INTRODUCTION
Lactate dehydrogenases (L-lactate: NAD oxidoreductase, EC 1.1.1.27; LDH) are glycolytic enzymes that initiate the pyruvates reduction to lactates under anaerobic cases (Li et al., 2013). LDHs are present in almost all living organisms since the enzyme was separated and described from animals, plants, and bacteria by diverse chromatographic techniques (Qian et al., 2014). LDH is detected in nearly whole creatures since it is playing a significant part in the metabolism of carbohydrates. Through situations when pyruvate manufacture in glycolysis surpasses the cell capability in pyruvate oxidizing, LDH transforms pyruvate to lactate and hence repeats the oxidized Nicotinamide adenine dinucleotide (NAD) necessary for more glycolysis. Furthermore, LDH permits the lactates diversion to pyruvates; and the obtained pyruvates and Nicotinamide adenine dinucleotide reduced (NADH) can then be employed for different operations (Kadiyala, 2015). In animal tissues, LDH is generated by two genes, called LDH A and LDH B. LDH A gene is strongly expressed in muscles and livers (M isozyme), while LDH B gene is strongly expressed in hearts (H isozyme). Nearly in whole kinds, the gene yields compose tetramer compounds with different properties slightly relying on the proportional quantities of various isoenzymes that exist in the tetramer (Schurr, 2017). The two common LDH subunits (LDH-M) and (LDH-H) found in circulation can compose five tetramers (isoforms): 4H, 4M, 3H1M, 2H2M, and 1H3M (Nadeem et al., 2014). High level of LDH in serum was found in myocardial infarction, liver disease, renal disease, malignant diseases, progressive muscle dystrophy, and certain forms of anemia (Holmes and Goldberg, 2009; Ketchum et al., 1984). LDH can be used as a marker for tumor, heart attack, hypothyroidism, anemia, preeclampsia, meninges and brain inflammations, severe pancreatitis, HIV, lungs and liver diseases, and tissues collapse (Butt et al., 2002; Kim et al., 2014; Xu et al, 2014). LDH and malate dehydrogenase (MDH) enzymes are major constituents in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) diagnosis kits that are very precise in the determination of AST and ALT enzymes levels in the blood, serum, and plasma. AST and ALT kits are at most employed as livers job marker tools in diagnostics, controlling, and treating hepatic illnesses (Darwish et al., 2018; Huijgen et al., 1997). Thus, the leading goal of this search is purifying, describing, and producing LDH enzyme from buffalo livers as the home available origin for use in constructing AST and ALT diagnosis kits.
MATERIALS AND METHODS
Liver tissues
Water buffalo (Bubalus bubalis) liver tissue samples of six different animals were gained from a local slaughter-house and stocked at −40ËšC.
Chemicals
Bovine serum albumin, DEAE cellulose, NADH, Nitroblue tetrazolium salt (NBT), Sodium pyruvate, Lithium lactate, Phenazine methosulfate (PMS), Sephacryl S-300, Standard proteins for molecular weights, and electrophoresis chemicals were bought from Sigma-Aldrich Chemical Co. All other chemical compounds were of analytical degree.
Assay of Lactate dehydrogenase activity
The activity LDH assay is measured by the method described by Nadeem et al. (2011). The test mixture consists of 3 ml 0.1 M phosphate buffer pH 7.0, 0.22 mM NADH, 0.2 mM sodium pyruvate, and enzyme solution. The variation in absorbance at 340 nm was followed for 5 minutes and LDH activity units were calculated by Beer–Lambert law, utilizing 6,220 M−1 cm−1extinction coefficient value of NADH. For LDH isoenzyme patterns on polyacrylamide gels, the gels were flooded in 50 ml 0.1M Tris-HCl, pH 8, 288 mg lithium lactate, 14 mg NAD, 3.5 mg NBT, and traces of PMS. In dark, gels were incubated in this staining mixture at 37ËšC until appearing of dark blue bands (Whitt, 1970).
Purification of buffalo liver lactate dehydrogenase (BLLDH)
Preparation of crude extract
Buffalo liver tissues were homogenized in 0.05 M Na phosphate buffer pH 7.4 including 10 mM Ethylenediaminetetraacetic acid (EDTA) and 1 mM β-mercaptoethanol by Omni mixer homogenizer. Cells debris and not soluble materials were segregated with centrifuging at 5,000 × g for 20 minutes with saving the supernatant as a crude extract.
Ammonium sulfate precipitation
Solid (NH4)2SO4 was progressively added to the crude extract till be 40% saturated and stirred for 30 minutes at 4 °C followed by 20 minutes centrifugation at 8,000 × g. The pellets from this stage were thrown out and the supernatant was 80% Solid (NH4)2SO4 saturated followed by 30 minutes centrifugation at 12,000 × g and the precipitate was gained and dissolved in 0.02 M Na phosphate buffer pH 7.4 including 10 mM EDTA and 1 mM β-mercaptoethanol and dialyzed extensively versus the same buffer.
