Materials
Dioscorea bulbifera aerial tubers were obtained from a farmland in Amichi, Nnewi South Local Government of Anambra State, Nigeria. The plant was identified in the IFE Herbarium of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria, where the specimen copy was deposited and voucher number IFE-14754 was given.
All chemicals and reagents used were purchased from either Sigma Chemical Co. (St. Louis, MO, USA), Pharmacia Chemicals (Uppsala, Sweden) or Bio-Rad Lab (Hercules, CA, USA).
Preparation of crude extracts
The crude extract of the aerial tubers of Dioscorea bulbifera was prepared at different pH and varying temperature, in order to ascertain conditions at which most of the proteins in the aerial tuber are solubilized.
Dioscorea bulbifera aerial tubers were peeled, sliced and homogenized with 4 volumes (w/v) of buffers at different pH: 0.5 M citrate/phosphate buffer (pH 4–6), 0.5 M Tris-HCl buffer (pH 7 and pH 8.3) and glycine-NaOH buffer (pH 9 and 10). The mixtures were stirred for 4 h and centrifuged at 13,500 rpm for 30 min at 4 °C. The supernatants collected were stored as crude extracts.
Also, approximately 100 g portions of yam slices were boiled in 1 L of water at 25, 30, 40, 50, 60, 70, 80, 90 and 100 °C for 10 min. The treated aerial tubers were drained, cooled, weighed and homogenized with 50 mM Tris-HCl (pH 8.3) at 1:4 (w/v). The mixture was stirred for 4 h and centrifuged at 13,500 rpm for 30 min at 4 °C. The supernatants were collected as crude extracts.
Protein content of extracts was determined by Lowry method using 1 mg/mL bovine serum albumin (BSA) as standard.
Purification of protein
The crude extract obtained at pH 8.3 and 25 °C (which had the highest protein concentration) was used for further studies. Purification of the storage protein of Dioscorea bulbifera aerial tuber was carried out following the method of Hou et al. [18] with a little modification.
The crude extract of the aerial tuber of Dioscorea bulbifera was subjected to 70% ammonium sulphate precipitation, stirred and kept overnight at 4 °C. The mixture was centrifuged at 13,500 rpm for 30 min and the precipitate recovered. The precipitate was dissolved in 10 volumes of 50 mM Tris-HCl buffer, pH 8.3 and dialyzed exhaustively against distilled water.
Ion-exchange chromatography on DEAE Sephadex A-25
The dialyzed protein solution (7.5 mg/mL; 2.5 mL) was loaded on DEAE Sephadex (A-25) ion exchange column (1.5 × 20 cm) previously equilibrated with 50 mM Tris-HCl buffer, pH 8.3. Unadsorbed proteins were eluted with 50 mM Tris-HCl buffer, pH 8.3, and adsorbed proteins were eluted stepwise with 150 mM NaCl in 50 mM Tris-HCl buffer, pH 8.3, at a flow rate of 15 mL/h. Fractions of 5 mL each were collected, and elution was monitored at 280 nm. The adsorbed protein fractions, which correspond to the major storage protein of the aerial tuber of D. bulbifera, were pooled and concentrated.
Gel filtration on Sephadex G-75
Adsorbed protein sample (1.5 mg/mL; 5 mL) obtained from ion-exchange chromatography was further purified by gel filtration on Sephadex G-75 column (1.5 × 40 cm) previously equilibrated with 50 mM Tris-HCl buffer, pH 8.3. The column was eluted with 100 mM Tris-HCl buffer (pH 7.9) containing 100 mM NaCl at a flow rate of 27 mL/h. Fractions of 3.6 mL each were collected. The purified protein was collected, concentrated and stored at − 20 °C for further use. Protein concentration was determined after each purification step.
Non-SDS polyacrylamide gel electrophoresis
The protein samples were subjected to polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulphate (SDS) according to the modified method of Shiu et al. [19] to monitor the purity of the protein obtained after each purification step. Electrophoresis was performed on a 10% discontinuous gel system under non-denaturing conditions and stained with Coomassie Brilliant Blue.
Determination of molecular weight
The native molecular weight of the protein was determined by gel filtration on a Bio gel P-200 column (1.5 × 63 cm) using the following protein markers: lysozyme (Mr 14,000), α-chymotrypsinogen A (Mr 25,000), egg ovalbumin (Mr 45,000) and bovine serum albumin (Mr 66,000). Each protein (5 mL) was applied on the column and run separately using 10 mM phosphate buffer pH 7.0 as eluant at a flow rate of 10 mL/h. Fractions of 5 mL were collected, and the elution was monitored at 280 nm. The void volume (Vo) of the column was determined using Blue dextran (elution monitored at 620 nm).
