A rapid and simple purification method of glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD) and glutathione reductase (GR) from camel kidney with a high yield was carried out. Camel kidney enzymes (G6PD, 6PGD, and GR) were purified homogeneously by one chromatographic step on 2′, 5′ ADP Sepharose 4B affinity column. After chromatography, the specific activity of G6PD was increased to 19.5 units/mg protein with 65.4% yield and 300-fold purification, while the specific activity of 6PGD was increased to 42.4 units/mg protein with 75.8% yield and 557.9-fold purification and the specific activity of GR was increased to 35.5 units/mg protein with 68.9% yield and 710-fold purification. G6PD, 6PGD and GR enzymes are appeared homogeneous on both native and 12% SDS-PAGE, with molecular weights of 60 kDa, 58 kDa and 65 kDa respectively. They displayed their optimum activity at pH 8.0, 8.2 and 7.8 respectively. The divalent cations MgCl2 and MnCl2 increased G6PD and 6PGD activity, while FeCl2, CuCl2 and ZnCl2 inhibited them. CuCl2, ZnCl2 and NiCl2 increased GR activity, while FeCl2 and MgCl2 inhibited it. NADPH inhibited G6PD and 6PGD activity while NADP inhibited GR activity. This study is the first report on the purification of G6PD, 6PGD, and GR from the camel kidney. A task for the future is the production of these enzymes for industrial medical applications
Keywords: 6-phosphogluconate dehydrogenase; Camel kidney; Glucose-6-phosphate dehydrogenase; Glutathione reductase
Glucose 6-phosphate dehydrogenase (G6PD) is the key and first enzyme of the pentose phosphate metabolic pathway that catalyzes the glucose 6-phosphate conversion to 6-phosphogluconate in the presence of + NADP . There are two main tasks of the pentose phosphate pathway in the cells, ribose 5-phosphate synthesis that is required for DNA, RNA and ribonucleotides and synthesis of phosphorylated carbohydrates that are important for synthesis of aromatic amino acids, vitamins and sedoheptulose-7-phosphate [1]. G6PD was purified from different mammalian organisms and tissues including rabbit liver [2], camel liver [3], mouse liver [4], dog liver [5], sheep erythrocytes [6], sheep kidney [7], rat kidney [8], from grass carp [1] and from bacteria [9]. 6-Phosphogluconate dehydrogenase (6PGD) is the second enzyme of the pentose phosphate metabolic pathway that catalyzes the transformation of 6- phosphogluconate to D-ribulose-5-phosphate and reduces NADP to NADPH [10]. 6PGD can be considered as an antioxidant enzyme due to yielding of NADPH, which protects the cell against oxidant agents by yielding reduced glutathione (GSH) and shares in the production of some molecules as fatty acids, steroids and amino acids [11, 12, 13]. The reactions catalyzed by G6PD and 6PGD in the pentose phosphate metabolic pathway are considered the major source of NADPH [12]. 6PGD enzyme has been purified from various sources from bacteria to mammals as rat erythrocytes [10], rat lung [14], chicken liver [15], from bacteria [16] and from grass carp [17]. Glutathione reductase (GR) is a member of the pyridine-nucleotide disulfide oxidoreductase family of flavoenzymes that performs a critical part to maintain a high intracellular GSH/GSSG ratio and converts NADPH + to NADP [18, 19]. GR has been purified from numerous various origins as diatom algae [20], rainbow trout liver [21], bovine erythrocytes [22], bovine liver [23], sheep liver [24, 25] and turkey liver [26]. The pentose phosphate pathway cycle is major for tumor cell metabolism and growth. G6PD deficiency is one of the haemolytic anaemia reasons and inhibitors of 6PGD and GR are used as anticancer and antimalarial drugs [27, 28]. Knowing the behaviour of these enzymes and their inhibitors will be helpful in the drug design and pharmaceutical investigations. Therefore, this study aims at purification and characterization of three important antioxidant enzymes G6PD, 6PGD and GR from the camel kidney using affinity chromatography. Such a study will be helpful to understand the regulatory mechanisms of these enzymes. The camel kidney is very large organ, available, safe, cheap and rich source for extraction and purification of G6PD, 6PGD and GR enzymes in comparison with the other sources and will allow large scale production of the three enzymes. G6PD enzyme is used in the preparation of glucose diagnostic kit (HK/G6PD; hexokinase/glucose-6-phosphate dehydrogenase) which is used for in vitro quantitative determination of glucose for diagnostic and medical purposes. G6PD, 6PGD and GR enzymes are included in antioxidant drugs preparations to protect the skin and hair against inflammatory reactions associated with chemical irritation.
