Source of pollen Material 50g of pollen were collected (hung fine net under the litchi trees) separately from the forest ranges of Nawabgang, Thanor, Rajshahi Town and Rajshahi University campus litchi garden in Bangladesh during the pollination period in March to April. These pollen grains were then processed for >95% purity by sieving through different grades of sieves (100, 200, and 300 mesh/cm2). All the samples were analyzed under the microscope which revealed pollen purity (shape & size) varying from 85% to 90%. To remove lipids and irritants of low molecular mass, the pollen sample was defatted with diethyl ether by repeated changes, until the ether become colorless. The defatted pollen powder was then completely vacuum-dried and stored at 40C in airtight containers until further use.
Protein Extraction The defatted dried pollen powder was extracted at 1:20(w/v) in 0.2M Tris- HCl buffer, pH 7.4 according to the method of Singh et al (1993) with slight modification (Mondal et al. 1997) by continuous stirring at 40C for 24 hours using a magnetic stirrer. The extract was clarified by centrifugation at 15,000 x g for 20 min at 40C. The supernatant was collected and was subjected to fractional precipitation by solid ammonium sulphate. It was made up to 80% saturation by slow addition of the salt and was stored for 4 hours at 40C in a freezer. After centrifugation the precipitate was re-suspended in 0.2M Tris-HCl buffer, pH 7.4 and desalted by dialyzing against distilled water for 48 hrs at 4 -100C by frequent changes of the water using dialysis sacs (MW cut off 9 kDa). Finally the supernatant was passed through a Millipore filter membrane (0.45µm), lyophilized in small aliquots, and stored at -200C until further use.
Purification of Litchi Flowers Pollen Protein (LFPP) Gel Filtration Chromatography: The crude protein solution (100mg/5ml) was applied to the column (1.5×29.0 cm) of Sephadex-G 75 (Uppsala, Sweden) and was pre-equilibrated with 50 mM phosphate buffer, pH 7.2-7.4, at 40C. The column was eluted with the same buffer at a flow rate of 25ml/h and each 2.5 ml fractions were collected. The absorbance of the column effluent was monitored at 280 nm using an Auto UV-Visible sprectophotometer, UV-280, Japan. Figure1 shows the purification pattern of pollen protein by gel chromatography.
Ion Exchange Chromatography on DEAE cellulose: A column (2.0X26.0cm) of DEAECellulose was equilibrated with 10 mM Tris-HCl buffer, pH 8.4, and operated at a flow rate of 25ml/h. After gel filtration protein from peak F-2 (fraction no. 21-35) combines together and reduces its volume to 5ml by freeze drying. Gel filtration protein fractions (5ml volume) containing Pectate lyase activity (around 60mg) were applied to the ion exchange column and washed by about 100 ml of 10mM Tris-HCl buffer. Enzyme protein was eluted with 0.2 M NaCl in 10mM Tris-HCl buffer pH 8.3. Following ion-exchange chromatography, the combined enzyme active fractions were stored in 50% (v/v) glycerol at -200C. Glycerol was removed by dialysis against Tris-HCl buffer before this enzyme solution is used for further enzyme assay, skin prick test and other allergen research studies.
Pectate Lyase (Pel) Activity Assays: The activity of pectate lyase (Pel)-LFPP was determined by monitoring the absorbance increase at 232 nm of a 1-ml reaction mixture containing sodium polypectate at 220C. To start the reaction, 5 μl of appropriately diluted protein were mixed with 870 μl of 50 mM Bis-Trispropane (BTP), pH 9.5, containing the optimal CaCl2 concentration and 125 μl of 1% sodium polypectate. [For preparation of stock solutions of 1% sodium polypectate, polygalacturonic acid (85-90% purity, Sigma) was dissolved in deionized water, boiled for 5 minutes, and centrifuged at 10,000×g for 10 minutes to remove sedimented materials. 50mM BTP, pH 9.5, containing the optimal CaCl2 concentration was prepared just prior to assays.] One unit of pectate lyase activity was defined as described (Wellhausen et al,. 1996). 1 μmol of unsaturated product formed/min, which equaled 1.73 absorbance unit’s min-1 and the specific activity, was expressed as unit’s mg1 protein. Protein concentration was determined by the method of Bradford.
