100 g of pigeon pea seeds were washed thoroughly with distilled water and soaked for 12 hr in pre cooled 200 mL Tris HCl buffer (100 mM, pH 8.0) at 4˚C. The seeds were homogenized in a kitchen blender for 2.5 min and filtered through a piece of double layered muslin cloth and centrifuged for 45 min at 4˚C at 13,000 g. About 176 mL of crude extract was collected and stored at 0˚C - 4˚C in a refrigerator. Crude extract was subjected to 40% - 70% ammonium sulphate precipitation by adding solid ammonium sulphate. The solution was stirred for at least 30 min at 0˚C - 4˚C and precipitated proteins were removed by centrifugation for 45 min at 4˚C at 13,000 g. The precipitate was collected and dissolved in Tris-HCl buffer (20 mM, pH 8.0). The enzyme obtained from ammo- nium sulphate fractionation was subjected to 30% polyethylene glycol (PEG-4000) precipitation. The protein precipitate was collected by centrifuging the content at 20,000 g for 45 min at 0˚C - 4˚C. The pellet was dis- solved in Tris-HCl buffer (20 mM, pH 8.0) and is stored at 0˚C - 4˚C in refrigerator. The enzyme solution was dialyzed against pre-chilled Tris-HCl buffer (20 mM, pH 8.0) at 0˚C - 4˚C with 5 - 7 repeated change of the same buffer by using dialysis membrane (50 KDa). The dialyzed sample was loaded on Carboxymethyl cellu- lose column, equilibrated with 100 mM Tris-HCl buffer of pH 8.0. The flow rate was maintained 30 - 40 mL/hr. The enzyme was collected from the unbound fraction (2 mL each). The unbound samples were tested for activ- ity and protein. The enzymically active fractions were pooled and precipitated out at 90% saturation of ammo- nium sulphate. The proteins were collected and dissolved in 20 mM Tris-HCl buffer, pH 8.0. The enzyme solu- tion was again dialyzed against pre-chilled Tris-HCl buffer (20 mM, pH 8.0) at 0˚C - 4˚C with 5 - 7 repeated change of the same buffer. The dialyzed sample was loaded on DEAE-cellulose column, equilibrated with 100 mM Tris-HCl buffer (pH 8.0). The flow rate was maintained 30 - 40 mL/hr. The column was washed with Tris- HCl buffer pH 8.0. The enzyme was eluted with same buffer containing 0.2 M KCl. The different eluted frac- tions were tested for activity and protein. The enzymically active fractions were pooled and precipitated out at 90% saturation of ammonium sulphate. The proteins were collected and dissolved in 20 mM Tris-HCl buffer, pH 8.0 and stored at −20˚C. The purified enzyme was stable with a minor loss of activity for at least 45 days when stored at −20˚C in 20 mM Tris-HCl buffer, pH 8.0.

The activity of Glucose-6-phosphate dehydrogenase enzyme has been assayed by determining the rate of forma- tion of NADPH at wavelength of 366 nm. The NADPH is formed as a result of oxidation of glucose-6-phos- phate to 6-phosphogluconolactone leading to reduction of NADP⁺ to NADPH. The reaction was started by add- ing 0.1 mL of appropriately diluted enzyme to 5.9 mL reaction mixture containing 0.2 mL glucose-6-phosphate (2 mM), 0.2 mL NADP⁺ (0.2 mM), 0.1 mL MgCl₂ (3.33 mM) and 5.4 mL assay buffer (55 mM Tris-HCl, pH 8.0). The rate of increase in absorbance at 366 nm was noted. The enzyme activity was calculated from ε_NADPH value = 3.11 × 10³ M⁻¹∙cm⁻¹ under mentioned conditions.

One unit of enzyme activity is defined as the amount of enzyme required to transform 1 µmole NADP⁺ to NADPH in one minute under our specific test conditions.

The UV spectrum of the purified enzyme was determined using UV-Vis spectrophotometer (Perkin-Elmer) in the wavelength range 240 - 360 nm.

The protein was estimated by the method of Lowry et al. [25] , using bovine serum albumin as a standard protein for the calibration of Folin-Ciocalteau phenol reagent.

Polyacrylamide gel electrophoresis in absence of any denaturing agent was carried out by the method of Laem- mli [26] .

