Purification and Biophysical Characterization of an ... - IngentaConnect

Send Orders of Reprints at [email protected] Protein & Peptide Letters, 2013, 20, 499-509

499

Purification and Biophysical Characterization of an 11S Globulin from Wrightia tinctoria Exhibiting Hemagglutinating Activity Pramod Kumar1, Dipak N. Patil1, Anshul Chaudhary1, Shailly Tomar1, Dinesh Yernool2, Nirpendra Singh3, Pushpanjali Dasauni3, Suman Kundu3 and Pravindra Kumar*,1 2

1

Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India; Department of 3 Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA; Department of Biochemistry and Central Instrumentation Facility, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India Abstract: Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS–PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of -sheets (44.2%) and moderate content of -helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 °C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

Keywords: Apocynaceae, hemagglutinating activity, Wrightia tinctoria, 11S globulin. INTRODUCTION Globulins are large globular proteins that constitute an indispensible part of the plant seed due to their ability to act as nutrient source for emerging embryos during seed germination [1]. Globulins along with albumins are major storage proteins in the seeds of food crops (cereals and legumes) and abundantly present in nuts [2]. These proteins have been isolated and characterized from a variety of plant species including Pisum sativum [3], Vicia faba [4], soyabean [5], oat [6], kiwifruits [7], wheat [8], rice [9], ginkgo [10], coffee [11], peanut [12], almond [13] and hazelnut [14] among others. Being one of the major storage proteins in various food sources, globulins play an important role in nutrition of humans and farm animals by providing essential amino acids. In addition to the role of globulins as seed storage proteins, they have been shown to have secondary activities such as protease inhibitory activity [7], insecticidal activity [15, 16] , chitin-binding [17] and lectin-like activity [18]. Due to these activities, globulins have also been implicated to play a defensive role in plant pathogenesis [11]. Globulins *Address correspondence to this author at the Department of Biotechnology, Indian Institute of Technology, Roorkee, Uttarakhand 247667, India; Tel: +91-1332-286312; Fax: +91-1332-286151; E-mail: [email protected] -5/13 $58.00+.00

are the most prevalent form of the seed storage protein present being distributed over a vast range from dicots to monocots and even in ferns, broadly classified as 2S, 3S, 7S and 11S. Globulins belonging to the class 11S are the most prevalent forms and identified as the major food allergen [14, 18, 19]. The 11S proteins are hexameric heterooligomers of molecular weight of ~320 kDa, with each subunit comprising of acidic and basic polypeptides that are attached by disulfide linkages [18, 20, 21]. The 11S globulins are synthesized as single precursor polypeptides which are post-transitionally processed proteolytically by signal peptidase and asparaginyl endopeptidase that results in formation of the acidic and basic polypeptides of the mature 11S hexamer subunits [20-22]. Transgenic crop plants generated for nutritional improvement or increased productivity are being potentially employed in the agriculture industry [23]. Genes having a role in plant defence are considered excellent candidates for construction of disease and pest resistant transgenic plants [24]. Proteins having both high nutritional-functional value and relation to plant pathogenesis [25] are considered excellent candidates to be overexpressed in transgenic cereal crops [26]. Various globulin proteins rich in nutritive value with characteristics of pathogenesis related proteins are being investigated and considered for generation of transgenic © 2013 Bentham Science Publishers

500 Protein & Peptide Letters, 2013, Vol. 20, No. 5

plants [11, 12, 14-17, 23, 24, 26, 27]. Therefore, it becomes necessary to study and characterize globulins from various plant families and sources. Several crystal structures of 11S seed globulin proteins from different plant sources are required for identification and characterization of the structural features that contribute to allergenicity. Wrightia tinctoria belongs to the family Apocynacea. Though Wrightia genus is distributed throughout the world, but the species tinctoria is confined majorly to India [28]. It is a well known medicinal plant having several applications that include properties like antinociceptive [29], antifungal [30], antiviral [31] and antioxidant [32]. Moreover, extract from Wrightia tinctoria also have applications in jaundice, toothache, psoriasis and other skin diseases [33]. In the present study, we report isolation, purification, characterization of secondary structure, partial amino acid sequencing, crystallization and preliminary crystallographic studies of 11SWTG from W. tinctoria seeds that exhibit hemagglutinating activity and may be considered as a lectin like protein. MATERIALS AND METHODS Materials Dry seeds of W. tinctoria were obtained from the local market. The reagents used for the purification and experimental assays were purchased from Sigma-Aldrich corporation, St. Louis, MO USA; BioRad Laboratories, Hercules, California, USA; Himedia Laboratories India Private Limited, Mumbai, India; Merck Limited, Worli, Mumbai, India. Hi-Trap DEAE FF (1 mL), Hiload superdex 200 16/60 columns and HMW Calibration kit were obtained from GE Healthcare, AB Uppsala, Sweden. Amicon ultra concentrator, PVDF membrane and millex syringe filter were from Millipore Corporation, Billerica, MA. Dialysis membrane with 3500 Da cutoff was from Pierce, Rockford, USA. Hampton crystal screens, 96-well crystallization plates and cryoloops were purchased from Hampton Research, CA, USA. Bio-Rad protein assay kit was from BioRad Laboratories, Hercules, California, USA. Extraction and Purification of WTG Seeds (5.0 g) were soaked overnight at room temperature in 20 mL of 50 mM Tris-HCl, pH 7.5. Seed coat was removed manually and seed kernels were obtained. A crude extract was prepared by homogenizing seed kernels in buffer A (50 mM Tris-HCl, pH 7.5, 10% glycerol, 10 mM MgCl2 , 0.1 mM PMSF) using a mortar and pestle. After stirring for 6 hr at 4 °C, the seed extract was centrifuged at 50,000 x g, 4 °C for 45 min. After centrifugation, the uppermost layer of fat was discarded and the sample was again centrifuged at 50,000 x g, 4 °C for 45 min. The clear supernatant collected after centrifugation was subjected to chromatography on 1 mL Hi-Trap DEAE FF column which had been preequilibrated with buffer A. Following removal of unbound proteins in the flow through, the column was washed with 25 mL of buffer A and bound material was subsequently eluted from the column using NaCl gradient of 0.0 – 1.0 M in buffer B (buffer A + 1 M NaCl). Eluted fractions were analyzed on 15% SDS-PAGE and fractions containing globulin were dialyzed three times in 1 L of buffer A in three rounds