Six fresh kidney samples of healthy adult camels Camelus dromedarius were obtained from a local governmental slaughter-house in Cairo and preserved at - 20 °C. The weights of kidneys were 900-1000 gm, and the dimensions were 9-10 cm width and 15-17 cm length. The health of camels was inspected by slaughter house veterinary doctors before slaughtering.
Glucose-6-phosphate (G6P), 6-phosphogluconic acid trisodium salt (6PG), β-Nicotinamide adenine + dinucleotide phosphate (NADP ), β-Nicotinamide adenine dinucleotide phosphate, reduced (NADPH) and all the fine chemicals were purchased from SigmaAldrich Chemical Co. (Germany). Molecular weight SDS marker proteins and 2′, 5′ ADP Sepharose 4B were products of GE Healthcare Fine Chemicals Co. (Sweden). The other chemicals were of analytical grade and purchased from Carl Roth Chemical Co. (Germany).
The G6PD assay reaction mixture contained in 1 ml 100 mM Tris-HC1 buffer pH 8.0, containing 0.2 mM NADP, 10 mM MgCl2 and 0.6 mM G6P. The reaction was started by enzyme addition and G6PD activity monitored by detecting the rate of NADPH production level spectrophotometrically at 340 nm [29]. The 6PGD assay reaction mixture contained in 1 ml 50 mM Tris-HC1 buffer pH 7.5, containing 3.3 mM MgC12, 0.25 mM NADP and 2 mM 6-phosphogluconate. The reaction was started by enzyme addition and 6PGD activity monitored by detecting the rate of NADPH production level at wavelength 340 nm [30]. One unit of G6PD or 6PGD was defined as the amount of enzyme catalyzing the reduction of 1 μmol of NADP/min. The GR assay reaction mixture contained in a total volume of 1 ml, 50 mM K-phosphate buffer, pH 7.0, 1 mM EDTA, 0.1 mM NADPH, 0.5 mM oxidized glutathione and the enzyme solution. One unit of GR activity is defined as the amount of enzyme which oxidizes 1 μ mole of NADPH per min [31].
For G6PD activity staining, the gel was submerged in 40 ml of 0.1 M Tris-HCI buffer pH 8, containing 4 mg of Phenyl methosulfate (PMS), 3.2 mg of Nitroblue tetrazolium (NBT), 4 mg of MgCI2, 13 mg of glucose 6- phosphate. The gels were incubated in the staining solution in dark at 37 °C for 60-90 min [32]. For 6PGD activity staining, the gel was submerged in 40 ml of 0.1 M Tris-HCI buffer pH 8, containing 4 mg of PMS, 3.2 mg of NBT, 4 mg of MgCI2, 13 mg of 6- phosphogluconate trisodium salt. The gels were incubated in the staining solution in dark at 37 °C for 60 min [33]. For GR activity staining, the gel was soaked in 25 ml of 50 mM Tris-HCl buffer, pH 7.9, containing 4.0 mM GSSG, 1.5 mM b-NADPH, and 2 mM DTNB with gentle shaking for 20 min. After a brief rinse with 50 mM TrisHCl buffer pH 7.9, the GR activity was negatively stained in darkness by 50 ml 1.2 mM MTT and 1.6 mM PMS for 5–10 min at room temperature with gentle shaking. Clear zones of GR activity against the purple background were found [34].
All of the procedures were performed at 4 ºC unless stated otherwise. Ten grams of fresh kidney were minced and homogenized by omni-mixer (Sorvall Dupont Instruments), with two volumes (2 ml/gm tissue) of 0.02 M K-phosphate buffer, pH 7.6 containing 1 mM DTT and 1 mM EDTA on ice. The mixture was centrifuged at 12.000 xg for 30 min at 4 ˚C and the pellet was discarded. The supernatant was saved and designated the crude extract.