Molecular weight was determined by gel filtration method. A thick slurry of preswellen and degassed Sepharose 6B was poured in a gel filtration column (1 × 50 cm). It was equilibrated with several bed volumes of extraction buffer (Tris HCl, 100 mM, pH 8.0). The column was calibrated by loading 1 ml each of lysozyme (14.7 KDa), ova albumin (45 KDa), Bovine Serum Albumin (130 KDa) and isocitrate dehydrogenase (141 KDa). After washing the column with same buffer, 1 mL of enzyme preparation was loaded. The elution was carried out at 0˚C - 4˚C with extraction buffer (flow rate 10 ± 2 mL/hr). Molecular weight of the enzyme was obtained from the calibration curve plotted with elution volume versus log molecular weight.

Subunit molecular weight for glucose-6-phosphate dehydrogenase was obtained by comparing its electropho- retic mobility in presence of sodium dodecyl sulphate (SDS) with mobilities of some known proteins, as the method described by Weber and Osborn [27] .

Effect of substrate concentration on purified glucose-6-phosphate dehydrogenase was seen at different concen- trations of glucose-6-phosphate varying from 0.5 mM to 35 mM and at fixed concentration of NADP⁺ (0.2 mM). Effect of coenzyme concentration on purified glucose-6-phosphate dehydrogenase was seen at different concen- tration of NADP⁺ varying from 0.1 mM to 45 mM and at fixed concentration of glucose-6-phosphate (2 mM) by determining the rate of formation of NADPH at 366 nm.

Effect of pH on the K_m and V_max values of substrate has been investigated in the pH range 7.5 - 9.0. The sub- strate concentration was varied from 0.5 mM - 10 mM for each pH value and the activity was assayed by deter- mining the rate of formation of NADPH at wavelength of 366 nm.

Effect of enzyme concentration on purified glucose-6-phosphate dehydrogenase was seen by adding different volume of enzyme in the reaction mixture [0.1 mL (2.7 U) - 1 mL (27 U)].

Effect of pH on purified glucose-6-phosphate dehydrogenase was seen by changing the pH of assay buffer in the range of 7.0 - 9.4.

Effect of temperature on purified enzyme was carried out by incubating the enzyme at different temperatures (ranging from 20˚C to 50˚C) for 5 min and then measuring the activity at 366 nm.

Thermal inactivation of purified NADP⁺ linked glucose-6-phosphate dehydrogenase from pigeon pea has been studied at different temperatures, i.e. 30˚C and 40˚C. For this experiment the enzyme solutions were incubated at 30˚C and 40˚C in water bath and aliquots were withdrawn at different time intervals and were chilled in the ice cold water and assayed for the activity of NADP⁺ linked glucose-6-phosphate dehydrogenase.

Substrate specificity of purified glucose-6-phosphate dehydrogenase was observed by using different substrates such as glucose-1-phosphate, glucose, fructose and galactose-6-phosphate. The reaction was started by adding 0.1 mL of appropriately diluted enzyme to 5.9 mL reaction mixture containing 0.2 mL, 2 mM different sub- strates (glucose-1-phosphate, glucose, fructose and galactose-6-phosphate), 0.2 mL NADP⁺ (0.2 mM), 0.1 mL MgCl₂ (3.33 mM) and 5.4 mL assay buffer (55 mM Tris-HCl, pH 8.0). The rate of increase in absorbance at 366 nm was noted.

Glucose-6-phosphate dehydrogenase was purified from pigeon pea by employing ammonium sulphate (40% - 70%) fractionation, polyethylene glycol precipitation, Carboxymethyl cellulose column chromatography and die- thylaminoethyl cellulose column chromatography. The finally purified enzyme obtained by diethylaminoethyl cellulose column chromatography was found to be 123.69 fold purified with a specific activity of 12.79 U/mg and 21.37% recovery. A sample protocol of purification and results obtained is documented in Table 1. An elu- tion profile of pigeon pea enzyme is given in Figure 1. 7.5% native Polyacrylamide gel electrophoresis yields a single protein band (Figure 2) indicating that the protein was obtained in pure form.

