Psoralea corylifolia L. (Bhavanchi, Fabaceae), an annual herb distributed
widely in tropical and sub-tropical regions, exhibits properties such as antitumor,
antibacterial, antifungal and antioxidative activities and is used in curing
stomach ache, anthelmintic, diuretic and diaphoretic in febrile conditions (Baskaran
and Jayabalan, 2007; Chanda et al., 2011).
The active principle of the plant consist commercially valuable psoralen and
isopsoralen. Psoralen is used for the photochemotherapy of vitiligo and skin
diseases such as psoriasis, mycosis fungoides and eczema due to photosensitizing,
photobiological and phototherapeutic properties (Frank et
al., 1998). Wild population of Psoralea has declined due to poor
rate of seed germination (Baskaran and Jayabalan, 2008,
2009), a process that requires acid phosphatase enzyme
to drive metabolic processes in the cell. Acid phosphatase catalyzes the hydrolysis
of tLuthrahe terminal phosphate of phosphomonoesters to release inorganic phosphate
and free energy required for seed germination (Guo and Pesacreta,
1997). The hydrolysis of phosphomonoesters by phosphatases in biological
systems is an important process. This process is linked to energy metabolism,
metabolic regulation and a wide variety of cellular signal transduction pathways.
The role of acid phosphatase in phosphorus metabolism has been extensively studied
in prokaryotic and eukaryotic systems (Al-Omair, 2010).
Plants use the enzyme to scavenge phosphate from organic sources under phosphate-limited
conditions (Lefebvre et al., 1990), pathogen
and salt stress (Parida and Das, 2004; Schweighofer
et al., 2004) and development (Luan, 1998).
Plant acid phosphatase contributes to the mobilization of phosphate from macromolecular
organic phosphates during seed germination and seedling growth (Duff
et al., 1994). The functioning of different metabolic processes and
growth of seedlings during germination are essentially dependent on the acid
phosphatase catalyzed solubilization and mobilization of organic phosphates
in soil (Bishnoi et al., 1993; Kawarasaki
et al., 1996).
In the present investigation, we have characterized the properties of acid phosphatase enzyme from the crude extract of cotyledons of medicinally valuable Psoralea corylifolia for the first time as a step towards understanding its properties. The optimum concentration of substrate p-NPP, pH, temperature and influence divalent metal ions, modulators and some of the phosphate substrate were determined. Further, the enzyme was partially purified and was analyzed by two dimensional gel electrophoresis followed by western blot.
MATERIALS AND METHODS
Materials: Seeds of Psoralea were obtained from local market of Delhi, India. p-NPP (p-Nitrophenyl phosphate), p-NP (p-Nitrophenol), NaF, NaMoO4, ATP, G-6-P, KH2PO4, NaH2PO4, BSA (bovine serum albumin), Coomassie brilliant blue G-250 and Phenylmethylsulphonyl flouride (PMSF) were purchased from Sigma Chemical Company (St. Louis, MO, USA). The HPLC solvents and chemicals were purchased from E-Merck.
Germination of Psoralea seeds: Seeds of Psoralea were surface sterilized with 2% NaClO solution for 10 min, washed with sterile water and germinated in autoclaved vermiculite in dark at 30°C incubator in July 2009. After 48 h of germination, plants were harvested, uprooted and thoroughly rinsed with distilled water. Cotyledons and embryonic axis were carefully detached, rinsed with distilled water, dried with filter paper, weighed and stored at -80°C.
Extraction of acid phosphatase: Cotyledons and embryonic axis (1 g)
were ground separately in a mortar (1 g 9 mL-1 buffer) with an extraction
mixture consisting of cold 0.1 M K2HPO4/KH2PO4
(K-Pi) buffer (pH 5.5), 2% nonyl phenyl ethylene glycol (NP- 40), 1 μg
mL-1 lysozyme 1 μg mL-1 each of protease inhibitor
cocktail (pepstatin, leupeptin and apoprotinin) along with 10 mM Phenyl Methyl
Sulphonyl Fluoride (PMSF) to give separate homogenous mixture of cotyledons
and embryonic axis (Luthra and Singh, 2010). Each homogenate
was centrifuged at 17000 rpm at 4°C for 20 min, producing a supernatant
designed as crude extract.