2 gm of 2′, 5′ ADP Sepharose 4B resin was suspended in 200 ml distilled water. The gel was degassed, suspended in 0.1 M K-phosphate buffer, pH 6.0, packed in the column (10 x 1 cm) and equilibrated with 0.05 M K- phosphate buffer including 1 mM DTT, 1 mM EDTA, pH 7.5, by means of a peristaltic pump. The flow rate was adjusted to 20 ml/h. The sample was loaded onto the column and washed with 25 ml of 0.1 M K-phosphate, pH 6 and 50 ml of 0.1 M K-phosphate, pH 7.8, containing 0.1 M NaCl. Then, the washing was continued with 0.05 M K-phosphate buffer including 1 mM EDTA, 1 mM DTT, pH 7.5, until the absorbance became zero at 280
†One unit of G6PD or 6PGD enzymes activity is identified as the amount of enzyme required to reduce 1 †One unit of GR enzyme activity is identified as the amount of enzyme required to oxidize 1 nm. The G6PD enzyme was eluted with 100 ml of 0.1 M K-phosphate buffer pH 7.8 containing 80 mM NaCl, 0.5 mM NADP and 1 mM EDTA. After elution of G6PD activity, the 6PGD enzyme was eluted with 100 ml of 0.1 M K-phosphate buffer pH 7.8 containing 80 mM NaCl, 5.0 mM NADP and 1 mM EDTA. After elution of 6PGD activity, the GR enzyme was eluted with 100 ml 0.1 M K- phosphate buffer pH 7.8 containing 1 mM GSH, 0.5 mM NADPH and 1 mM EDTA [8]. The activities of all fractions were measured and graphed.
Native gel electrophoresis was carried out using 7% PAGE according to Smith [35]. SDS-PAGE was performed with 12% polyacrylamide gel according to Laemmli [36]. The molecular weights of the purified G6PD, 6PGD and GR enzymes were determined using SDS-PAGE as described by Weber and Osborn [37].
Proteins were stained with 0.25% coomassie brilliant blue R-250.
Protein was quantified by the dye binding assay method using bovine serum albumin (BSA) as a standard protein [38].
The major aim of this study is to establish a high-yield and fast procedure for the purification to homogeneity of the three antioxidant enzymes G6PD, 6PGD and GR.
Here, a fast, simple and very convenient purification ‡Specific activity is expressed as units/mg protein.
method for G6PD, 6PGD and GR from camel kidney by one chromatographic step on 2′, 5′ ADP Sepharose 4B affinity column was carried out. The purification scheme of G6PD, 6PGD and GR is summarized in (Table 1). After chromatography on 2′, 5′ ADP Sepharose 4B column (Figure 1), G6PD specific activity was increased to 19.5 units/mg protein representing 65.4% yield and 300-fold purification over the crude extract, 6PGD specific activity was increased to 42.4 units/mg protein representing 75.8% yield and 557.9-fold purification over the crude extract and GR specific activity was increased to 35.5 units/mg protein representing 68.9% yield and 710-fold purification over the crude extract (Table 1). Different specific activities and purification folds have been stated for these enzymes. G6PD from sheep kidney had a specific activity of 27.69 units/mg protein, 1348- fold purification and 16.96% yield [2] and from sheep erythrocytes had a specific activity of 4.6 units/mg protein and 37.1% yield [6]. 6PGD from rat erythrocytes had a specific activity of 5.15 units/mg protein, 2575-fold purification and 78.4% yield [10], 6PGD from chicken liver had a specific activity of 61 units/mg protein, 344- fold purification and 5.5% yield [15] and from grass carp with a specific activity of 5.2 units/mg protein, 309-fold purification and 68% yield [17]. GR from human erythrocyte had a specific activity of 20.7 units/mg protein, 8600-fold purification and 26% yield [18]. GR from bovine liver has been purified 5456-fold purification and 38.4% yield [23], from rainbow trout liver had a specific activity of 27.7 units/mg protein, 1654-fold purification and 41% yield [21] and from turkey liver had a specific activity of 606.6 units/mg protein, 2476-fold purification and 10.7% yield [26].