The UV spectrum of the purified enzyme was determined using UV-Vis spectrophotometer (Perkin-Elmer). The purified glucose-6-phosphate dehydrogenase shows a typical characteristic protein absorption spectrum in ultra- violet region with maximum absorbance of 0.39 (protein concentration 2.1 mg/ml) at 280 nm. The A₂₈₀/A₂₆₀ ra- tio of purified enzyme was found to be 1.5 suggesting that the enzyme preparation was free from nucleotides. The absorption spectrum of pigeon pea glucose-6-phosphate dehydrogenase is shown in Figure 3.

Molecular weight of pigeon pea glucose-6-phosphate dehydrogenase was determined by using gel filtration chromatography. Molecular weight of the enzyme was obtained from the calibration curve plotted with elution

volume versus log molecular weight (Figure 4). The molecular weight of purified glucose-6-phosphate dehy- drogenase was found to be 113 KDa. The molecular weight of glucose-6-phosphate dehydrogenases from hu- man erythrocyte was reported to be 105 KDa [28] , Pseudomonas W6 native enzyme was reported to be 123 ± 5 KDa [29] , isozymes of spinach was found to have a molecular weight of 105 ± 10 KDa [21] , Methylomonas M15 glucose-6-phosphate dehydrogenase was found to have a molecular weight of 108 ± 5 KDa [30] . A 112 KDa dimeric protein was reported from Schizosaccharomyces pombe [31] .

Subunit molecular weight for pigeon pea glucose-6-phosphate dehydrogenase was obtained by the method of Weber and Osborn [27] . After quantitating the relative mobility in SDS-PAGE, the molecular mass of purified glucose-6-phosphate dehydrogenase was found to be 55 KDa (Figure 5) indicating that the pigeon pea enzyme is a homodimer. The subunit molecular weight of pigeon pea glucose-6-phosphate dehydrogenase was found to be quite close to the molecular weight of enzyme obtained from Cryptococcus neoformans (50 KDa) [32] , A. vinelandii (52 KDa) [33] , dog liver (52.5 KDa) [34] , A. aculeatus (52 ± 1.1 KDa) [15] , goat erythrocyte (52 KDa) [35] . However, the value is lower than as reported from Pseudomonas W6 [29] , pig liver [20] , turkey erythrocyte [36] , coriander [24] and rainbow trout [37] . The result revealed that pigeon pea glucose-6-phosphate dehydrogenase is a homodimer with molecular weight of 113 KDa and subunit molecular weight approximately equals to 55 KDa. Active glucose-6-phosphate dehydrogenase from various sources has been reported as dimer, tetramer or hexamer except that of rainbow trout where the protein is active in its monomeric form [37] .

Effect of varying concentration of glucose-6-phosphate on the oxidation of glucose-6-phosphate was studied in Tris-HCl buffer (55 mM, pH 8.0). The substrate concentration was varied from 0.5 mM to 35 mM. The results (Figure 6) revealed that at low substrate concentration (0.5 mM to 2 mM), the increase in rate of reaction was directly proportional to the concentration of substrate. However, further increase in glucose-6-phosphate con- centration leads to insignificant increase in enzyme activity (Figure 6). In order to determine the Michaelis con- stant (K_m) and maximum velocity (V_max), the observations of Figure 6 were replotted into Lineweaver-Burk (LB) plot (Figure 7). The K_m and V_max value for glucose-6-phosphate was found to be 2.68 mM and 0.11 U/ml re- spectively. The K_m value was found to be higher than other reported K_m values in plant sources such as pea leaves (2000 µM) [23] , potato tuber (260 µM) [11] and coriander (116 µM) [24] . The K_m value was also greater

than reported from microbial and animal sources [15] [35] [38] . The V_max value was found to be greater than the value reported from coriander (0.038 U/ml) [24] . The V_max value from other plant sources has not been reported. The V_max value of glucose-6-phosphate was less than that of reported from animal sources, such as goose eryth- rocyte (0.28 U/ml) [39] , turkey erythrocyte (0.5 U/ml) [36] and rainbow trout erythrocyte (1.352 U/ml) [37] .