Kumar et al.

and then concentrated to 10 mg/mL using Amicon Ultra 15. The concentrated protein sample was finally loaded onto preequilibrated HiLoad 16/60 Superdex 200 size-exclusion column using 1 mL sample loop at a flow rate of 0.5 mL/min on ÄKTA purifier (GE Healthcare) and the protein elution profile was monitored by measuring absorbance at 280 nm. The size-exclusion column was calibrated with gel filtration HMW calibration kit containing thyroglobulin, (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa) and ovalbumin (44 kDa) for determination of the void volume, construction of the standard curve and estimation of the molecular weight of purified protein. Fractions of the major peak containing pure protein were pooled and concentrated using Amicon ultra 15. The homogeneity of the concentrated protein sample was determined by 15% non-reducing SDS– PAGE stained with Coomassie brilliant blue. Protein concentration was determined with Bio-Rad protein assay kit using BSA as standard and the protein yield was subsequently estimated. Purified and concentrated protein was dialyzed overnight against the dialysis buffer (50 mM Tris-HCl, pH 7.5) and stored at -20 °C. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS-PAGE analysis of WTG was performed according to Laemmli [34] using 5% stacking and 15% separating gels under reducing and non-reducing conditions. The gels were stained with Coomassie brilliant blue and the molecular weight of WTG was estimated using molecular weight references of myosin (210 kDa), galactosidase (125 kDa), phosphorylase b (101 kDa), bovine serum albumin (56.2 kDa), ovalbumin (35.8 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (21 kDa) and aprotinin (6.5 kDa). Intact Mass Determination Purified protein was diluted in 50% acetonitrile and 0.1% TFA at the concentration of 10 pmol/L. The diluted protein was spotted on MALDI target plate with -cyano 4-hydroxy cinnamic acid (CHCA). Instrument was calibrated with BSA using ProteoMass Protein MALDI/MS calibration kit (Sigma Aldrich). The spectra were acquired in linear ion mode for intact mass estimation. Amino Terminal and Mass Spectrometry-based Internal Sequencing The N-terminal amino acid sequencing of larger subunit of WTG was performed by Edman degradation on an automated protein sequencer (model 494; Applied Biosystems) at the protein sequencing facility of Columbia University, New York, USA. Pure protein was subjected to a 15% SDSPAGE under reducing condition and was electro blotted onto a polyvinylidene fluoride (PVDF) membrane using 10 mM CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) buffer, pH 11 in 10% methanol [35]. The upper band corresponding to the larger acidic subunit was excised from PVDF membrane and the first 10 amino acid residues at the N-terminal of the polypeptide were determined. Sequence homology search was performed using NCBI database (http://www.ncbi.nlm.nih.gov/) for identification of purified protein based on sequence homology.

Purification and Biophysical Characterization

For internal sequencing, the Coomassie blue-stained protein bands were excised from the corresponding SDSpolyacrylamide gels and destained with 50% acetonitrile in 50 mM ammonium bicarbonate (ABC) for 1 h with three intermittent changes of the solution [36]. The supernatants were replaced with 10 mM DTT (Sigma) in 50 mM ABC solution to reduce the protein over 15 min at 56 0C. The final supernatant was discarded and 20 mM iodoacetamide (Sigma) in 50 mM ABC was added and incubated for 15 min at room temperature (RT) in the dark to alkylate the proteins. The gel pieces were collected, washed three times with 200 L of 50 mM ABC for 15 min at RT and then dried in vacuo. The dried gel pieces were rehydrated with 3.0 L of 12.5 ng/L of sequencing grade modified trypsin gold (Promega) in 50 mM ABC and incubated for 60 min at RT. Further, 50 L of 10% acetonitrile in 50 mM ABC was added and the digestion of proteins was continued for 18 h at 37 0C with agitation. The supernatant was collected and the gel pieces were extracted over 40 min at 37 0C with successive 50 L aliquots of 0.1% TFA and 50% acetonitrile. The combined extracts were concentrated using Speed Vac. The peptide extracts were reconstituted in 10 L of 50% acetonitrile and 0.1% TFA. Reconstituted peptides were spotted on 384 well MALDI plate after mixing with cyano-4- hydroxyl cinnamic acid matrix in 1:1 (v/v) ratio. The peptides were analyzed on AB Sciex 4800 Plus TOF/TOF analyzer in reflector ion mode. To identify the peptides, Protein Pilot 2.0 software was used to inspect peptide masses obtained from the mass spectrometric analysis against given translated EST sequence (GenBank: HS559044.1) obtained from the NCBI database blast search of translated nucleotide database using protein query with search set of EST against selected organism W. tinctoria. In the Protein Pilot search, biological modification and amino acid substitution was incorporated as ID focus factor along with the gel based ID search. Detection protein threshold was fixed at a confidence score of 99.9%. The peptide sequences derived from the MS/MS analysis of trypsin digested peptides were searched for homology in open database. Chemical Modification of Trypsin Digested Protein by SPITC for De novo Sequence To obtain independent, de novo sequence of trypsin digested peptides of WTG, SPITC modification of peptides was performed following an earlier described method [37]. In brief, trypsin digested peptides were diluted with 0.1% TFA in water and adsorbed on ziptip. Ziptip with bound peptide were washed with 5% methanol and water containing 0.1% TFA. The desalted peptides were then incubated for 1 h at 55 0C with 5 mg/mL of SPITC (Sigma Aldrich, St.Louis,USA) in 20 mM sodium bicarbonate. After the reaction, ziptip was washed 3 times with 0.1% TFA and peptides were eluted in 3 L of 50% acetonitrile and 0.1% TFA. The eluted peptides were deposited in CHCA on MALDI target plate. SPITC derivatised (+215 Da) arginine-terminated peptides was manually selected for MS/MS.