The purified storage protein was subjected to SDS-polyacrylamide gel electrophoresis for subunit molecular weight determination following the modified method of Shiu et al. [19] using the following protein markers: ovalbumin (Mr 45,000), carbonic anhydrase (Mr 29,000), trypsinogen (Mr 24,000), trypsin inhibitor (Mr 20,000) and α-lactalbumin (Mr 14,200).
Detection of protein-bound carbohydrate
The presence of covalently-bound carbohydrate in the storage protein was investigated by staining the gels with periodic acid-Schiff’s reagent (PAS) after electrophoresis, as described in the Pharmacia Manual of Laboratory Techniques, revised edition. The protein sample was subjected to electrophoresis under non-denaturing conditions using phosphate-buffered system. After electrophoresis, the gel was fixed in 7.5% acetic acid at room temperature for 1 h. The fixed gel was transferred into a beaker containing 0.2% aqueous periodic acid and kept at 4 °C for 45 min. Afterwards, the gel was removed and transferred into a beaker containing Schiff’s reagent, kept at 4 °C for 45 min. The gel was destained in 10% acetic acid. Glycoprotein band (if present) will stain purplish red.
Amino acid composition of the protein
The storage protein was subjected to amino acid content analysis using methods described by Ekeanyanwu [20]. The sample was hydrolysed, evaporated in a rotary evaporator and loaded into the Technicon Sequential Multi-Sample Amino Acid Analyzer (TSM).
Enzymatic activities of storage protein of Dioscorea bulbifera
Determination of carbonic anhydrase activity
Carbonic anhydrase activity of the protein was measured by hydrolysis of 4-nitrophenyl acetate resulting in an increase of absorbance at 348 nm [21]. The activity of the tuber storage protein was compared with that of carbonic anhydrase from bovine erythrocytes. The reaction mixture contained 0.3 mL of freshly prepared 3 mM 4-nitrophenyl acetate in aqueous 3% acetone and 0.7 mL of 15 mM Tris sulphate buffer, pH 7.6. Exactly 10 μL purified protein solution (1 mg/mL) was added, and the catalyzed reaction was monitored by measuring the increase in absorbance at 348 nm for 5 min.
Determination of dehydroascorbate reductase activity
Dehydroascorbate (DHA) reductase activity of the protein was carried out according to the method of Hou et al. [18]. In this reaction, 10 mg of DHA was dissolved in 5 mL of 100 mM phosphate buffer of different pH values (pH 6.0, 6.5 and 7.0). The reaction was carried out at 30 °C; 100 μL purified protein solution (1 mg/mL) was added to 0.9 mL DHA solution with or without 4 mM glutathione. Increase in absorbance at 265 nm was recorded for 5 min. Non-enzymatic reduction of DHA in phosphate buffer was measured in a separate cuvette.
Determination of monodehydroascorbate reductase activity
Monodehydroascorbate (MDA) reductase activity of the protein was assayed according to the method described by Hou et al. [18] by monitoring the decrease in absorbance at 340 nm due to NADH oxidation. MDA free radicals were generated by ascorbate oxidase in the assay system. The reaction mixture contained 50 mM phosphate buffer (pH 6.0, 6.5 and 7.0); 0.33 mM NADH; 3 mM ascorbate, ascorbate oxidase (0.9 U); and 200 μL purified protein solution (200 μg protein) in a final volume of 1 mL. Distilled water was used to replace protein solution in blank solutions. One unit of MDA reductase is defined as the amount of protein required to oxidize 1 μmol of NADH per min.
Determination of trypsin inhibitory activity
Trypsin inhibitory activity of the protein was determined according to the method of Xue et al. [22] by monitoring the inhibition of trypsin-catalyzed hydrolysis of N-benzoyl-L-arginine-4-nitroanilide (substrate) in 0.1 M Tris-HCl buffer (pH 8.2). Different concentrations of the protein were pre-incubated with 20 μM trypsin at room temperature for 15 min. The substrate (100 μg/mL) was added to give a final volume of 1 mL for an additional 20 min. The absorbance at 405 nm was measured. The inhibitory activity is calculated as the percentage decrease in substrate hydrolysis rate, which is directly proportional to increase in absorbance at 405 nm. The result was expressed as micrograms of trypsin inhibited.