The pigeon pea glucose-6-phosphate dehydrogenase shows absolute specificity towards coenzyme NADP⁺, whereas it shows negligible activity with NAD⁺. Thus the detailed kinetics experiments have been carried out with NADP⁺ only. The initial rate of reaction was determined at various concentrations of NADP⁺. The results are shown in Figure 8. The K_m and V_max for NADP⁺ using Lineweaver-Burk plot was found to be 0.75 mM and 0.13 U/ml which was greater than that of values reported from pea leaves (500 µM) [23] , potato (6 µM) [11] co- riander (26 µM, 0.035 U/ml) [24] , rat liver (100 µM) [40] , Acetobacter hansenii (340 µM) [41] , goose erythro- cyte (7.4 µM, 0.286 U/ml) [40] , turkey erythrocyte (17.1 µM, 0.37 U/ml) [36] and rainbow trout erythrocyte (166 µM, 0.275 U/ml) [37] . However, the K_m value of NADP⁺ was found to be less than that of glucose-6-phos- phate which indicates that glucose-6-phosphate dehydrogenase shows more affinity towards NADP⁺. Similar results have been reported by various workers [11] [15] [23] [24] [35] [36] [38] -[42] . The initial rate of reaction was also determined at various concentrations of NADP⁺. The double reciprocal plot converges at a point above the abscissa (Figure 9) indicating a sequential binding mechanism in which both substrates must bind to enzyme simultaneously before product formation can occur. The results (Figure 9, Figure 10) depicted that the is larger than indicating that the binding of substrate enhances the affinity of the enzyme towards co- enzyme [15] .

Influence of pH on the K_m and V_max values of substrate has been investigated in the pH range 7.5 - 9.0. The double reciprocal plot gives a family of lines which converges at a point on the X-axis (Figure 11). The data of Figure 11 shows that the activity of pigeon pea glucose-6-phosphate dehydrogenase decreases as pH is lowered. Thus, suggests that at pH below 8.5, proton behaves as “non-competitive inhibitor”. The pK_a value was found to be 10.41, indicating that the amino acid residue at the active site might be lysine.

Effect of enzyme concentration on purified pigeon pea glucose-6-phosphate dehydrogenase was seen by adding different volumes of enzyme in the reaction mixture. The rate of formation of NADPH increases with increasing enzyme concentration (Figure 12).

The effect of pH on glucose-6-phosphate dehydrogenase activity was examined using Tris-HCl buffer (55 mM) of varying pH values ranging from 7.0 to 9.4. The result (Figure 13) shows that the pigeon pea glucose-6- phosphate dehydrogenase has a pH optimum of 8.2. The pH optima is in accordance to that reported from Neu- rospora crassa (pH 7.4 - 8.2) [19] and very close to that of pea leaves (pH 8.0) [23] . However, pH optimum is less as compared to two isozymes of spinach (pH 9.0 and 9.2) [21] .

Effect of temperature on pigeon pea glucose-6-phosphate dehydrogenase was carried out by incubating the en- zyme solution in a temperature range of 20˚C - 50˚C for 5 minutes (Figure 14). The result revealed that pigeon pea glucose-6-phosphate dehydrogenase is highly thermosenstive and the activity of the soluble enzyme starts decreasing after 30˚C which might be due to thermal denaturation of the enzyme above 30˚C. Optimum tem- perature of glucose-6-phosphate dehydrogenase from coriander and D. radiophilus was found to be 30˚C [24] [43] . The activities of the two isoforms of D. radiophilus decrease above 40˚C [43] .

Thermal inactivation studies of purified NADP⁺ linked glucose-6-phosphate dehydrogenase from pigeon pea

revealed that there is a progressive inactivation of enzyme with time at different temperatures. Semilog plot of the thermal inactivation (Figure 15) of enzyme was found to be linear, indicating that the thermal inactivation of pigeon pea enzyme follows simple first order kinetics at 30˚C and 40˚C with half life of 6 and 1.5 minutes re- spectively.

To determine the substrate specificity of enzyme, glucose-1-phosphate, glucose, fructose and galactose-6- phosphate were used as substrates. Out of these only galactose-6-phosphate was found to be oxidized by glu- cose-6-phosphate dehydrogenase. However, the relative rate of oxidation was low. The Lineweaver-Burk plot of galactose-6-phosphate (Figure 16) shows higher K_m value (K_m = 3.23mM) and lower V_max value (V_max =

0.008879 U/ml) than glucose-6-phosphate. The higher K_m value directly indicates that glucose-6-phosphate de- hydrogenase have lower affinity towards galactose-6-phosphate. Similar results were reported in case of Neuro- spora crassa [19] , however, in case of human placental enzyme affinity towards glucose-6-phosphate and ga- lactose-6-phosphate was found to be nearly equal [44] .