SUPPLEMENTARY MATERIAL

Transcription

1 SUPPLEMENTARY MATERIAL Purification and biochemical characterization of acid phosphatase-i from seeds of Nelumbo nucifera Sanaullah Khan a*, Shahnaz Asmat c, Sajida Batool a, Mushtaq Ahmed b a Department of Bioscience, COMSATS Institute of Information Technology Islamabad, Pakistan b Department of Biotechnology, University of Science and Technology, Bannu, Pakistan c Department of Chemistry, University of Science and Technology, Bannu, Pakistan * Author to whom correspondence should be addressed: Tel: Fax: sanaullah.khan@comsats.edu.pk Abstract Acid phosphatase-i (Apase-I) from seeds of Nelumbo nucifera was purified to electrophoretic homogeneity by combined use of ammonium sulfate (NH4)2SO4 precipitation, size-exclusion and ion exchange chromatography. SDS-PAGE of purified Apase-I gave a single band with molecular mass of 80 kda under reducing and non-reducing conditions, indicating that the enzyme was a monomer. Upon biochemical characterization, the enzyme showed maximum activity at 50ºC and at ph 5. The Km, Vmax and Kcat for p-nitrophenyl phosphate were 132 µm, 10 µmol/min/mg and 6.7/sec respectively. Apase-I activity was strongly inhibited by Zn 2+, W 2+ ; weakly inhibited by Cu 2+, Mo 2+ and Cr 6+ and moderately activated by Mg 2+. The enzyme was shown to be thermolabile as it lost 50% of its activity at 50ºC after incubation for 1 hour. The amino acid analysis of enzyme revealed high proportion of acidic amino acids, which is very similar to that of tomato Apase-I and lower than potato Apase. Keywords: Nelumbo nucifera seeds; acid phosphatase-i; purification; biochemical characterization Experimental

2 Chemicals p-nitrophenyl phosphate (p-pnp) was purchased from Merk, Sephadex G-50 from Pharmacia and rest of the chemicals and reagents were from Sigma and of analytical grade. Commercial N. nucifera dry seeds were obtained from local market. Seeds extraction About 100 g dry seeds of N. nucifera free of seedcoat were ground to fine powder and suspended in 10 mm Tris-HCl buffer ph 7.4 containing 0.01% sodium azide, gently stirred for 8 hours and kept overnight at 4ºC. The seed mixture was centrifuged at 5,000 rpm for 15 minutes and supernatant was used as seed extract for further analysis. Protein quantification and enzyme assay Protein concentration was determined at each step of purification by Bradford assay (Bradford 1976) using bovine serum albumin (BSA) as standard. Apase-1 activity was measured using p-pnp as a substrate. Briefly, in total volume of 2.5 ml, 5 μg enzyme was incubated with 4 mm p-pnp in 50 mm sodium acetate buffer (ph 5.0) at 37ºC for 5 min. The reaction was stopped by the addition of 0.1 M NaOH and the amount of free p-nitrophenol was calculated by measuring absorbance at 405 nm. One unit of enzyme activity was defined as 1 μmol of p-nitrophenol released per minute under these experimental conditions. Enzyme purification Enzyme extraction was carried out at 4ºC except chromatography (size-exclusion and ionexchange) which was done at room temperature. At each step of purification, enzyme activity was measured using 4 mm p-pnp as a substrate. For precipitation, solid ammonium sulfate was slowly added (up to 67%) to the seed extract with constant stirring at 4 o C. The mixture was allowed to stand for 1 hour at 4 o C and then centrifuged at 5000 rpm for 20 minutes at 4 o C. The pellet was dissolved in 10 mm Tris-HCl buffer (ph 7.4) and again centrifuged at 5000 rpm for 20 minutes at 4 o C. Protein extract were then loaded on Sephadex G-50 gel filtration column (2.5 54cm) equilibrated and eluted with 10 mm Tris-HCl buffer (ph 7.4). Flow rate was maintained at 12 ml/hour and 3 ml elute was collected as fraction; and protein concentration in each fraction was determined by taking absorbance at 280 nm. Apase-I activity of pooled fractions corresponding to individual protein peaks was measured as described earlier in enzyme assay. The enzyme-active fractions were then pooled and loaded onto an anion exchanger i.e. DEAE-cellulose column (2.5 x 24 cm) equilibrated with 10 mm Tris-HCl (ph 7.4). The bound proteins were eluted by applying linear gradient of M NaCl in the same buffer. The flow rate was adjusted to 24 ml/hour. Column elute was collected as fractions and used for measurement of proteins by taking optical density at 280

