 0.1 M Gly-NaCl-NaOH, pH 8.3. In this reaction mixture, the gels were incubated at 37°C for 20–30 min. Then, they were immersed into 7.5% (v/v) acetic acid to stop the reaction. To detect lactate dehydrogenase isoenzyme zones on the electrophoreogram, we avoid using Tris-HCl buffer as it produced quite an intensive background on the gel. PhastImage system (Pharmacia LKB, Sweden) served for densitometric scanning (613 nm) of the pattern, and for the determination of relative distribution (%) of the isoenzyme fractions, GEL LOGIC 100 IMAGING SYSTEM with Kodak 1D Image Analysis Software (Japan) was used as well. To identify individual bird LDH isoenzymes on the electrophoreogram, a principle commonly used for the identification of mammalian LDH isoenzymes was applied:

Lactate Dehydrogenase Isoenzyme Electrophoretic Pattern in Serum and Tissues of Mammalian…

Four to five isoenzymes of LDH are usually present in normal sera of animal and human beings as a result of natural degradation of cells of various tissues/organs. Their quantitative distribution in the serum is different and relatively characteristic for a particular biological species (**Figure 1**) [8]. After their separation in a concentration gradient of polyacrylamide (10–15%) and at pH 8.8, mammalian lactate dehydrogenases were separated with a good resolution, whereas bird serum (chicken) produced only one, somewhat diffuse enzymatic

A good and clear resolution of bird LDH (chicken and pheasant) isoenzymes with all five isoenzymes zones was achieved using isoelectric focusing technique in a pH range of 3–9 [8]. A similar pattern was also produced by turkey isoenzymes [27]. A comparison of LDH catalytic activity and relative distribution (%) of its isoenzymes in the sera of the investigated animals (**Figure 1**) revealed that the predominant portion of chicken serum LDH activity was con-

and that nearest to the cathode LDH5

http://dx.doi.org/10.5772/intechopen.76322

) (66%), followed by LDH<sup>4</sup>

(23%). LDH<sup>1</sup>

to

.

85

the fraction nearest to the anode was designated LDH1

centrated in the muscle form of the enzyme (LDH5

**Figure 1.** Lactate dehydrogenase isoenzyme patterns in the serum of some birds and mammals.

zone [8].

**6. Patterns of LDH isoenzymes and their interpretation**

Chicken embryonic organs/tissues were taken from 9-day-old chicken embryo (n = 6), washed in buffer saline, and homogenated in 50 mM Tris-HCl, pH 7.5, with the content of 0.01% EDTA using Precellys 24—Dual homogenizer (Bertin Technologies, France). The homogenates were centrifuged at 25,000 × *g* for 30 min at 4°C, and the supernatants served as the source of the enzyme.

Catalytic activity of LDH was assayed colorimetrically at 37°C using lactate as the substrate with NAD+ as the coenzyme and brownish-red pyruvate hydrazone as the measured product (A505). Protein concentration for the calculation of specific enzyme activity (U/g) was determined using Bradford method [26]. The activity of LDH in erythrocytes was expressed in enzyme units per gram of hemoglobin (U/g of hemoglobin).
