3. PURIFICATION AND BIOPHYSICAL CHARACTERIZATION OF SECRETED
3.3 PARTIAL PURIFICATION OF ACID PHOSPHATASE
3.3.2 Methods
3.3.2.1 Ion-exchange chromatography
Using the optimized extraction protocol described in Section 3.2, a large scale extraction was conducted, where samples were filtered with cotton muslin cloth to remove large particles. In this study, cocktails of protease inhibitors (Roche) and phenylmethylsulfonyl fluoride (PMSF) were used to inhibit the protease enzyme. The protease inhibitors were added in the form of
94 tablets following the manufacture‟s instruction. After 30 min centrifugation (14, 000x g), the clear supernatant was filtered through a 0.22 µM disc (Millipore, Germany). The filtrate was concentrated by ultra-filtration Viva spin column (Millipore) (50 000 molecular cut-off membrane).
The enzyme fraction was purified using a DEAE column, connected to an AKTA HPLC.
Equilibration of the DEAE column was carried out with 5 ml column volumes of binding buffer A (20 mM Tris-HCl, pH 8.0). Absorbance at 280 nm was monitored at a flow rate of 1 ml/min.
The filtered and concentrated samples were applied to the DEAE column using a 200 µl sample loop. Elution was undertaken by using a stepwise gradient of buffer B (20 mM Tris HCl and 1.0 M NaCl, pH 8.0). Non-bound protein fractions collected during the washing step as well as fractions eluted by the increased salt concentrations were collected and further analyzed.
3.3.2.2 Gel-filtration chromatography
The concentrated fraction was injected in an AKTA purifier HPLC, Superdex column (10/300 GL, Amersham Biosciences), with the elution buffer. The elution buffer normally used contains 50 mM sodium phosphate,150 mM NaCl, 0.2 mM Na2EDTA and 1.0 mM sodium azide (pH 7.2). To avoid any interference of phosphorus, the elution buffer only consisted of 150 mM NaCl (pH 7.2). Absorbance at 280 nm was monitored at a flow rate of 0.5 ml/min.
The molecular mass of the purified enzyme was estimated by calibration with the standard protein, thyroglobin (670 kDa), IgG (150 kDa), Ovalbumin 44 (kDa), Myoglobin (17 kDa) and vitamin B12 (13.5 kDa) (Bio-Rad). Fractions with high activity were pooled and concentrated using a Viva tube (Millipore) with a molecular cut-off of 50 000. The eluted fractions were assayed for apase activity using p-NPP as a substrate by assay A. Native Mr was calculated by plotting the Kd (partition co-efficient) against log Mr using the protein standards. The void volume was estimated by dextran blue 2000.
95 3.3.2.3 Electrophoretic analysis of apase enzyme
In this study, electrophoresis was carried out using Native PAGE and SDS-PAGE which were commercially sourced (Invitrogen) and also manually prepared (Appendix A, Table A1). The Native-PAGE was mainly used to detect the active enzyme. SDS-PAGE electrophoresis was used to assess the protein composition of fractions from purification steps and to estimate the molecular masses of apase and its subunits.
3.3.2.4 Detection of enzyme activity by non-denaturing PAGE
Unless otherwise stated 5-20% polyacrylamide gradient gels with 3% stacking gel were used or 7% gels with 3% stacking gel were used. SDS-PAGE was performed as described by LAEMMLI (1971) using the electrophoresis apparatus from Invitrogen and manually-made gels according to Appendix A, Table A1. For preparation of native gels, SDS was omitted.
Non-denaturing (native) one-dimensional PAGE was carried out using 8% NativePAGE™
Novex® polyacrylamide gel (Invitrogen) or made as described in Appendix A, Table A1. To detect apase activity, the gels were equilibrated for 30 min at 37 °C in 100 ml of 100 mM sodium acetate buffer (pH 4.8). One ml of 1.0 M MgCl2, and 100 mg Fast Garnet GBC Salt (Sigma) were added. The gel was immersed in the mixture and 3.0 ml substrate (1.0% ß- naphthyl acid phosphate in 50% (v/v) acetone) was added. Incubation times varied from 5 min to 5 h, at a temperature of 37 °C.
A plot of relative mobility versus log (molecular mass) was constructed with native unstained protein standard (Invitrogen). The standard consisted of IgM Hexamer (1236 kDa), IgM pentamer (1048 kDa), Apoferritin band (1), Apoferritin band 2 (480 kDa), B-phycoerythrin (242 kDa), Lactate Dehydrogenase (146 kDa), Bovine Serum (66), Soybean Trypsin Inhibitor (20). Pre-stained protein standards were Myosin (250 kDa), phosphorylase (148 kDa), BSA (98 kDa), Glutamic dehydrogenase (64 kDa), alcohol dehydrogenase (50 kDa), carbonic anyhydrase (36 kDa), myoglobin red (22 kDa), lysozyme (16 kDa), aprotinin (6 kDa) and insulin (4 kDa).
96 3.3.2.5 Estimation of molecular weight by SDS-PAGE
Denaturing one-dimensional SDS-PAGE was performed using either NuPAGE® Novex® Bis- Tris precast gels (Invitrogen) or manually prepared (7-9%) SDS-PAGE gels which were stained as described by LAEMMLI (1971). For estimation of molecular weight mixtures of pre-stained standards (Invitrogen) were used: Myosin (250); phosphorylase (148); BSA (98); Glutamic dehydrogenase (64); Alcohol dehydrogenase (50); carbonic anhydrase (36); myoglobin red (22); lysozyme (16); aprotinin (6) and insulin B chain (4). The separated protein bands were stainedwith SimplyBlue™ SafeStain (Invitrogen) or the SilverQuest™ Staining Kit (Invitrogen).