Colorimetry assay for acid phosphatase: Activity of acid phosphatase
(crude extract) was assayed at 37°C. The assay started after adding 15 μL
of total homogenate to reaction mixture (985 μL) containing 2 mM p-NPP
in 0.1 M sodium acetate buffer (pH 5.5). After 60 min the reaction was stopped
by adding 100 μL of 2 N NaOH; the absorbance of the solution was measured
at 405 nm using a Multimode microplate reader (Biotek Synergy, USA) (Senna
et al., 2006).
|| Epsilon values of p-NP at different pH
|- Reach Buffer stopped by 2N NaoH
The results were expressed in μ mole min-1 mg-1,
where μ mole represents the one International Unit of enzyme (One unit
is defined as the amount of the enzyme that catalyzes the conversion of 1 μ
mole of substrate per minute). Zero time blank containing all reagents, except
plant extract, was always included in the assay. The linearity was evaluated
by determining standard working solution containing various concentration of
p-NP. The activity of acid phosphatase was measured in μ mole min-1
mg-1 protein versus p-NPP hydrolysis. Absorbance and concentration
were subjected to least square linear regression and correlation co-efficient.
Molar extinction coefficient of p-NP was measured at different pH to correlate
the data obtained by colorimetric assay (Table 1).
HPLC: Samples of acid phosphatase catalyzed reaction were also analyzed by HPLC (model series 200 pump; Perkin Elmer) using C18 column (ODS, 250x4.8 mm, 5 μm; Hypersil). The mobile phase consisted of sodium acetate buffer (0.1 M, pH 5.5; at a flow rate of 1 mL min-1). The sample (20 μL) was detected using UV/vis detector model 200 (Perkin Elmer) at λ max 320 nm. The retention time (Rt) of p-NPP and p-NP were at 2.3 min and 18.0 min, respectively.
Influence of pH: Total homogenate (15 μL) from cotyledons was
used to carry the pH optimum study for the enzyme. Various buffers were prepared
at different pH such as 0.1 M sodium citrate (pH 3, 4), 0.1 M sodium acetate
(pH 5.0, 5.5, 6.0), 0.1 M Tris (pH 7.0) and 0.1 M glycine (pH 8.0). p-NPP (2
mM) was used as a substrate for measuring the pH optimum at 37°C.
Effect of temperature: The optimum temperature for maximum acid phosphatase activity was determined by measuring the enzyme activity at various temperatures (10, 20, 30, 40, 50, 60, 70 and 80°C). The enzyme reaction buffer containing 2 mM p-NPP was incubated for 5 min at each temperature. The supernatant from cotyledons was then added to measure the enzyme activity.
Influence of various metal ions and modulators: The influence of metal ions (MnO2, NiCl2, FeCl2, 7 H2O, CoCl2 6H2O, ZnSO4, MgSO4 7H2O, CuSO4 5H2O) and modulators (NaMoO4 and NaF) on catalytic activity of acid phosphatase was assessed by adding different concentrations (0-2000 μM and 0-16 mM, respectively) of metal/modulator to the enzymatic reaction. The mixture was incubated at 37°C for 60 min. The results were expressed as relative activity in respect to the control reaction without metal/modulator added.
Influence of inorganic and organic phosphates on p-NPP hydrolysis: Various inorganic (NaH2PO4 and KH2PO4) and organic (ATP and G-6-P) phosphates (0-16 mM) were used along with 2 mM p-NPP to assess their influence on p-NPP hydrolysis. The reduction in p-NPP hydrolysis was measured in the presence of inorganic/organic phosphates and compared to control containing 2 mM of p-NPP only.
Isolation and purification of acid phosphatase: All procedures were carried out at 4°C. Enzyme activity was determined using the ELISA plate reader. K-Pi buffer (section 2.3) was used throughout the purification process.