Protein & Peptide Letters, 2013, Vol. 20, No. 5 501

Hemagglutination Assay The hemagglutination activity (H.U.) of purified protein was tested against human erythrocytes by standard serial dilution technique in a multi-well microtiter plate. Blood samples of 1 mL volume of different blood types (A, B, O) were taken in centrifuge tubes containing 6% EDTA as anticoagulant in 3 mL of phosphate buffer saline (PBS), pH 7.2. The mixture was then centrifuged at 1000 g at 4 °C for 5 min. Supernatant was discarded and the cell pellet was diluted and resuspended in 10 mL of PBS buffer. Dilution and centrifugation was repeated until the supernatant was clear. Finally, erythrocytes were resuspended in PBS buffer to make a 3% (v/v) blood suspension. Fifty L of protein (between 10 g/mL and 100 g/mL) sample was added to 50 L of 3% suspension of human red blood cells. The agglutination reaction was assessed after 1 h incubation at 37 °C. Specific activity was expressed as the minimum WTG concentration (g/mL) having detectable hemagglutination activity [38, 39] (Table 1). Negative control containing BSA instead of WTG was used for the assay. Far-UV Circular Dichroism Spectrum For estimation of secondary structure elements, purified WTG was subjected to CD analysis using Chirascan circular dichroism spectrometer (Applied Photophysics Ltd, Surrey, United Kingdom). CD spectra was collected between 190 to 260 nm in 0.5 nm wavelength steps and an average time of 3.0 s at 25 ºC using 1 mm quartz cell under constant nitrogen purge. Protein samples at concentrations of 0.1 mg/mL, 0.2 mg/mL and 0.3 mg/mL were used in 20 mM potassium phosphate buffer, pH 7.5 [40, 41]. All solutions were prepared fresh, filtered through 0.45 m Millex syringe filter and degassed to avoid noise in CD spectrum. For each protein sample, 3 scans were collected, averaged and the baseline corresponding to the above buffer was subtracted to obtain the final values. The CD data is expressed in terms of mean residue ellipticity. The data thus obtained was analyzed using the web based DichroWeb software [42] and the neuronal network program CDNN . Effects of chemical denaturants on the secondary structure conformation of WTG were determined by incubation of 0.2 mg/mL of purified WTG with 8 M urea or 6 M GdnHCl for 4 h at 25 °C followed by the far-UV CD measurements. Intrinsic Fluorescence Spectrum For the emission fluorescence measurements, purified protein (50 g/mL) in 20 mM phosphate buffer, pH 7.5 filtered through 0.45 m Millex syringe filter was used. Protein was excited at 280 nm and emission wavelength spectra were recorded at 290-400 nm using Varian Cary Eclipse fluorescence spectrometer (Varian, Inc., Walnut Creek, CA) at constant temperature (25 °C). Emission slits were at 5 nm and quartz cuvette of 1 cm path length was used. Fluorescence measurements were performed to study the effect of chemical denaturants (urea and GdnHCl), pH and DTT on WTG. The protein samples were incubated overnight with urea (1 to 8 M) or GdnHCl (1 to 6 M), 2 h with DTT (0.1 to 10 mM) and 2 h in the pH range from 2 to 13 at room temperature.


Table 1.

Kumar et al.

Hemagglutination activities of WTG obtained at different stages of purification. Fractions

Total Protein (mg)

H.U.

Specific Activity (H.U/mg)

Fold Purification

Crude extract

385

1540

4

1

DEAE

62.7

697

11.1

2.8

Gel filtration

40

615

15.4

3.85

Crystallization Crystallization was performed by the sitting-drop vapour diffusion method in 96-well crystallization plates at 293 K. A 2 L droplet of protein mixed in a 1:1 ratio with reservoir solution was equilibrated against 100 L reservoir solution. Final protein concentration in the drops was 20 mg/mL. Initial crystallization conditions were obtained using Hampton Crystal Screen I. Initially, small crystals were obtained from the 13th condition containing 0.2 M sodium citrate tribasic dihydrate, 100 mM Tris hydrochloride pH 8.5, 30% v/v PEG 400. Crystals suitable for X-ray diffraction were obtained by optimizing the condition by varying the pH and precipitant concentration.

matography using AKTA purifier. The target protein eluted in a major peak while other impurities showed up as minor peaks (Fig. 1B). The purity of protein in the major peak fractions was analyzed on non-reducing SDS-PAGE and they showed a single band indicating homogeneity (Fig. 1B). The pure protein fractions were pooled and concentrated to about 40 mg/mL. The estimated yield of purified WTG protein was about 8 mg/gram of seeds.

Data Collection and Preliminary Crystallographic Analysis Crystals were soaked in cryoprotectant solution for 2 min prior to freezing in a nitrogen cooled stream. Crystals were mounted in cryoloops and flash-cooled by liquid nitrogen prior to X-ray diffraction analysis. Data were collected with a MAR 345 imaging-plate system using Cu K radiation generated by a Bruker Microstar H rotating-anode generator operated at 45 kV and 60 mA and equipped with Helios optics. Data were collected as 200 images with a crystal to detector distance of 200 mm with 0.5° oscillation per image and time of exposure was 5 min. The diffraction data were processed using the HKL-2000 suite of programs [43]. The structure solution was attempted by the molecular replacement method with the program MOLREP [44, 45] using poly-Ala model of available 11S globulin structures. The best solution was obtained using the poly-Ala model of Pru du amandin, an allergenic protein from Prunus dulcis (PDB ID: 3EHK) as a search model. This solution revealed acceptable crystal packing and no clashes between symmetry related molecules. WTG model was built into the electron density map using the program COOT [46] and the refinement is in progress using REFMAC5 [47]. RESULTS Purification and Sequencing of WTG WTG was purified successfully to homogeneity in two steps by anion-exchange and size-exclusion chromatographies. In the first step, crude extract was applied on to a Hi-Trap DEAE FF column. After extensive washing, WTG was eluted from the column using NaCl gradient. Partially purified protein having hemagglutinating activity was eluted in the NaCl concentration range of 0.45 M to 0.6 M, which was analyzed on non-reducing 15% SDS-PAGE (Fig. 1A). Subsequently, the DEAE fractions were pooled and concentrated to 10 mg/mL. In the second step, concentrated partially purified protein was subjected to size-exclusion chro-

Figure 1. Chromatographic purification profiles and SDS-PAGE of WTG: (A) DEAE chromatography profile and non-reducing SDSPAGE in 15% gel: Lane 1, molecular weight marker; lane 2, crude extract; lane 3 DEAE eluted fraction. (B) Size-exclusion chromatography profile and non-reducing SDS-PAGE in 15% gel: Lane 1, molecular weight marker; lane 2 and 3, Superdex 200 peak fractions.