DEAE-cellulose and Sephacryl S-300 column chromatography
The 40%–80% (NH4)2SO4 portion was loaded on DEAE cellulose column (12 cm × 2.4 cm i.d.) already equilibrated with 0.02 M sodium phosphate buffer pH 7.4 containing 10 mM EDTA and 1 mM β-mercaptoethanol. For further purification, the DEAE cellulose portions retaining LDH activity were pooled and concentrated by lyophilization. The concentrated DEAE-cellulose material was loaded over Sephacryl S-300 column (142 cm × 1.75 cm i.d.) equilibrated by 0.02 M sodium phosphate buffer pH 7.4 including 10 mM EDTA and 1 mM β-mercaptoethanol.
Electrophoretic analysis
Electrophoretic analysis of the purified LDH enzyme was performed on 7% PAGE (Smith, 1969) and 12% SDS-PAGE (Laemmli, 1970), while its molecular mass was detected according to Weber and Osborn (1969).
Staining of proteins was carried out utilizing 0.25% Coomassie Brilliant Blue R-250.
Protein determination
Proteins concentration was detected by Bradford method (1976) utilizing albumin from bovine serum as a standard.
Preparation of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) diagnostic kits
The purified buffalo liver LDH and the previously purified sheep liver MDH (Darwish et al., 2018) are employed in constructing AST and ALT diagnosis kits. The AST kit consists of two solutions; 80 mM Tris-HCl buffer pH 7.8, 240 mM L-aspartate, 900 U/L LDH and 600 U/L MDH and 12 mM 2-oxoglutarate and 0.18 mM NADH. ALT kit consists of two solutions; 80 mM Tris-HCl buffer pH 7.5, 500 mM L-Alanine, 1200 U/L LDH and 15 mM 2-oxoglutarate and 0.18 mM NADH (Thomas, 1998).
RESULTS
Purification of buffalo liver lactate dehydrogenase (BLLDH)
The procedure of the major buffalo liver LDH purification is summarized in Table 1. The LDH specific activity of buffalo liver crude extracts was detected as 1.1 units/mg protein. The chromatographic pattern of the buffalo liver LDH on DEAE cellulose matrix showed one major LDH activity peak collected by 0.05 M NaCl and designated BLLDH and other two minor LDH activity peaks (Fig. 1a). The major BLLDH fractions were gathered, lyophilized, and furthermore loaded on Sephacryl S-300 column (Fig. 1b) which showed one peak of LDH. After chromatography, BLLDH specific activity was elevated to 17.6 units/mg showing 16-folds and 32% outputs (Table 1).
 | Table 1. A typical purification scheme of BLLDH. [Click here to view] |
Molecular weight, electrophoretic analyses, and Optimum pH of BLLDH
In this study, the BLLDH pureness was inspected by analyzing on 7% native-PAGE. BLLDH isoenzyme demonstrated a unique protein band identical with the LDH isozyme band corroborating the pureness of the preparation (Fig. 2a and 2b). The molecular weight of BLLDH was deduced from both gel filtration column as well as SDS-PAGE to be 35 kDa (Fig. 1b and 3a). The maximum activity of BLLDH enzyme was investigated with Tris-HCl buffer, pH (7.0–9.0) since BLLDH enzyme manifested its optimum activity at pH 7.6 (Fig. 3b).
 | Figure 1. (a) A typical elution profile for the chromatography of the buffalo liver ammonium sulfate fraction on DEAE-cellulose column (6 cm × 2.4 cm i.d.) previously equilibrated with 0.02 M sodium phosphate buffer pH 7.4 containing 10 mM EDTA and 1 mM β-mercaptoethanol. (b) A typical elution profile for the chromatography of the buffalo liver DEAE-cellulose fraction (BLLDH) on Sephacryl S-300 column (142 cm × 2.4 cm i.d.) previously equilibrated with 0.02 M sodium phosphate buffer pH 7.4 containing 10 mM EDTA and 1 mM β-mercaptoethanol. [Click here to view] |
.png) | Figure 2. Electrophoretic analysis of BLLDH on 7% native PAGE: (1) crude extract, (2) ammonium sulfate fraction, (3) 0.05M NaCl DEAE-cellulose fraction, and (4) Sephacryl S-300 purified fraction (a) protein pattern and (b) LDH isoenzyme activity pattern. [Click here to view] |
.png) | Figure 3. (a) Subunit molecular weight determination by Electrophoretic analysis of BLLDH on 12% SDS-PAGE (1) Low molecular weight marker proteins and (2) purified BLLDH. (b) Effect of pH on the purified buffalo liver lactate dehydrogenase (BLLDH) using phosphate buffer, pH 7.2–9.0. [Click here to view] |
Influence of divalent cations and different inhibitors on BLLDH
The influence of metal cations on BLLDH activity was tested. MgCl2 and ZnCl2 elevated the activity of BLLDH, while FeCl2, CuCl2, and NiCl2 minimized it (Table 2). The BLLDH inhibition was performed by pre-incubating inhibitors with BLLDH at 37ËšC for 5 minutes and inhibition percent was derived as a rate of a non-inhibited specimen. SDS and N-ethylmaleimide inhibited the purified enzyme vigorously (Table 3).