Ammonium sulphate precipitation: The crude extract from cotyledons was brought to 30% saturation with ammonium sulphate in an ice bath under slow stirring for 1 h and centrifuged at 5000 rpm for 15 min. The treatment was repeated to give a final saturation of 90%. The saturated (90%) extract was centrifuged and the supernatant was discarded. The 20 fold active pellet was re-suspended in the extraction buffer and retained for further purification.
Chromatographic purification: The re-suspended pellet in extraction buffer (2 mL) was loaded on a sepharose 6 B column (2.5x10 cm) equilibrated with same buffer. The enzyme was eluted at a flow rate of 40 mL h-1. Fractions of 2 mL were collected and the fractions with acid phosphatase activity (colorimetric assay) were retained for further analysis.
Two dimensional gel electrophoresis: Active fractions obtained after partial purification of the enzyme (226.08 fold) were treated with ice-cold acetone (1:5 v/v) at -20°C overnight, centrifuged at 4°C, 5000 rpm for 15 min to precipitate the proteins. K-Pi buffer (0.1 M, pH 5.5) with protease inhibitor cocktail (1 μg mL-1) was used to dissolve the proteins and dialyzed overnight against water. The dialyzed protein sample was centrifuged at 15000 rpm for 10 min. The precipitated proteins were washed with ice-cold acetone containing 0.07% 2-mercaptoethanol to remove pigments and lipids until the supernatant was colorless. The pellet was vacuum-dried, re-suspended in re-solubilization buffer, followed by sonication (10 secx3 cyclex50% power) and centrifugation (15000 rpm for 20 min).
The pellet obtained from centrifugation was dissolved in re-hydration buffer
(Dong et al., 2009) and IEF (Protein IEF Cell
BIO-RAD) was performed as previously reported by Xu et
al. (2008) on a 11 cm immobilized pH (3-10) strip. Focused proteins
and pre-stained broad range protein markers (New England Lab) were resolved
by SDS-PAGE using 12% (w/v) of acrylamide. Gel was stained with colloidal coomassie
blue. Western blot analysis of partially purified protein were carried out with
potato polyclonal antibody as described previously (Abdelmeguid
et al., 2008).
Protein quantification: Total protein concentration was determined by the 2-D quanta kit (GE Amersham Biosciences).
Statistical analysis: The Mean±S.E.M. was expressed for values obtained from a minimum of three experiments. Data were analyzed by one-way ANOVA; post method students t test (Graph pad Prism Software). Differences with a p-value less than 0.05 were considered significant.
Acid phosphatase activity in cotyledons and embryonic axis of Psoralea: Enzyme activity was evaluated using p-NPP as synthetic substrate in cotyledons and embryonic axis of Psoralea seedlings (Fig. 1). Variation in enzyme activity was observed in cotyledons and embryonic axis of Psoralea. Crude extract from cotyledons showed 2.5 fold higher acid phosphatase activity (2412±35.86 μ mole min-1 mg-1) as compared with activity observed in the crude extract from embryonic axis (1028±40.71 μ mole min-1 mg-1 Fig. 1).
HPLC quantification of acid phosphatase catalyzed p-Nitrophenol synthesis: HPLC quantification of acid phosphatase induced (catalyzed) hydrolysis of p-NPP (2 mM; Rt 2.3 min) showed that the formation of 880 μM p-NP (Rt 18.0 min) after 60 min (Fig. 2a) However, conversion of p-NPP (2 mM) to p-NP was very low (138 μM) after 10 min of incubation (Fig. 2b).
Measurement of Km and Vmax for acid phosphatase: The rate of hydrolysis of the substrate p-NPP (0.015-8 mM) in the presence of Psoralea acid phosphatase showed that optimum concentration of p-NPP was 8 mM for acid phosphatase catalyzed hydrolysis in to p-NP (Fig. 3a); thereafter stability in hydrolysis was observed. The apparent Km value for p-NPP was 0.775 mM and the maximum reaction rate (Vmax) was 3.48 x 103 μ mole min-1 mg-1 protein at pH 5.5 (Fig. 3b).