WTG protein isolated from seeds of W. tinctoria was also analyzed on 15% SDS-PAGE in the presence and absence of a reducing agent (-mercaptoethanol). In the absence of reducing agent, WTG showed a single band with estimated molecular weight of ~56 kDa (Fig. 2A). While in the pres-


ence of reducing agent the WTG contains heterogenous populations of two major subunits. The estimated molecular weight of these polypeptides was ~32-34 kDa and ~22-26 kDa. An attempt to estimate the intact mass of WTG by MALDI-TOF confirmed the presence of heterogenous population of subunits since multiple peaks with a range of mass/charge ratio was observed (Fig. 2B). Assuming that the peak with the highest mass/charge ratio represents a population with intact subunits, the molecular weight of WTG is likely to be 49, 570 Da.


To analyze the oligomerization state, the molecular weight of purified protein was estimated by applying the sample to a gel filtration column. Purified protein showed a major peak at an elution volume of 62.1 mL in 50 mM TrisHCl buffer, pH 7.5 (Fig. 1B). Using a standard curve based on molecular weight markers, the molecular weight of WTG was calculated and estimated to be ~320 kDa. This suggests that WTG exists in the hexameric form. Purified WTG was electro blotted onto PVDF membrane from reducing SDS-PAGE gel and N-terminal sequence of the globulin was determined. The sequence obtained was

Figure 2. SDS-PAGE and MALDI-TOF analysis of WTG. (A) SDS-PAGE. Lane 1, molecular weight marker; lane 2, purified protein in the absence of reducing agent; lane 3, purified protein in the presence of reducing agent. Subunits are designated as and , respectively. (B) Intact mass determination by MALDI-TOF indicates the presence of multiple subunits in WTG that vary in their concentration. In the intact mass spectra of the native protein three singly charged molecular ion peaks of protein subunits at the molecular weight of approximately 33.1 kDa, 40.9 kDa and 49.5 kDa were observed. The +2 charged peaks indicated molecular weights of approximately 16.55 kDa, 20.45 kDa and 24.80 kDa, respectively.


LRTPQLNEAQ. This sequence was used for homology search by applying blast tool in NCBI database against the expressed sequence tags in the search of translated nucleotide database using a protein query with the option of organism as W. tinctoria. The N-terminal residues of WTG matched with 100% identity to the ESTs of W. tinctoria developing embryos having gene identities as GenBank HS561676.1, HS558020.1, HS572228.1, HS563045.1, HS571038.1, HS557004.1, HS562364.1 and HS560184.1. Among all these ESTs, HS572228.1 has the maximum length of 256 amino acids and was used for the identification of the protein, which confirms that it belongs to the 11S seed storage globulin (Supplementary Fig. 1). Moreover, to verify the EST sequence identified on the basis of N-terminal sequence, we performed partial internal sequencing using mass spectrometry. MS and MS/MS spectra were acquired using trypsin digested protein (Supplementary Fig. 2). The MS/MS spectra were used to generate the sequence of some peptides. The obtained partial sequences (Supplementary Fig. 2B and 2C) were compared to the N-terminal amino acid based translated EST, which shows 37% identity to the GenBank HS572228.1 and a maximum identity of 69% to the GenBank: HS559044.1. De novo amino acid sequence of one internal peptide of WTG was also obtained (Supplementary Fig. 2D); IQHEAGYSEIWDPTSR) after chemical modification of the peptide that helped suppress the “y” ions 37. Apart from these, non redundant NCBI blast were used against the obtained internal sequence and the results (Fig. 3) showed that all the sequences belonged to 11S seed storage globulin having bi-Cupin conserved domain, a characteristic feature of 11S seed storage globulins. Hemagglutinating Activity of WTG Hemagglutinating activity was detected in seed extracts of W. tinctoria and measured after each step of purification (Table 1) using human erythrocytes from three blood groups (A, B, O). Purified protein showed lattice formation of the erythrocytes of all the blood groups with significant specific activity of ~65g/mL (Table 1). Absence of such activity for BSA used as a negative control confirmed that WTG was indeed responsible for the hemagglutinating activity. Circular Dichroism Studies of WTG The secondary structure of purified WTG protein was measured by far-UV CD spectroscopy. CD spectra show maximal negative mean residual ellipticities [] near 210 nm for WTG. The CD spectral data were analyzed and deconvoluted by CDNN and other algorithms available through the DichroWeb website. CDNN yields a structural content of 44.2% sheets, 10.3% -helix, 20.1% turns, and 26.6% random coil. Further, the CD spectra obtained for WTG after incubating the protein with high concentrations of denaturants (8 M urea and 6 M GdnHCl) showed very different ellipiticity values at 210 nm and altered shape of the CD spectra (Fig. 4B) indicating denaturation. Fluorescence studies of WTG Fluorescence emission spectra of WTG protein were recorded in 20 mM phosphate buffer pH 7.5 by exciting the

Kumar et al.