Estimation of Protein The protein concentrations in the crude extract, as well as in the various eluted enzymatically active fractions, was estimated by the method of Lowry et al. (1951) with a slight modification, as the protein was precipitated with phosphotungestic acid and then dissolved in 2% sodium hydroxide. The experiment was repeated five times within 10% variability, and the mean value was taken for protein concentration. A calibrated solution of lipid-free bovine serum albumin (BSA) was used as a standard.
Protein Gel Electrophoresis
The protein sample was heated with an equal amount of sample buffer [0.06M Tris HCl (pH 6.8), 1% SDS, 10% sucrose, 0.5% β-mercaptoethanol, 0.01% Bromophenol blue] at 1000C for 3 min. 10µl of the sample containing 85µg of protein was loaded in the well of a 12% T mini-gel (8 x7 cm gel) Mini-Protean II slab gel apparatus (Bio-Rad, Hercules, CA, USA) and the gel was run using the method of Laemmli (1971) [0.05M Tris, 0.192 M Glycine, 0.1% SDS, pH 8.4] at room temperature for 2 hours 30 min, at 70 V. The molecular mass of the fractions was calculated by calibrating with standard marker protein, lysozyme (14 kDa), trypsin inhibitor (20 kDa), carbonic anhydrase (29 kDa), ovalbumin (45 kDa), albumin (BSA, 67 kDa) [Pharmacia, Uppsala, Sweden]. After electrophoresis, the gel was stained with 0.1% coomassie brilliant blue R-250 and destained with methanol: acetic acid:water (4:1:5) mixture for 12 hours.
Recovery protein from gel Protein was eluted from the gel following the method of Wilson and Goulding (1986). After electrophoresis, one side of the gel (covering 2 lines) was cut vertically and stained to ascertain the banding positions and only those portions corresponding to the protein bands to be recovered were cut out from the other half of the unstained gel. The gel pieces were transferred to pretreated dialysis bags filled with electroelution buffer (containing 0.05M Tris and 0.192M Glycine, pH 8.4). These dialysis bags were immersed in the electrophoresis tank of a horizontal gel apparatus containing the same buffer and electroelution was carried out for 3h by passing electric current (120V) through the bags. At the end, the current was reversed for 30seconds in order to release the protein from the wall of the sacs. The buffer containing the eluted proteins were then transferred through cotton plugged Eppendrof tube and centrifuged for 2 min to remove contaminating gel particles. The protein in each fraction was quantified and each fraction was again electrophoresed to check its homogeneity.
Skin Prick Test The skin tests were performed on patients suffering from nasobronchial allergy as well as healthy volunteers (120 no. healthy volunteers for 4 different location allergen protein skin tests and 50 no. nasobronchial patients, age between 9 – 60 years; male & female for purified litchi allergen protein) at Rajshahi University Medical center and Rajshahi Medical Collage Hospital, Bangladesh. Each patient was tested by placing 10µl of each allergen; at least 5 cm apart on the volar surface of his/her forearm and each site was then pricked with a disposable hypodermic needle. Negative and positive controls were also performed. The negative control solution used was the buffer saline in which the allergen was resuspended and the positive control solution was histamine acid phosphate injection diluted with buffered saline to 1:10,000 ie 1µg of histamine acid phosphate. The patients were prohibited from using antihistamine, steroid and ephedrine for 48 hrs before the skin prick tests. The skin reactions were read after 15 to 20 min from the commencement of the test. The test was quantified on the basis of the wheel diameter and graded 1 + to 3+.