Influence of pH: The effect of pH (Fig. 4a) was determined with crude extract using p-NPP as a substrate, the enzyme showed significant acid phosphatase activity in the range of pH 4.0-5.5, with maximal activity at pH 5.5. Lower than 4.0 pH and upper than 5.5 pH value strongly inhibited the activity of the enzyme.
To evaluate whether lower (3.0) and higher (8.0) pH range inhibited or inactivated
the enzyme; the enzyme was incubated with p-NPP at these pH for 1 h. The reaction
mixture with either lower or higher pH range was separately brought to optimal
pH. p-NPP hydrolysis was not observed at optimal pH after 1 h incubation of
the reaction mixture. Addition of fresh crude extract to the above reaction
mixture showed normal enzyme activity.
||Acid phosphatase activity of germinating seedlings of Psoralea
corylifolia a. Seedlings after 48 h of germination in dark showing cotyledons
and embryonic axis. Specific activity of acid phosphatase in cotyledons
and embryonic axis. The results given are Mean±S.E.M; n = 3
||HPLC chromatograms of acid phosphatase catalyzed dephosphorylation
of p-NPP (flow rate 1 mL min-1, λmax 320 nm). (a) Reaction
mixture and enzyme extract with 2 mM of p-NPP after 10 min of incubation
(p-NPP, Rt 2.3 min; p-NP, Rt 18.0 min) and (b) after
60 min of incubation
These results indicated that high or low pH of medium inactivated the acid
phosphatase enzyme from Psoralea.
In the time course study, the maximum de-phosphorylation of p-NPP (2613±8.8 μ mole min-1 mg-1) was observed after 90 min of incubation and remained constant in extended time intervals (Fig. 4b).
Effect of temperature on the enzyme activity: The optimum temperature
for maximum acid phosphatase activity was determined by measuring the enzyme
activity at various temperatures from 0 to 80°C. The optimum temperature
for de-phosphorylation of p-NPP was found to be 50°C with catalytic activity
of 8297±4655 μ mole min-1 mg-1. Acid phosphatase
was quite stable at higher temperature range of 70 to 80°C with significant
catalytic activity of 5790±48.60 and 4852±37.27
μ mole min-1 mg-1, respectively (Fig.
||Kinetic constant of acid phosphatase with p-NPP from cotyledons
of Psoralea. (a) Gradual increase in activity of acid phosphatase
was observed over a range of substrate concentrations. (b) Line-weaver Burk
plot was used to calculate the kinetic constants (Km of 0.775
mM and a Vmax of 3.48x103 μ mole min-1
mg-1). The results given are Mean±SE (n = 3)
Influence of metal ions on acid phosphatase activity: The effect of metal ions (Mg++, Co++, Zn++, Ni++, Mn++, Fe++ and Cu++) as possible activators or inhibitors of acid phosphatase showed that Mg++ was observed least (2175.34±5.6 μ mole min-1 nkat mg-1) and Cu++ most (452.6±2.6 μ mole min-1 nkat mg-1) toxic metal (at 2000 μM) as compared to control (2419.61±11.73 μ mole min-1 nkat mg-1 Table 2). The maximum inhibition in enzyme activity was observed at the concentration of 2000 μM of metal in the order of Cu++>Zn++>Ni++>Fe++>Co++>Mn++> Mg++.
Influence of modulators: Influence of two important modulators NaF and NaMoO4 on acid phosphatase was studied. NaF influenced the activity of acid phosphatase in dose dependent manner. Gradual reduction in p-NPP hydrolysis was observed with increasing concentrations of NaF and at lower concentration of NaF, no significance inhibition was observed. Lower concentration of NaMoO4 (0.03 mM) strongly reduced the activity of acid phosphatase from cotyledons of Psoralea. The results demonstrate that NaMoO4 is a strong inhibitor of acid phosphatase extracted from cotyledons of Psoralea (Fig. 6).