tryptophan residues at 280 nm (Fig. 5). The emission max value for native protein was 330 nm. Upon addition of 1-8 M urea, a redshift was observed and the max of protein in 8 M urea was 348 nm (Fig. 5A). A redshift was also observed in the presence of GdnHCl and the max of protein in 6 M GdnHCl was 356 nm (Fig. 5B). The redshift of 18 nm and 26 nm observed for WTG in the presence of 8 M urea and 6 M GdnHCl, respectively, suggests a significant gain in tryptophan solvent accessibility in presence of denaturants. Effect of pH over a range of 2 to 13 on the emission fluorescence spectra of native WTG protein was studied (Fig. 5C). Between pH 5 to 10, the emission spectra resembled the native spectrum at pH 7.5 while strong shift was observed at less than pH 5 and higher than pH 10. Protein samples that were incubated for two hours with varying concentration of DTT were used for studying the shift in emission max values. A small shift of 1 nm in 10 mM DTT and decrease in the fluorescence emission intensity with increasing DTT concentration was observed (data not shown). Crystallization and X-ray Diffraction Studies of WTG For X-ray diffraction studies, crystallization was attempted using purified protein concentrated to about 40 mg/mL. Diffraction quality crystals of WTG were obtained after 20 days using 100 mM Tris pH 7.8, 0.2 M sodium citrate tribasic dihydrate and 28% PEG 400 (v/v) (Fig. 6). Crystals were soaked in cryoprotectant solution containing 20% glycerol, 100 mM Tris pH 7.8, 0.2 M sodium citrate tribasic dihydrate and 28% PEG. Crystals belonged to the orthorhombic space group P212121 and diffracted to 2.2 Å resolution in-house (Supplementary Fig. 3). The unit-cell parameters were found to be a =109.9 Å, b = 113.2 Å, c = 202.2 Å with six molecules per asymmetric unit, which corresponds to a crystal volume per unit molecular weight (VM) of 1.94 Å3 Da-1, given the molecular weight of 56 kDa for the protein. The preliminary structure of WTG was determined by molecular replacement method. The data-collection statistics are summarized in Table 2. After collection of the diffraction pattern some of the crystals were crushed and silver stained to verify the homogeneity of the crystallized protein (Supplementary Fig. 4). The bands present corresponded to the expected molecular weight and indicated homogeneity. DISCUSSION The seed globulin from W. tinctoria is unique in its ability to hemagglutinate erythrocytes. On this basis the fractions containing the activity were purified in two steps, first by anion exchange and then by gel filtration chromatography. SDS-PAGE reveals that in the absence of reducing agent the protein exists as a homogeneous species while in the presence of reducing agent WTG contains heterogeneous populations of two major subunits that are linked by disulfide bridges. The higher and lower molecular weight polypeptides are referred to as acidic () and basic () subunits respectively in 11S globulins isolated from oat [48], pea [49, 50] and broad bean 4. We have adopted the same nomenclature for WTG. In general, 11S globulins have been reported



Figure 3. Sequence alignment of the obtained internal sequence with GenBank: HS559044.1 of W. tinctoria followed by other homologues of globulins of 11S nature.

Figure 4. Far UV circular dichroism analysis of WTG 11S globulin from W. tinctoria at 25 °C. (A) CD spectra of the protein at concentrations of 0.1 mg/mL, 0.2 mg/mL, and 0.3 mg/mL in 20 mM potassium phosphate buffer, pH 7.5. (B) CD spectra of the protein in the same buffer containing 6 M GdnHCl and 8 M urea.


Kumar et al.

Table 2. Data-Collection Statistics Obtained for 11S Wrightia tinctoria Globulin. Space group

P212121

Unit-cell parameters (Å)

a =109.9, b = 113.2 Å, c = 202.2 Å

Resolution range (Å)

50-2.2 (2.25-2.21)

Completeness (%)

97.9 (96.4)

Rsym† (%)

8.5 (30.4)

Mean I/(I)

13.5 (2.3)

No. of observed reflections

538437

No. of unique reflections

122995 (6201)

Molecules per ASU

6

Mathews coefficient (Å3 Da-1 )

1.94

Solvent content (%)

37

Redundancy

4.4 (2.4)

Mosaicity (º)

1.1

(† Rsym = h l |Ihl- |/ h l , where Il is the lth observation of reflection h and is the weighted average intensity for all observations l of reflection h. Values in parentheses are for the highest resolution shell (2.25–2.21Å))

as very heterogeneous proteins that contain no identical acidic and basic subunits. The acidic and basic subunits randomly unite to form heterodimers which further associate and produce heterogeneity in globulin oligomers [48, 51]. For example, buckwheat globulin which exists as a heterooligomer has been reported to be composed of a large number of polypeptides which differ in molecular weight and relative concentrations [52]. Such observation is likely to be true for WTG as well since it shows multiple subunits under reducing conditions on SDS-PAGE. MALDI-TOF-based intact mass determination further supports the presence of heterogeneous population in the sample as evidenced by the observation of multiple peaks of mass/charge ratio with the highest mass/charge ratio likely to be the molecular weight of WTG. The oligomerization state of the protein was revealed by gel filtration analysis, which indicates that WTG exists in the hexameric form. The N-terminal and internal sequencing results confirm that WTG belongs to the 11S seed storage class of the proteins and assessed by the “nr BLAST” of NCBI showed that all the sequences belong to 11S seed storage globulin having bi-Cupin conserved domain, a characteristic feature of 11S seed storage globulins. The lattice formation obtained against erythrocytes reveals the hemagglutinating activity of WTG, which may be further correlated to its lectin like activity. To the best of our knowledge this is the first report of 11S globulin possessing hemagglutinating activity and it may lead to lectin like

Figure 5. Fluorescence emission spectra of the purified protein (50 g/mL) in 10 mM phosphate buffer pH 7.5 under native and denatured conditions. (A) Fluorescence spectra in the absence and in the presence of increasing concentration of urea. (B) Fluorescence spectra in the absence and in the presence of increasing concentration of guanidium hydrochloride (GdnHCl). (C) Effect of pH on the intrinsic fluorescence of WTG.

behavior of the protein. Some plant lectins were reported to possess insecticidal and fungicidal activities and have been suggested to play a defensive role against insects and pathogens [53, 54, 55, 56]. Further investigations into the antiinsect and antimicrobial activities of the protein are needed to decipher the possible role for lectin-like property of WTG. The CD analysis reveals that WTG protein is primarily composed of -sheets with small amounts of -helices. The maximal negative mean residue ellipticities [] near 210 nm



mined by molecular replacement method, shows that it is present in a hexameric form. Due to unavailability of complete amino acid sequence of WTG, the refinement calculations are still in progress. The presence of bands on SDS-PAGE corresponding to the expected molecular weight when crushed crystals were used indicates that the crystals were homogenous combination of subunits. This is in sharp contrast to the observation of heterogeneous combination of subunits seen for reducing SDS-PAGE and MALDI-TOF experiments described previously. The apparent anomaly can probably be explained by the fact that the heterogeneous population contains one combination of the subunits of WTG at a sufficiently high concentration that allows it to form crystals of homogenous protein. CONCLUSION Figure 6. Crystals of WTG 11S globulin from W. tinctoria. The longest dimension of a typical crystal is between 120 - 160 m.

