PURIFICATION AND CHARACTERIZATION OF ACID PHOSPHATASE FROM A GERMINATING BLACK GRAM ( VIGNA MUNGO L . ) SEEDLING

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55°C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

Acid phosphatase is believed to be important for many physiological processes, including regulation of phosphorus efficiency.Exuded and internal acid phosphatase activities change in response to phosphorus availability.Low phosphorus availability increases acid phosphatase secretion to the rhizosphere in a number of plant species, including maize (Zea mays), tomato (Lycopersicon esculentum) wheat (Triticum aestivum) and clover (Trifolium spp.), lupin (Lupinus spp.), rice (Oryza sativa), and soybean (Glycine max).From seeds and seedlings, the physiological function of the acid phosphatases is to provide inorganic phosphate to the growing plant during germination, and many different phosphate esters of sugars and substrates stored in the seed and seedling need to be hydrolyzed during germination and growth (Gahan and McLean, 1969;Schultz and Jensen, 1981;Akiyama and Suzuki, 1981;Hoehamer et al., 2005).
In this study, different chromatographic methods were employed to purify acid phosphatase from black gram seedlings, and their properties were examined.

Collection of black gram seeds
Black gram seeds were collected from Bangladesh Agriculture Research Institute (BARI), sub-station of Iswardi, Pabna, and brought to the protein and enzyme laboratory of Biochemistry and Molecular Biology, University of Rajshahi.After collection, the seeds were cleaned, dried in sunlight and stored in airtight desiccators in a polyethylene bag for experimental purpose.

Black gram seed germination
Black gram seeds were sterilized with 1% (V/V) sodium hypochloride solution for 10 min, washed with water three times and soaked in water for 24 h.After hydration, germination of black gram seed was performed on a moist cotton cloth during 5 days in a room at ambient temperature of 28°C with a relative humidity of 85%.Germinating seeds were watered daily.The seedling was separated to cotyledons for enzyme activity assays.

Preparation of crude enzyme extract
In order to purify enzyme from the seedling of black gram in a biologically active form, all the operations were performed at 4°C.50 gm of black gram was put in a mortar and ground uniformly into a fine paste.The paste was mixed uniformly with pre-cooled citrate buffer, pH 4.8 in a homogenizer and homogenized uniformly.The extract was then transferred into a beaker and kept overnight at 4°C with occasional gentle stirring.The homogenate was then filtered through a muslin cloth.The filtrate was collected and clarified further by centrifugation at 8×10 3 r.p.m. for 30 min at 4°C.After centrifugation, the clear supernatant was used as crude extract containing the partially purified that was precipitated with ammonium sulfate.

Enzyme assay
Acid phosphatase activity was based on conversion of para-nitrophenyl phosphate (pNPP) into p-nitrophenol (pNP).The reaction mixture was incubated at 37 0 C for 10 min, then 2 ml of sodium carbonate (2% w/v) were added to stop the reaction and absorbances were measured at 410 nm using a spectrophotometer.pNP was used as standard.Under experimental conditions, one unit of enzyme activity was defined as 1µmol of pNP released per min.Specific activity was defined as the units of enzyme activity per mg of protein.

Buffer selection
After initial supernatant and 70% (NH 4 ) 2 SO 4 saturation by a different buffer (100 mM sodium acetate buffer, 20 mM citrate buffer and water fraction), the highest activity of acid phosphatase was found in citrate buffer (pH 4.8).This buffer was selected for the purification step.

Protein estimation
Protein elution profiles from chromatographic columns were monitored by measuring fractional absorbance at 280 nm.The concentration of purified enzyme was determined according to Lowry et al. (1951).BSA was used as the standard protein.

Gel filtration
After dialysis with 20mM citrate buffer, the sample at pH 4.8 was loaded on a Sephadex G-75 gel column which was equilibrated with the same buffer.The column was washed with 20 mM citrate buffer, pH 4.8, containing 1 M sodium chloride.

DEAE-cellulose chromatography
The enzymatically active protein fractions obtained after gel filtration were dialyzed with distilled water for 12 h and against 20 mM Tris-HCl buffer, pH 7.4 overnight.After centrifugation, the clear supernatant was applied to the DEAE-cellulose column which was previously equilibrated with the same buffer.The protein was eluted stepwise with increasing concentrations of the NaCl from 0 to 0.2 M with the same buffer.

Molecular weight determination by SDS-PAGE
Proteins were analyzed by sodium dodecyl-sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli.