Substrate specificity of Psoralea acid phosphatase: The substrate
specificity of acid phosphatase was tested with various potential competitive
substrates with 2mM p-NPP at a concentration of 0.03-16 mM by measuring the
reduction in p-NPP hydrolysis. A gradual reduction in p-NPP hydrolysis was observed
with the increasing concentration of inorganic or organic phosphates (Table
||Effect of pH and time course study of Psoralea acid
phosphatase of (a).To ascertain the pH optima of the crude enzyme, p-NPP
hydrolysis activity was measured at different pH varying from 3-8 in different
buffers (0.1 M) H2PO4/K2HPO4 (K-Pi),
sodium acetate, glycine, Tris-HCl and sodium citrate at 37°C under standard
assay condition (b). To assess the time course study, after a specific time
interval, the reaction was stopped with 100 μL of 2 N NaOH; total p-NP
formed was read at 405 nm. The results given are Mean±S.E.M (n =
||Thermal stability of acid phosphatase from crude extract of
cotyledons of Psoralea corylifolia. The crude extracts from cotyledons
were incubated at 10-80°C for 60 min. The residual enzyme activity was
analyzed by colorimetric assay. The results given are Mean±S.E.M
(n = 3)
|| Effect of divalent metal ions on in vitro acid phosphatase
|aEach value represents the average and standard
deviation (students t test)
|| Effect of competitive substrates on p-NPP hydrolysis
|aConditions are described in materials and methods.
Each value represents the average and standard deviation (students
||Effect of modulators on acid phosphatase activity from crude
extract of cotyledons of Psoralea. The results given are Mean±SEM
(n = 3)
The results established that acid phosphatase in Psoralea cotyledons
||Purification profile of Psoralea cotyledon acid phosphatase
activities and protein concentration through Sepharose 6B column chromatography
|| Summary of purification procedure of acid phosphatase from
cotyledons (3 g) of Psoralea
Isolation and purification of acid phosphatase: The cotyledons were used for the purification of enzyme because cotyledons showed higher activity of acid phosphatase as compared to embryonic axis. Ammonium sulphate precipitation and chromatographic purification of crude extract from cotyledons of Psoralea gave partially purified (226.08 fold) enzyme as a single peak with a yield of 28.26% (Fig. 7). The specific activity of the partially purified acid phosphatase was 264211.157 μmole min-1 mg-1 of protein. The results of the purification are summarized in Table 4.
Two Dimensional gel electrophoresis and Western-blot analysis of partially purified acid phosphatase: The iso-electric focusing and SDS-PAGE analysis of the single peak obtained from gel filtration showed partially purified acid phosphatase protein (Fig. 8a). The focused proteins on Western blot analysis displayed five major protein spots with a molecular mass of ~28 and ~30 kDa, respectively (Fig. 8b).
The acid phosphatase maintains an adequate phosphate level for seed germination
and enzyme activity in the cells which is directly related to the phosphate
content (Prazeres et al., 2004). We observed very
high acid phosphatase activity in the cotyledons of germinating seedlings of
Psoralea as compared to embryonic axis. Increased acid phosphatase activity
could be related to either de novo protein synthesis or to the activation of
a pre-existing protein (Garcia et al., 2004).
The acid phosphate activity has been reported in several plant species and
large variation in activity was found in different plant parts (Panara
et al., 1990; Waymack and Van Etten, 1991;
Duff et al., 1991; LeBansky
et al., 1991; Ullah and Gibson, 1988; Penheiter
et al., 1997). Psoralea cotyledons showed relatively higher
Km (0.775 mM) at pH 5.5 from crude extract. The plants (Glycine
max, Phaseolus vulgaris, Triticum vulgare and Zea maize) generally
with high rate of germination possessed low Km higher Km for the acid phosphatase
for could be responsible for poor rate of seed germination in Psoralea.
||2-D gel electrophoresis and western blot of partially purified
acid phosphatase from cotyledons of Psoralea. (a) Coomassie blue-stained
protein, (200 μg) was loaded on a 11 cm (pH 3-10) immobiline dry strip
for IEF followed by SDS-PAGE (12 %). Pre-stained broad range protein markers
were used for SDS-PAGE. (b) Immunoblot of total protein from cotyledons
probed with purified polyclonal potato acid phosphatase antibody reacted
with five spots of ~28 kDa and 30 kDa protein bands of acid phosphatase
using potato acid phosphatase polyclonal IgG antibody
In general, the optimum pH value for acid phosphatase has been found to be
in pH range 3-6 (Juma and Tabatabai, 1988), the optimum
pH for acid phosphate extracted from cotyledons of Psoralea was 5.5.