for WTG are characteristic for 11S globulin proteins [57, 58]. The far-UV CD spectra obtained for WTG after incubating the protein with high concentrations of denaturants (8 M urea and 6 M GdnHCl) results both in loss of ellipiticity values at 210 nm and change in the shape of the CD spectra indicating the loss of secondary structure. Tryptophan, among other aromatic amino acids, is quite sensitive to the change of the local environment and roughly acts as an indicator that correlate the extent of solvent exposure to the chromospheres [59]. The emission max value for native protein was 330 nm. This is a characteristic emission profile of tryptophan residues in a relatively hydrophobic environment indicating that WTG is natively folded [60]. A significant gain in tryptophan solvent accessibility in presence of denaturants indicates tryptophan solvent accessibility and additional randomization of the tertiary structure in presence of 6 M GdnHCl compared to 8 M urea. Similar results for 11S globulin from Brazil nut in presence of urea have been reported [61], and is expected since GdnHCl is a stronger denaturant than urea.

A globulin was purified and crystallized for structural and functional studies from W. tinctoria seeds. Protein was identified as 11S globulin based on N-terminal and partial internal sequencing. The 11S WTG protein is hexameric with a molecular weight of ~320 kDa composed of monomers of ~56 kDa. The subunits of the monomer consist of acidic (~32 kDa) and basic (~24 kDa) polypeptides, which are linked by disulfide bonds. CD spectral analysis of the protein shows predominance of -sheets similar to known 11S globulins. Moreover WTG displays hemagglutinating activity that may impart the unique ability to it to act as lectin like protein. Purified protein was crystallized and Xray diffraction data has been collected. Molecular cloning of WTG protein is in progress from which complete amino acid sequence will be deduced for crystal structure refinement, modeling and analysis. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

ACKNOWLEDGEMENTS

The behaviors of WTG at different pH indicate that the protein is structurally stable in the pH range of 5 to 10. However, the spectra at pH greater than 10 and pH less than 5 showed distinct decrease in the emission fluorescence intensity and pronounced redshift (18 nm at pH 2, 13 nm at pH 13) in max. The shift of 18 nm at pH 2.0 was similar to that in 8 M urea. Moreover the strong reducing nature of DTT is predicted to reduce the disulfide bond that holds the different subunits of 11S globulin hexamer of WTG. Thus, tryptophan residues are rendered more accessible to the solvent resulting decrease in the emission fluorescence intensity. Similar observation for fluorescence emission spectra in presence of DTT has been reported for phaseolin, a globulin from red kidney bean [62].

Protein purification and X-ray diffraction experiments were performed at the Macromolecular Crystallography Unit (MCU), Institute Instrumentation Centre, IIT Roorkee. We thank Dr. Ashwani K. Sharma and Sonali Dhindwal for helpful discussions and critical reading of the manuscript. Pramod thanks CSIR, Dipak thanks Ministry of Human Resource Development (MHRD), Shailly thanks CSIR and Pravindra thanks DST and DBT for the financial support. Suman appreciates support from Central Instrumentation Facility at UDSC and funding from University of Delhi (R&D funds) and UGC (SAP programme), Government of India.

The initial crystallization studies showed that WTG corresponds to a crystal volume per unit molecular weight (VM) 3 -1 of 1.94 Å Da , given the molecular weight of 56 kDa for the protein. This is within the range of the values expected for most protein crystals [63] and corresponds to a solvent content of 37%. The preliminary structure of WTG, deter-

Supplementary material is available on the publishers Web site along with the published article.

SUPPLEMENTARY MATERIAL

ABBREVIATIONS ABC

=

ammonium bicarbonate


BSA

=

bovine serum albumin

CD

=

circular dichroism

DEAE

=

diethyl amino ethyl

DTT

=

dithiothreitol

EDTA

=

ethylene diamine tetraacetic acid

GdnHCl

=

guanidine hydrochloride

PEG

=

polyethylene glycol

PMSF

=

phenyl methanesulfonyl fluoride

PVDF

=

polyvinylidene fluoride

SDS-PAGE =

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TFA

=

trifluoroacetic acid

WTG

=

Wrightia tinctoria globulin

SPITC

=

4-sulpho phenyl isothiocyanate

REFERENCES [1] [2]

[3]

[4] [5]

[6] [7]

[8] [9] [10]

[11]

[12]

[13]

[14]

Shewry, P.R.; Halford, N.G. Cereal seed storage proteins: structures, properties and role in grain utilization. J. Exp. Bot., 2002, 53(370), 947-958. Mandal, S.; Mandal, R.K. Seed storage proteins and approaches for improvement of their nutritional quality by genetic engineering. Curr. Sci., 2000, 79(5), 576-589. Rangel, A.; Domont, G.B.; Pedrosa, C.; Ferreira, S.T. Functional properties of purified vicilins from cowpea (Vigna unguiculata) and pea (Pisum sativum) and cowpea protein isolate. J. Agric. Food. Chem., 2003, 51(19), 5792-5797. Wright, D.J.; Boulter, D. Purification and subunit structure of legumin of Vicia faba L. (broad bean). Biochem. J., 1974, 141(2), 413-418. Yagasaki, K.; Takagi, T.; Sakai, M.; Kitamura, K. Biochemical characterization of soybean protein consisting of different subunits of glycinin. J. Agric. Food Chem., 1997, 45(3), 656-660. Shotwell, M.A.; Afonso, C.; Davies, E.; Chesnut, R.S.; Larkins, B.A. Molecular characterization of oat seed globulins. Plant physiol., 1988, 87(3), 698-704. Rassam, M.; Laing, W.A. The interaction of the 11S globulin-like protein of kiwifruit seeds with pepsin. J. Plant Sci., 2006, 171(6), 663-669. Burgess, S.R.; Shewry, P.R. Identification of homologous globulins from embryos of wheat, barley, rye and oats. J. Exp. Bot., 1986, 37(12), 1863-1871. Komatsu, S.; Hirano, H. Rice seed globulin: a protein similar to wheat seed glutenin. Phytochem., 1992, 31(10), 3455-3459. Jin, T.; Chen, Y.W.; Howard, A.; Zhang, Y.Z. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2008, 64(7), 641-644. Coelho, M.B.; Macedo, M.L.R.; Marangoni, S.; Silva, D.S.; Cesarino, I.; Mazzafera, P. Purification of legumin-like proteins from Coffea arabica and Coffea racemosa seeds and their insecticidal properties toward cowpea weevil (Callosobruchus maculatus)(Coleoptera: Bruchidae). J. Agric. Food Chem., 2010, 58(5), 3050-3055. Marsh, J.; Rigby, N.; Wellner, K.; Reese, G.; Knulst, A.; Akkerdaas, J.; van Ree, R.; Radauer, C.; Lovegrove, A.; Sancho, A. Purification and characterisation of a panel of peanut allergens suitable for use in allergy diagnosis. Mol. Nutr. Food Res., 2008, 52(1), S272-S285. Gaur, V.; Sethi, D.K.; Salunke, D.M. Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2008, 64(1), 32-35. Beyer, K.; Grishina, G.; Bardina, L.; Grishin, A.; Sampson, H.A. Identification of an 11S globulin as a major hazelnut food allergen