3 nm. Fractions corresponding to individual protein peaks were again assayed for Apase-I activity and enzyme-active fractions were pooled and kept at 20ºC. Enzyme units were calculated at each purification step. Determination of molecular mass and activity staining SDS-polyacrylamide gel-electrophoresis (SDS-PAGE) consisting of 4% stacking and 8-10% resolving gels were run according to Laemmli method (Laemmli 1970) under both reducing and non-reducing conditions. The molecular mass of the enzyme was determined by comparing its relative mobility with known molecular markers ( kda). Native polyacrylamide gel electrophoresis (4% stacking and 8% resolving gel) was performed on the slab gel to determine enzyme activity as described above. Apase-I activity was then detected with β-naphthyl acid phosphate and fast black potassium salt as reported earlier (Vallejos 1983, Cap & Roberts 1992). Determination of optimum ph and temperature For determination of optimum ph, the purified Apase-I activity was assayed in 50 mm sodium acetate (ph range ) and 50 mm Tris-HCl (ph 7.2 to 8.5) using p-pnp as a substrate at 37ºC. To determine the optimum temperature of the enzyme, reaction mixture was incubated at various temperatures ranging from 17ºC to 90 C under standard assay conditions. Thermal stability of the enzyme For determination of thermal stability, the purified Apase-I was first incubated at 26ºC, 40ºC, 50ºC, 60ºC and 80ºC for 1 hour. After incubation, p-pnp solution was added to start the reaction and enzyme activity was measured. Effect of metals ions on enzyme activity The effect of various metals ions on enzyme activity was assayed in the presence of various metals under standard assay conditions (Table S2). Amino acid analysis 1.5 mg of purified Apase-1 was hydrolyzed with 6 N HCl for 24 hours at 110 C. The acid was then evaporated using a rotary evaporator at 50 C after 24 hours. After removal of HCl, the hydrolysate was dissolved in 2 ml of sample application buffer i.e. 0.2 M sodium citrate (ph 3.0). Aliquots of 20 µl were analyzed with a Shimadzu HPLC amino acid analysis system (LC 10A). References

4 Bradford M Soluble proteins and multiple enzyme forms in early growth of wheat. Anal. Biochem. 72:248. Cap GB, Roberts PA A rapid and efficient method for the screening of acid phosphatase 1in young tomato seedlings, and for the identification of root-knot nematode species using miniaturized polyacrylamide gel electrophoresis. Electrophoresis. 13: Laemmli, U. K Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature. 227: Vallejos CE Enzyme activity staining. In: Tanksley, S. D., Orton, T. J. (Eds.), Isozymes in Plant Genetics and Breeding Part A, Elsevier, Amsterdam, pp. 469.

5 Supplementary results Table S1. Summary of N. nucifera Apase-I activity at various purification steps measured against p-pnp Purification Steps Activity Units/ml Total activity Protein mg/ml Specific Activity (Units/ mg ) Purification fold Recovery % Units Crude (NH4)2SO Precipitation Sephadex G DEAE Cellulose Chromatography

6 Table S2. Effect of various metal ions on N. nucifera Apase-I activity Metals Ions Concentration Relative activity No metals 100 CaCl2 2 mm 101 MgCl2 2 mm 168 CuSO4 2 mm 30.4 ZnCl2 2 mm 02.2 NaF 2 mm 29.4 Na2WO4.H2O 2 mm 05.8 Na2Mo.2H2O 2 mm 44.1 K2Cr2O7 2 mm 70.4 KCl 2 mm 102 LiCl 2 mm 100 NaCl 2 mm 100

7 Table S3. Amino acid composition of purified N. nucifera Apase-I in comparison to Tomato and Potato enzyme. Amino acids Apase-I Apase-I Apase (N.nucifera) (Tomato) (Potato) % residue Lys His Arg Cys n.d Asp Thr Ser Glu Pro Gly Ala Val Met n.d Ile Leu Tyr Phe

8 Fig. S1. (a) 8% SDS-PAGE of purified Apase-I. Lane 1: dissociating condition, lane 2: nondissociating condition and lane 3: protein markers ( kda). (b) 8 % Native-PAGE and activity staining of purified Apase-I.

9 Fig. S2. (a) Optimum ph of Apase-I. The activity of enzyme was assayed against p-pnp in 50 mm sodium acetate (ph ) and 50 mm Tris-HCl (ph 7.2 to 8.5). (b) Optimum temperature of Apase-I. The activity of enzyme was assayed against p-pnp in 50 mm sodium acetate ph 5.0 at various temperatures (17-90 C). (c) Thermal stability of the N.nucifera Apase-I. The residual activity of the enzyme was determined using standard enzyme assay after 1 hour pre-incubation at various temperatures (26-80 C). (d) Michaelis-Menten enzyme kinetics for Apase-I. The enzyme activity was assayed with p-pnp concentrations (0.1-4 mm).