+ μmol of NADP per minute.
μmol of NADPH per minute.
The purity of G6PD, 6PGD and GR eluted from the 2′, 5′ ADP Sepharose 4B column was tested by analysis on 7% native PAGE. The three enzymes G6PD, 6PGD and GR showed one protein band in each (Figure 2a) corresponding the isoenzymes activity bands (Figure 3) revealed a homogeneous preparation and indicating the purity of these enzymes. The molecular weights of G6PD, 6PGD and GR were determined by SDS-PAGE to be 60 ± 1.22, 58 ± 1.43 and 65 ± 1.37 kDa respectively (Figure 2b). Similar molecular weights were reported; 62 kDa of bovine lens G6PD [39], 59 kDa of rat erythrocytes 6PGD [10], 64 kDa of sheep brain GR [40] and 55 kDa of bovine liver GR [23] while diatom algae GR was 118
12% SDS-PAGE, (1) molecular weight marker proteins,
GR.
The effect of pH on the purified G6PD, 6PGD and GR activities were examined in 0.02 M K-phosphate buffer, pH (7.2 - 9.0). The pH profile of G6PD, 6PGD and GR displayed their optimum activities at pH 8.0, 8.2 and 7.8 respectively Figure (4). Similar pH optimum ranges were
The purified G6PD, 6PGD and GR enzymes were preincubated with 2 mM and 5 mM of each cation for 5 min at 37 ˚C and the activity was assayed. A control test without any cation was taken as 100% relative activity. The activity of G6PD was inhibited in presence of FeCl2, CuCl2 and ZnCl2 while increased with MnCl2 and MgCl2 (Table 2), this is in good agreement with G6PD of rainbow trout liver [41]. Also, 6PGD was inhibited in presence of FeCl2, CuCl2 and ZnCl2 and increased with MnCl2 and MgCl2. While GR activity increased by CuCl2, ZnCl2 and NiCl2 and inhibited by FeCl2 and MgCl2 in agreement with rainbow trout liver GR [21].
The purified G6PD, 6PGD and GR enzymes were preincubated with 2 mM and 5 mM of each inhibitor (0.1 mM and 0.2 mM of NADPH for G6PD and 6PGD and 0.1 mM and 0.2 mM of NADP for GR) for 5 min at 37 ˚C and the residual activity was calculated as a ratio of a control lacking inhibitor (Table 3).
found to be very weak inhibitors for all enzymes. 1, 10 Phenanthroline and ADP are weak inhibitors for all enzymes. β-Mercaptoethanol, DTT and PMSF are moderate inhibitors for all enzyme activity. NAD has no inhibitory effect on G6PD and 6PGD but inhibited GR activity. β-Mercaptoethanol and DTT inhibited G6PD, 6PGD and GR indicating that SH groups in the active site of these enzymes important for their activity. PMSF inhibited G6PD, 6PGD and GR indicating that serine is involved in the active site of these enzymes.
In Conclusion, this study presents a simple, rapid and convenient method for the purification of G6PD, 6PGD and GR from camel kidney with a high recovery. The procedure is adaptable for enzymes production by scaling up; the characterization of the purified enzymes will allow their uses with maximum efficiency.
The authors declare that there are no conflicts of interest. All authors have approved the manuscript and agree with its submission to the journal of applied pharmaceutical science.
This article does not contain any studies with human participants or animals performed by any of the authors. We get our samples from slaughtered camel in slaughterhouse.
G6PD: Glucose-6-phosphate dehydrogenase, 6PGD: 6- Phosphogluconate dehydrogenase, GR: Glutathione reductase, G6P: Glucose-6-phosphate, GSSG: Oxidized +: glutathione, GSH: Reduced glutathione, NADP β- Nicotinamide adenine dinucleotide phosphate, NADPH: Reduced β-Nicotinamide adenine dinucleotide phosphate, NBT: Nitroblue tetrazolium, PAGE: Polyacrylamide gel electrophoresis, PMS: Phenazine methosulfate, ROS: Reactive oxygen species, MTT: 3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide, DTNB: 5,5′- Dithiobis(2-nitrobenzoic acid).