Molecular characterization of the storage protein of Dioscorea bulbifera
Genomic DNA extraction
The aerial tuber was peeled, cut into bits and ground into fine powder with a mortar and pestle under liquid nitrogen. Genomic DNA was extracted using QIAGEN DNeasy Plant Mini Kit. DNA concentrations were determined with a Nanodrop spectrophotometer (Beckman Coulter) and adjusted to 25 ng/μL for PCR amplification.
Primer design for polymerase chain reaction
Sequences of some Dioscorin genes from various Dioscorea sp. were obtained from NCBI nucleotide database (http://www.ncbi.nlm.nih.gov/nuccore). These sequences were inserted into the input window of the web-based polyacrylamide chain reaction (PCR) primer designing program, Primer3 (https://primer3plus.com/cgi-bin/dev/primer3plus.cgi). The primer minimum and maximum sizes were set to 100 and 900 nucleotides, respectively. The DNA was subjected to PCR amplifications using the designed Dioscorin-specific primers (5′-CTCCTCTCCTCCCTCCTCTT-3′ (forward primer) and 5′-GGGGGTACAATGGAGAAGT G-3′ (reverse primer)). The amplification was conducted in a final reaction volume of 25 μL containing 5 μL of DNA sample, 2.0 μL MgCl2, 0.2 μL Taq polymerase, 2.5 μL 10 X reaction buffer, 1 μL dNTPs, 1 μL each of forward and reverse primers, 2.0 μL Tween 20 and sterile deionized water in a 96-well microtiter plate and carried out in a GeneAmp PCR System 9700 (Applied Biosystems). The PCR cycles were made up of initial denaturation of DNA template at 94 °C for 3 min, followed by 36 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min and extension at 72 °C for 2 min. The final extension step was at 72 °C for 7 min.
Electrophoresis of PCR products
The PCR products obtained were detected by agarose gel electrophoresis. An aliquot (3 μL) of 5 × loading dye (0.25% bromophenol blue, 0.25% xylene cyanol FF and 13% Ficoll in water) was added to the 10 μL of the PCR product, and 6 μL of the mixture was loaded onto a 1.5% agarose gel pre-stained with ethidium bromide. TBE (0.5X) was used as running buffer, and DNA ladder (markers) was loaded for fragment sizing. Electrophoresis was conducted at 1500 V for 3 h, and the gels were viewed under ultraviolet rays.
Gel extraction, DNA sequencing
The resulting DNA fragments generated from amplifications were purified by excising bands from the agarose gel after electrophoresis. The DNA was recovered using QIAquick gel extraction kit (Qiagen). The nucleotide sequences of the purified dioscorin genes were obtained with a genetic analyser. The sequencing amplifications were performed in a 20-μL reaction mixture consisting of 400 ng of DNA to be sequenced, 10 pmole of dioscorin-specific primers 5′-CTCCTCTCCTCCCTCCTCTT-3′ (forward primer) and 10 pmole of 5′- GGGGGTACAATGGAGAAGTG-3′ (reverse primer) and 4 μL of Reaction Dye Terminator Premix (Qiagen) with standard sequencing conditions. Amplification was performed in a thermowell microtitre plate (Costa Corporation) using Perkin Elmer programmable Thermal Controller model 9600. The cycling program was 36 cycles of 94 °C for 1 min for denaturation, 60 °C for 1 min for annealing of primers and 72 °C for 2 min for extension. Amplification products were stored at 4 °C before use. One microlitre of 125 mM EDTA, 1 μL 3 M sodium acetate (pH 4.8), 25 μL 100% ethanol (− 20 °C) and 50 μL 70% ethanol (− 20 °C) were added to the amplification products, mixed and centrifuged for 10 min at 10,000 rpm at 4 °C. DNA pellet was dried at room temperature and re-suspended in 5 μL sterile deionized distilled water. One microlitre of the re-suspended DNA was added to 9 μL Hi formamide, mixed and denatured for 3 min at 94 °C. It was placed inside ABI PRISM 3130 X1 genetic analyser, which carried out the automated sequencing analysis using a standard sequencing module with Performance Optimized Polymer and 50-cm array.
Sequence analysis
The nucleotide sequences of the purified dioscorin genes were subsequently translated to protein sequence using bioinformatic resource tool from CLC Genomics Workbench software (CLC Bio Denmark). The nucleotide and translated protein sequences were further subjected to computer-based homology search with NCBI BLAST program. Phylogenetic analysis was carried out to compare the relationship of the major storage protein of D. bulbifera with the storage proteins of other Dioscorea spp.