Kumar et al.

[15]

[16]

[17]

[18] [19]

[20]

[21]

[22] [23] [24]

[25]

[26] [27]

[28] [29]

[30]

[31]

[32]

[33]

in hazelnut-induced systemic reactions. J. Allergy Clin. Immunol., 2002, 110(3), 517-23. Macedo, M.L.R.; Coelho, M.B.; Freire, M.G.M.; Machado, O.L.T.; Marangoni, S.; Novello, J.C. Effect of a toxic protein isolated from Zea mays seeds on the development and survival of the cowpea weevil, Callosobruchus maculatus. Protein Pept. Lett., 2000, 7(4), 225-232. Soares, E.L.; Freitas, C.D.T.; Oliveira, J.S.; Sousa, P.A.S.; Sales, M.P.; Barreto-Filho, J.D.M.; Bandeira, G.P.; Ramos, M.V. Characterization and insecticidal properties of globulins and albumins from Luetzelburgia auriculata (Allemao) Ducke seeds towards Callosobruchus maculatus (F.)(Coleoptera: Bruchidae). J. Stored Prod. Res., 2007, 43(4), 459-467. Moura, F.T.; Oliveira, A.S.; Macedo, L.L.P.; Vianna, A.L.B.R.; Andrade, L.B.S.; Martins-Miranda, A.S.; Oliveira, J.T.A.; Santos, E.A.; Mauricio, P. Effects of a chitin-binding vicilin from Enterolobium contortisiliquum seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus) and phytopathogenic fungi (Fusarium solani and Colletrichum lindemuntianum). J. Agric. Food Chem., 2007, 55(2), 260-266. Shewry, P.R.; Napier, J.A.; Tatham, A.S. Seed storage proteins: structures and biosynthesis. Plant Cell, 1995, 7(7), 945-956. Jin, T.A.; Albillos, S.M.; Guo, F.; Howard, A.; Fu, T.J.; Kothary, M.H.; Zhang, Y.Z. Crystal structure of prunin-1, a major component of the almond (Prunus dulcis) allergen amandin. J. Agric. Food Chem., 2009, 57(18), 8643-8651. Adachi, M.; Okuda, E.; Kaneda, Y.; Hashimoto, A.; Shutov, A. D.; Becker, C.; Müntz, K.; Utsumi, S., Crystal structures and structural stabilities of the disulfide bond-deficient soybean proglycinin mutants C12G and C88S. J. Agric. Food Chem., 2003, 51(16), 46334639. Jung, R.; Scott, M.P.; Nam, Y.W.; Beaman, T.W.; Bassuner, R.; Saalbach, I.; Muntz, K.; Nielsen, N.C. The role of proteolysis in the processing and assembly of 11S seed globulins. Plant Cell, 1998, 10(3), 343-358. Müntz, K. Proteases and proteolytic cleavage of storage proteins in developing and germinating dicotyledonous seeds. J. Exp. Bot., 1996, 47(5), 605-622. Shewry, P.; Jones, H.; Halford, N. Plant biotechnology: transgenic crops. Adv. Biochem. Eng. Biotechnol., 2008, 111, 149-186. Murdock, L.L.; Shade, R.E. Lectins and protease inhibitors as plant defenses against insects. J. Agric. Food. Chem., 2002, 50(22), 6605-6611. Tandang-Silvas, M.R.G.; Fukuda, T.; Fukuda, C.; Prak, K.; Cabanos, C.; Kimura, A.; Itoh, T.; Mikami, B.; Utsumi, S.; Maruyama, N. Conservation and divergence on plant seed 11S globulins based on crystal structures. Biochim. Biophys. Acta, 2010, 1804(7), 14321442. Bright, S.W.J.; Shewry, P.R.; Kasarda, D.D. Improvement of protein quality in cereals. Crit. Rev. Plant Sci., 1983, 1(1), 49-93. Rascon-Cruz, Q.; Sinagawa-Garcia, S.; Osuna-Castro, J.A.; Bohorova, N.; Paredes-Lopez, O. Accumulation, assembly, and digestibility of amarantin expressed in transgenic tropical maize. Theor. Appl. Genet., 2004, 108(2), 335-342. Ngan, P.T. A revision of the genus Wrightia (Apocynaceae). Ann. Missouri Bot. Gard., 1965, 52(2), 114-175. Reddy, Y.S.R.; Venkatesh, S.; Ravichandran, T.; Murugan, V.; Suresh, B. Antinociceptive activity of Wrightia tinctoria bark. Fitoterapia, 2002, 73(5), 421-423. Ponnusamy, K.; Petchiammal, C.; Mohankumar, R.; Hopper, W. In vitro antifungal activity of indirubin isolated from a South Indian ethnomedicinal plant Wrightia tinctoria R. Br. J. Ethnopharmacol., 2010, 132(1), 349-354. Selvam, P.; Murugesh, N.; Witvrouw, M.; Keyaerts, E.; Neyts, J. Studies of antiviral activity and cytotoxicity of Wrightia tinctoria and Morinda citrifolia. Indian J. Pharm. Sci., 2009, 71(6), 670672. Kumar, L.; Rao, K.N.V.; Madhvi, b.; Kumar, S.; Banji, D. Anti oxidation activity of Wrightia tinctoria Roxb bark and Schrebera swietenoides Roxb bark extract. J. Pharm. Res., 2011, 4(2), 396397. Bigoniya, P.; Singh, C.S.; Shukla, A. Pharmacognostical and physicochemical standardization of ethnopharmacologically important seeds of Lepidium sativum Linn. and Wrightia tinctoria R. Br. Indian J. Nat. Prod. Resour., 2011, 2(4), 464-471.