3.3.2.6 Estimation of Isoelectric point
Isoelectric focusing was performed with polyacrylamide gel (30% acrylamide/0.8% Bis- acrylamide), with 3% Ampholytes 3-10 (Bio-Rad) and 2% (w/v) CHAPS. Ten percent ammonium persulfate (APS) (60 µl) and 1% TEMED (6 µl) and the volume was made to 10 ml.
The cathode buffer (upper chamber) consisted of 20 mM NaOH (0.4 g in 500 ml). The anode buffer (lower chamber) consisted of 10 mM phosphoric acid (0.7 ml in 1L). The loading buffer IEF (2.5x) consisted of 5% CHAPS and 5% ampholytes. The electrophoresis was conducted at 1000 V for 1 h 30 min, followed by 250 V for 1 h and lastly at 500 V for 30 min. All the experiments were conducted in a cold room (10 °C).
After IEF, the gel was rinsed with distilled water. After the addition of an IEF marker protein standard to confirm the protein pI, the pH values were measured by cutting the gel slab into 0.5 cm segments. The slices were incubated for at least 4 h in dH20 (0.5 ml) and the pH were measured. The rest of the gel was equilibrated in sodium acetate buffer and stained for apase activity as already described (Section 3.3.2.4). Alternatively, the gel was stained with Coomassie stain. The pI of acid phosphatase was determined by plotting the standard curve versus pI versus distance of standard markers (Bio-Rad) from the anode. The standard proteins (pI in parentheses) consisted of the following proteins at a concentration of 3.6 mg/ml:
Phycocyanin Blue (3 bands) pI 4.45, 4.65,4.75, ß-Lactoglobulin B pI 5.1, Bovine carbonic pI 6.0, anhydrase, Human carbonic pI 6.5, Equine myoglobin (2 bands) pI 6.8, 7.0, Human
97 hemoglobin A pI 7.1, Human hemoglobin C pI 7.5 , Lentil lectin 3 pI 7.8, 8.0, 8.20 and Cytochrome c pI 9.6
3.3.2.7 Deglycosylation N-Glycosidase F
Deglycosylation of acid phosphatase was done using N-Glycosidase F (PNGase F). The glycoprotein of apase was deglycosylated using typical reactions guidelines according to the manufacturer‟s instructions (New England Biolabs, Beverly, MA). The deglycosylated proteins were analyzed in a 9 % SDS-PAGE as described in Section 3.3.2.3. The gel was stained with silver.
Carbohydrate staining
The detection of the carbohydrate moiety of glycoproteins was performed by the periodic acid- Schiff reagent (PAS). The method is based on the oxidation of hexose vicinal 1,2-diol groups to aldehydes using periodate with subsequent staining by Schiff base (JAY et al., 1990). After electrophoresis, the gel was soaked with 200 ml of fixative (50% methanol) solution for 16 h at 25 °C. The fixative was changed once and the gel was left for 60 min with gentle agitation to remove SDS. The gel was rinsed in three changes of dH2O for 20 min each. The gel was then replaced with 2% (m/v) periodic acid. After two brief washes with 200 ml dH2O for 2 min, the gel was immersed in Schiff‟s Reagent until it turned magenta. After metabisulfite reduction, the gel was rinsed with several changes of dH2O until the water remained clear, indicating complete removal of unreacted pararosanile.
3.3.2.8 Enzyme thermal stability and determination of optimal pH
The thermostability of acid phosphatase was determined at different temperatures (0, 15, 37, 60, 80 and 100 °C). These studies were carried using pNPP as substrate by assays A described in Section 3.2.2.3
98 The acid phosphatase activity was determined at different pH‟s (2.2; 2.5, 3.6, 4.8, 5.6, 6.6 and 7.6) using citric acid and 100 mM glycine-NaOH (8.6, 9.6 and 10.6) using assay A as mentioned in Section 3.2.2.3.
3.3.2.9 Enzyme modulators and inhibitors
Using standard assay A, enzyme modulators and inhibitors were carried-out with various divalent metal cations such as: MgCl2, MnCl2, CaCl2, ZnCl2, CuNO3, CuSO4, and EDTA.
Known inhibitors of apase such as sodium fluoride, vanadate, molybdate, and tartrate were tested. The standard assay A was used and enzyme activity in the presence of metal cations or EDTA was expressed relative to the control.
3.3.2.10 Kinetics properties
The KM and Vmax values were obtained from Lineweaver-Burk (LINEWEAVER and BURK, 1934)) plot using various phosphatase substrates, determined using 0.4 µM to 0.5 mM concentration ranges. Substrates specificity was determined by a standard assay A and for other phosphorylated substrates such as α-naphthyl phosphate, β-glycerophosphate (GLOP), D- glucose-6-phosphate, ADP and AMP, assay B was used. The apases activity was determined at pH 2.5 measured at 37 °C. All kinetics parameters are the means of duplicate determination performed on two separate preparations of the purified enzyme and they are reproducible to within +10% SE.