Comparison of the constructed AST and ALT kits with commercially available kits
BLLDH is employed in constructing the ALT diagnosis kit. Also, BLLDH and the purified sheep liver malate dehydrogenase SLMDH previously purified in our lab (Darwish et al., 2018) are employed in constructing the AST diagnosis kit. The manufactured kits have been compared with commercially available kits utilizing 30 different individual samples (Tables 4 and 5).
 | Table 2. Effect of divalent cations on the purified BLLDH. [Click here to view] |
 | Table 3. Effect of inhibitors on the purified BLLDH. [Click here to view] |
DISCUSSION
LDH enzyme can be considered as a bio-indicator for liver, muscular, and cancer troubles (Rao et al., 2017; Zheng et al., 2017). LDH and MDH enzymes are predominant constituents of AST and ALT diagnosis kits, which are used as liver function biomarkers (Darwish et al., 2018; Huijgen et al., 1997). In this study, the purification process of buffalo liver LDH (BLLDH) included crude extraction, (NH4)2SO4 fractionation, DEAE-cellulose column, and Sephacryl S-300 column. This purification method is considered to be appropriate and proportionally short and consists only of two chromatographic steps. Diverse LDH purification processes were reported, LDH from liver of Uromastix hardwickii (Javed et al., 1995), LDH from yak Hypoderma sinense larva (Li et al., 2013), LDH from pig heart (Karamanos, 2014), LDH from heart ventricles of river buffalo (Nadeem et al., 2011), and LDH from bovine heart (Labrou and Clonis, 1995). The BLLDH enzyme obtained from the gel filtration column with 17.6 units/mg protein specific activity, 16 purification folds, and 32% output (Table 1). Large diversity of LDH purification folds and yield ratios were notified. LDH of H. sinense larva by 280-folds and 195 U/mg protein (Li et al., 2013), LDH of pig heart by 54.96 folds and 26.93 U/mg protein (Karamanos, 2014), and LDH of river buffalo heart ventricles with 48-fold and 410 U/mg protein (Nadeem et al., 2011).
 | Table 4. Comparison of the constructed AST kit with a commercially available kit. [Click here to view] |
In this study, the BLLDH pureness was inspected on 7% native-PAGE that manipulated single protein band matched enzyme activity band supporting homogeneity and pureness of BLLDH (Fig. 2). The intact molecular weight of BLLDH obtained from gel filtration was established as 35 kDa. The molecular mass of BLLDH isoenzyme was affirmed by SDS-PAGE revealing that BLLDH is a monomer 35 kDa protein (Fig. 3a). Consistent with BLLDH, LDH from the liver of U. hardwickii is a monomer protein of 34 kDa (Javed et al., 1995), from H. sinense larva is 36 kDa (Li et al., 2013), from pig heart is a monomer protein of 36 kDa (Karamanos, 2014), and from river buffalo heart ventricles is 36 ± 2 kDa (Nadeem et al., 2011). BLLDH showed its maximum activity at pH 7.6 (Fig. 3b) consistent with optimum pH of LDH from the liver of U. hardwickii at pH 7.5 (Javed et al., 1995), while river buffalo heart ventricles LDH was at pH 7 (Nadeem, et al., 2011). In this study, MgCl2 and ZnCl2 increased the activity of the purified BLLDH. FeCl2 was a potent inhibitor of BLLDH activity while CuCl2 and NiCl2 inhibited BLLDH activity moderately (Table 2). Consistent with this, Cu2+, Co2+, and Mn2+ have shown inhibitory effects toward LDH from the liver of U. hardwickii (Javed et al., 1995). SDS was the potent inhibitor for the purified BLLDH which is also inhibited by PMSF suggesting that BLLDH active site contains a serine residue. BLLDH was inhibited by iodoacetamide indicating the role of methionine, cysteine, and histidine residues on structure and activity of the enzyme. N-Ethylmaleimide inhibited BLLDH indicating the presence of -SH group in enzymatic action (Table 3).
 | Table 5. Comparison of the constructed ALT kit with a commercially available kit. [Click here to view] |
Both AST and ALT diagnosis kits are used to observe the liver job to follow the diverse hepatic diseases (Darwish et al., 2018; Zheng et al., 2017). Here, BLLDH is employed in the manufacturing of ALT diagnosis kit. Also, BLLDH and the previously purified sheep liver malate dehydrogenase SLMDH (Darwish et al., 2018) are utilized in constructing the AST diagnosis kit. The two manufactured kits were found sensitive when compared to ready trade kits. The distinction between structured and trade kits was noticed somewhat within the empirical error.
CONCLUSION
This study presents the first description of BLLDH. It was purified with simple and suitable purification steps from local source contain a large amount of the enzyme. Both BLLDH and SLMDH enzymes were included in the construction of ALT and AST diagnostic kits. The two prepared kits were found sensitive, accurate, and competent with the purchased kits. Production of these two kits on a large scale for commercial use will be our task for the future.
CONFLICTS OF INTEREST
The authors state that there are no conflicts of interest.
ACKNOWLEDGMENT
National Research Centre, Egypt is greatly appreciated for funding of this work, Agreement No. 11010403.
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