Purification and Biophysical Characterization [34]

[35] [36]

[37]

[38] [39]

[40]

[41] [42]

[43] [44]

[45] [46]

[47] [48]

[49]


Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227(5259), 680685. Matsudaira, P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem., 1987, 262(21), 10035-10038. Rosenfeld, J.; Capdevielle, J.; Guillemot, J.C.; Ferrara, P. In-gel digestion of proteins for internal sequence analysis after one-or two-dimensional gel electrophoresis. Anal. Biochem., 1992, 203(1), 173-179. Joss, J.L.; Molloy, M.P.; Hinds, L.A.; Deane, E.M. Evaluation of chemical derivatisation methods for protein identification using MALDI MS/MS. Int. J. Pept. Res. Ther., 2006, 12(3), 225-235. Moreira, R.D.A.; Perrone, J.C. Purification and partial characterization of a lectin from Phaseolus vulgaris. Plant Physiol., 1977, 59(5), 783. Horta, A.N.A.C.G.; De Azevedo, M.R. Isolation and partial characterization of a lectin from Bauhinia pentandra (bong) vog. Ex. Steua. Braz. J. Plant Physiol., 2001, 13(3), 262. Matsuura, J.E.; Manning, M.C. Heat-Induced gel formation of lactoglobulin: A study on the secondary and tertiary structure as followed by circular dichroism spectroscopy. J. Agric. Food Chem., 1994, 42(8), 1650-1656. Zemser, M.; Friedman, M.; Katzhendler, J.; Greene, L.L.; Minsky, A.; Gorinstein, S. Relationship between functional properties and structure of ovalbumin. J. Protein Chem., 1994, 13(2), 261-274. Whitmore, L.; Wallace, B.A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res., 2004, 32(suppl 2), W668-W673. Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol., 1997, 276(Macromolecular Crystallography, Part A), 307-326. CCP4, Collaborative Computational Project Number 4, The CCP4 Suite: Programs for Protein Crystallography. Acta Crystallogr. D Biol. Crystallogr. 1994, 50(1), 760-763. Vagin, A.; Teplyakov, A., MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr., 1997, 30(6), 10221025. Emsley, P.; Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr., 2004, 60(12), 2126-2132. Murshudov, G.N.; Vagin, A.A.; Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr., 1997, 53(3), 240-255. Brinegar, A.C.; Peterson, D.M. Separation and characterization of oat globulin polypeptides. Arch. Biochem. Biophys., 1982, 219(1), 71-79. Peterson, D.M. Subunit structure and composition of oat seed globulin. Plant physiol., 1978, 62(4), 506-509.

Received: June 06, 2012

Revised: July 14, 2012

Accepted: July 15, 2012

[50]

[51] [52]

[53] [54]

[55] [56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

Casey, R. Genetic variability in the structure of the -subunits of legumin from Pisum—A two-dimensional gel electrophoresis study. Heredity, 1979, 43(2), 265-272. Langston-Unkefer, P.J.; Gade, W. A seed storage protein with possible self-affinity through lectin-like binding. Plant physiol., 1984, 74(3), 675-680. Choi, S.M.; Ma, C.Y. Extraction, purification and characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) seeds. Food Res. Intl., 2006, 39(9), 974-981. Chen, Y.; Barkley, M.D. Toward understanding tryptophan fluorescence in proteins. Biochemistry, 1998, 37(28), 9976-9982. Pohleven, J.; Brzin, J.; Vrabec, L.; Leonardi, A.; Cokl, A.; trukelj, B.; Kos, J.; Saboti, J. Basidiomycete Clitocybe nebularis is rich in lectins with insecticidal activities. Appl. Microbiol. Biotechnol., 2011, 91(4), 1141-1148. Lam, S.K.; Ng, T.B. Lectins: production and practical applications. Appl. Microbiol. Biotechnol., 2011, 89(1), 45-55. Banerjee, N.; Sengupta, S.; Roy, A.; Ghosh, P.; Das, K.; Das, S. Functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status. PLoS ONE, 2011, 6(4), e18593. Zirwer, D.; Gast, K.; Welfle, H.; Schlesier, B.; Dieter Schwenke, K. Secondary structure of globulins from plant seeds: A reevaluation from circular dichroism measurements. Int. J. Biol. Macromol., 1985, 7(2), 105-108. Clara Sze, K.W.; Kshirsagar, H.H.; Venkatachalam, M.; Sathe, S.K. A circular dichroism and fluorescence spectrometric assessment of effects of selected chemical denaturants on soybean (Glycine max L.) storage proteins glycinin (11S) and -conglycinin (7S). J. Agric. Food Chem., 2007, 55(21), 8745-8753. Chen, J.; Liu, B.; Ji, N.; Zhou, J.; Bian, H.; Li, C.; Chen, F.; Bao, J. A novel sialic acid-specific lectin from Phaseolus coccineus seeds with potent antineoplastic and antifungal activities. Phytomedicine, 2009, 16(4), 352-360. Dufour, E.; Hoa, G.H.B.; Haertle, T. High-pressure effects on lactoglobulin interactions with ligands studied by fluorescence. Biochim. Biophys. Acta, 1994, 1206(2), 166-172. Sharma, G.M.; Mundoma, C.; Seavy, M.; Roux, K.H.; Sathe, S.K. Purification and biochemical characterization of Brazil nut (Bertholletia excelsa L.) seed storage proteins. J. Agric. Food Chem., 2010, 58(9), 5714-5723. Yin, S.W.; Tang, C.H.; Wen, Q.B.; Yang, X.Q. Conformational and thermal properties of phaseolin, the major storage protein of red kidney bean (Phaseolus vulgaris L.). J. Sci. Food Agric., 2011, 91(1), 94-99. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol., 1968, 33(2), 491-497.