Effects of pH and temperature
The effect of pH on the enzyme activity was determined by measuring the hydrolysis of the substrate pNPP in a series of buffer at various pH values ranging from pH 3.0-9.0.20 mM citrate buffer was used.The pH value of buffer was determined at 37 0 C. The effect of temperature on acid phosphatase activity was performed in 20 mM citrate buffer pH 5.0 over a temperature range of 10-90 0 C using pNPP under standard test conditions.

Effect of incubation period on the activity of acid phosphatase
In order to ascertain the effect of the incubation period on the enzyme activity, the enzyme solutions were kept at 55°C, pH 5.0 for 20, 40, 60, 80 and 120 min, respectively, and the residual activity was assayed.

Effect of some chemical agents
To determine the effect of various compounds (metal ions, detergents and dithiol-reducing agents) as possible activators or inhibitors of the purified acid phosphatases, the enzymatic solutions were preincubated at 37 0 C for 30 min with the compounds and then the activity was assayed.The substrate pNPP was added to the medium and incubated at 37 0 C for 10 min.

Kinetic parameter of acid phosphatase
The initial velocity was equal to the amount of product formed per unit time.The initial velocity (Vi) was determined by quantitatively measuring the amount of one of the products at various time.

Substrate specificity of acid phosphatase
The substrate specificity of black gram acid phosphatase was measured using the following substrates: pNPP, phenylphosphate, galactose-1-phosphate, glucose-1-phosphate.In order to determine substrate specificity, 0.25 ml of 20 mM citrate buffer substrate pH-5.0placed in different test tubes.Then 0.1 ml of enzyme solution was added to each tube and incubated at 37°C for 30 min, and the acid phosphatase was stopped by adding 4 ml of 0.1 N NaOH.The hydrolysis of these substrates was determined by titration of inorganic phosphate according to method.

Chemicals
Acrylamide, magnesium chloride, SDS were purchased from Sigma Chemicals Ltd., USA.DEAEcellulose was purchased from Pharmacia Chemicals Ltd., Sweden.All other chemicals were analytical grade.

Acid phosphatase activity in germinating black gram seedlings
During the germinating stage the acid phosphatase activity in black gram ranged from 0.08-4.9unit/L.

Buffer selection
Enzymatic activity of acid phosphatase from black gram seedlings by different buffer is shown in Fig. 1.

Gel filtration chromatography
The crude protein extract was eluted as two main peaks, namely F-1 and F-2.It was found that the F-1 fraction contained acid phosphatase activity while the F-2 peak showed no activity (Fig. 2).The fraction F-1 containing the acid phosphatase activity as indicated by a solid line was pooled, concentrated and further purified by DEAE-cellulose column chromatography.

DEAE-cellulose chromatography of F-1 fraction
The components of the F-1 fraction were separated into four peaks F-1a, F-1b, F-1c and F-1d, with the buffer containing 0.02 M, 0.05 M, 0.1 M and 0.2 M NaCl gradients, respectively.The enzymatic activity of all these fractions was investigated and it was found that the fractions from peak F-1c contained acid phosphatase activity while the fractions F-1a, F-1b and F-1d possessed no acid phosphatase activ- ity (Fig. 3).The fraction containing acid phosphatase activity was pooled separately and dialyzed against 20 mM Tris-HCl buffer, pH 7.4 at 4°C overnight, and then concentrated.

Determination of molecular weight by gel filtration method
The molecular weight of the enzyme was determined by comparing their elution volume on sephadex G-75 with those of the marker proteins under the same experimental conditions.The molecular weight was calculated from the standard curve (Fig. 4) of the reference proteins, constructed by plotting a log of molecular weight against elution volume on gel filtration.The molecular weight of purified enzyme was found to be 25 kDa.

Molecular weight determination by SDS-PAGE
The molecular weight of the enzyme was also determined by SDS slab gel electrophoresis (SDS-PAGE) method (plate-1) using β-galactosidase (MW 116,000), Bovine Serum Albumin (MW 66,000), Egg white Albumin (MW 45,000), Trypsin Inhibitor (MW 20,000), Lysozyme (MW 14,600) as standard proteins.The molecular weight of purified enzyme was found to be 25 kDa.

Effects of pH and temperature
The effect of the pH and temperature on acid phosphatase activity is shown in Figs 5 and 6.The enzyme

Determination of incubation period
Purified acid phosphatase activity at various incubation periods is shown in Fig. 7. Incubation at the assay temperature for acid phosphatase activity (55°C) for various incubation periods showed that the enzyme gradually loses with time.The highest acid phosphatase activity was found when the homogenate was assayed after heat treatment for 30 min.After 1 h or even 2 h, the activity of the acid phosphatase remained at 44%.The decrease in activity with time was probably due to denaturation of the enzyme or the self-degradation properties of enzyme.