The enzyme displayed significant activity in narrow pH range (4.0-5.5) i.e.,
below pH 4.0 and above pH 5.5, the activity diminished strongly and 80% reduction
in p-NPP hydrolysis was observed at pH 6.0. Acid phosphatase from Psoralea
was quite stable to heat demonstrating significant activity at 80°C, however,
possessed maximum activity at 50°C. Similar result of acid phosphatase activity
was observed by Matinizadeh et al. (2008). Heat
stable acid phosphatase has been reported in several plants such as bean (Garcia
et al., 2004), banana (Zhou et al., 2003),
tomato and lupin roots (Li and Tadano, 1996).
Enzyme reactions are inhibited by metals which may form complex with the substrate,
or combine with the protein-active groups of the enzymes, or react with the
enzyme-substrate complex (Hinajosa et al., 2004).
Acid phosphatase activity of wheat roots was inhibited in the presence of Cu++,
Fe+++, Zn++ and Co++, respectively (Hasegawa
et al., 1976). The metals Cu++, Fe+++ and Zn++,
inhibited the acid phosphatase activity of White Lupine seedlings (Newmark
and Wenger, 1960) and tobacco leaves (Roberts, 1956).
Present results showed significant inhibition of the acid phosphatase activity
from Psoralea cotyledons in the presence of divalent metal cations in
the order of Cu++>Zn++>Ni ++>Fe++>Co++>Mn++>Mg++.
NaF and NaMoO4 are strong inhibitors of the acid phosphatase (Senna
et al., 2006; Smith and Walker, 1996; Van
Etten et al., 1974), similar results were found in Psoralea
acid phosphatase. Although, NaMoO4 inhibited the enzyme at all concentrations
but NaF did not influence the enzyme activity at lower concentrations (Fig.
6). Acid phosphatase from Psoralea has been found to be non-specific.
KH2PO4 and ATP reduced the hydrolysis of p-NPP to 60%
at maximum concentration of 16 mM, however, NaH2PO4 and
G-6-P did not affect the hydrolysis of synthetic substrate p-NPP.
Low acid phosphatase activity has been reported in several plants such as 418-nkat
mg-1 (25.07 μmole min-1 mg-1) protein
from Barley coleoptiles (Pasqualini et al., 1997),
1500 nkat mg-1 (89.98 μmole min-1 mg-1)
from apple leaves (Zhang and McManus, 2000) however,
acid phosphatase purified from roots of white clover (Cirkovic
et al., 2002) showed reasonably good activity (9000 nkat mg-1
protein or 539.89 μmole min-1 mg-1). Crude extract
from cotyledons of Psoralea possessed low enzyme activity (2412±35.86
μmole min-1 mg-1) however, high specific activity
(264211.157 μmole min-1 mg-1 protein) was found in
partially purified protein. The isoforms of acid phosphatase in root nodule
of P. vulgaris (Garcia et al., 2004) and
pollen extract of Artemisia vulgaris (Cirkovic et
al., 2002) have been reported previously. In western blot analysis of
partially purified acid phosphatase from cotyledons of Psoralea, the
spots with a molecular mass of ~28 kDa and 30 kDa could be isoforms of the same
enzyme (Fig. 8b).
In conclusion, the acid phosphatase from crude extract of cotyledons of Psoralea has been found to possess relatively low affinity for the substrate which may be responsible for poor rate of seed germination. The enzyme activity was strongly inhibited by divalent metal Cu++ and NaMoO4 at low concentrations. Further, characterization of the isoforms from the purified enzyme will provide a scaffold leading to define its role during seed development.
Satendra Singh is thankful to University Grant Commission for financial support, New Delhi, India.