Effect of metal ions and chelating agents
The influence of various metal ions and chelating agents on the purified acid phosphatase is presented in Tables 2 and 3. Black gram seedling acid phosphatase was activated by K + , Cu 2+ and Ba 2+ , while Mg 2+ , Sr 2+ and Zn 2+ had an inhibitory effect on the activity of acid phosphatase.EDTA, a metal chelating agent, had an inhibiting effect on the acid phosphatase of the seedlings of black gram .The decrease in activity may be due to the removal of metal ions located on or near the active site.

Kinetic properties of acid phosphatase
The activity of purified acid phosphatase was analyzed using different concentrations of para-nitrophenyl phosphate as substrate.As shown in Fig. 8, the extrapolated Km value for acid phosphatase from black gram seedlings was estimated to be 0.49 mM.

Substrate specificity of acid phosphatase
Activity of the acid phosphatase was assessed using the different phosphate compounds as substrate and the result is presented in Table 4.The acid phosphatase was found to be capable of hydrolyzing pNPP, phenyl phosphate and glucose-1-phosphate.The hydrolysis activities of these three substrates varied from 21.4 to 100 for the respective acid phosphatase.But the enzyme had no effect on galactose-1-phosphatase.This indicated that the activity of the enzyme was not restricted to a single substrate.

CONCLUSION
Germination is accompanied by the synthesis or activation of enzymes responsible for the degradation of seed reserves.Among these enzymes, acid phosphatases are involved in the metabolic proc- esses of germination and maturation of plants.
They are constitutively expressed in seeds during germination, and their activities increase with germination to release the reserve materials for the growing embryo (Biwas and Cundiff, 1991;Thomas, 1993).In the present study, acid phosphatase activities increased and reached the maximum on the 5 th day of black gram seeds germination at temperatures below 28 0 C. Prazeres et al.(2004) reported that when pNPP was used as a substrate the maximum acid phosphatase activity of soybean seedlings was detected on the 6 th and 9 th days, for germination temperatures of 28 0 C and 20 0 C, respectively.These results suggest that temperature and seed species have an influence on germination phenomena.
Several studies have been devoted to the purification of acid phosphatases from the cotyledons of germinating seeds (Ullah and Gibson, 1988;Basha, 1984;Granjeiro et al., 1999).However, few works concerning the purification of seedlings' acid phosphatase have been reported so far (Bhargava and Sachar, 1987;Yenigum and Guvenilir, 2003).To better understand the role played by acid phosphatase during black gram seed germination, acid phosphatases from the crude extract of black gram seedlings were purified to homogeneity by chromatographic process and their properties were examined.
The purified acid phosphatase was sensitive to ions at various degrees depending on the ions' nature and isoenzymes.The requirement of metallic ions for acid phosphatase activity has also varied according to plant species, development stage and isoenzymes heterogeneity (Scandalois, 1974;Panara et al., 1990;Tso and Chen, 1997).However, Mg 2+ , Zn 2+ and EDTA were inhibitors and, K + , Cu 2+ and Ba 2+ were activators for the enzyme.The inhibition of acid phosphatase activity by Zn 2+ has been previously reported in other plant species such as pea (Mizuta and Suda, 1980), tobacco (Pan et al., 1987) and peanut seedlings (Gonnety et al., 2006).
For the enzymes, kinetic study correlates well with substrate specificity and showed a Lineweaver-Burk plot.The purified acid phosphatase showed the highest catalytic efficiency toward pNPP.The Km value (0.49 mM) of acid phosphatase was similar to the Km value of acid phosphatase from soybean seeds and peanut seedlings toward pNPP (Ferreira et al., 1998;Gonnety et al., 2006).The purified enzymes hydrolyzed broad phosphorylated substrates to various degrees.Similar observations have been reported for the acid phosphatase from sweet potato (Kusudo et al., 2003) and peanut seedlings (Gonnety et al., 2006).This indicates that the activity of enzyme was not restricted towards a single substrate.

Fig. 4 .Fig. 5 .Fig. 6 .
Fig. 4. Effect of temperature on the activity of acid phosphatase from the germinating black gram seedling

Table 1 .
Acid phosphatase activity in germinating black gram seedling Table.2. Effect of EDTA on the activity of acid phosphatase enzyme

Table 3 .
Effect of various metallic salts on acid phosphatase activity

Table - 5
: Acid phosphatase purification from germinating black gram seedling

Table - 4
: Substrate specificity of acid phosphatase