Characterization of Soluble Amaranth and Soybean Proteins Basedon Fluorescence, Hydrophobicity, Electrophoresis, Amino AcidAnalysis, Circular Dichroism, and Differential Scanning CalorimetryMeasurements

Shela Gorinstein,*, Efren Delgado-Licon, Elke Pawelzik, Herry Heriyati Permady,Moshe Weisz, and Simon Trakhtenberg

Department of Medicinal Chemistry and Natural Products, School of Pharmacy, The HebrewUniversity-Hadassah Medical School, P.O.B. 12065, Jerusalem 91120, Israel, Institute of AgriculturalChemistry, Georg-August-University Goettingen, D-37075 Goettingen, Germany, and Kaplan Medical

Center, Rehovot, Israel

Intrinsic fluorescence (IF), surface hydrophobicity (So), electrophoresis, amino acid analysis, circulardichroism (CD), and differential scanning calorimetry (DSC) were used to study folded and unfoldedsoluble proteins from Amaranthus hypochondriacus (A. h.) and soybean (S). Globulin (Glo) andalbumin subfractions (Alb-1 and Alb-2) were extracted from A. h. and S and denatured with urea.Electrophoretic and functional properties indicated a significant correlation between soluble proteinfractions from soybean and amaranth. The protein fractions shared some common electrophoreticbands as well as a similar amino acid composition. The larger percent of denaturation in proteinfractions, which is associated with enthalpy and the number of ruptured hydrogen bonds, correspondsto disappearance of R-helix. The obtained results provided evidence of differences in their secondaryand tertiary structures. The most stable was Glo followed by the Alb-2 fraction. Predicted functionalchanges in model protein systems such as pseudocereals and legumes in response to processingconditions may be encountered in pharmaceutical and food industries. These plants can be asubstitute for some cereals.

Keywords: Amaranth; soybean; amino acids; proteins; electrophoresis; amino acids; fluorescence;calorimetry; denaturation; spectroscopy

INTRODUCTION

Amaranth belongs to pseudocereals and containsabout 13.2-18.2% protein. Soybean represents legumeswith a high percentage of proteins of three times incomparison with amaranth. A better balanced aminoacid composition (lysine from 3.2 to 6.4%) was found inboth plants in comparison with the major cereals (2.2to 4.5%). This makes amaranth a promising plant as afood or source of dietary proteins and as potentialmatrixes for drug release even in comparison with thesoybean proteins (1-3). The most important task in theintroduction of this plant as a new protein source is theprevention of the destruction of lysine. Some studieshave shown that the popping process causes minimaldestruction to the quality of amaranth protein. How-ever, other studies reveal significant destruction oflysine, thus decreasing the protein quality of the grainand its dietary supply of lysine (3). Nowadays, a ton ofamaranth is produced annually in Mexico for specialfood purposes such as candies, flakes, and flours dis-tributed by stores specialized in nutraceutical foods (3).According to our investigations and literature data,

amaranth and soybean have albumins and globulins asstorage proteins (4-6). Our recent papers were focusedon the structure and functional properties of plantglobulins (2, 7-9). To our knowledge, there are noreferences in the recent literature about the differencesand identity of electrophoretic patterns in isolated seedprotein fractions such as albumins and its subfractions,and globulins of amaranth and soybean.

This paper reports on the characterization of albuminand globulin fractions from Amaranthus hypochondria-cus and soybean (Glycine max) by electrophoresis, aminoacid analysis, fluorescence, hydrophobicity, circulardichroism, and differential scanning calorimetry. Theidentity and differences between pseudocereal andlegume were based on amino acid analysis, electro-phoretic patterns, and functional properties of proteinfractions.

MATERIALS AND METHODS

Sample Preparation. Whole mature seeds from A. hypo-chondriacus (Mexico) and soybean (G. max) with low oilcontent (Brazil) were ground on a mill through a 60-meshscreen. The meal was defatted in a Soxhlet extractor withn-hexane for 10 h and then was stored at 5 C after removalof hexane.

Protein Extraction. Proteins were extracted stepwiseaccording to the following methods (5, 10, 11). The meal (1 g)was extracted with a solvent: sample ratio of 10:1 (v/w) andvigorously shaken. The extracts were separated by centrifugingat 10000g for 10 min. Each step was repeated twice. The

* To whom correspondence should be addressed. Tele-phone: 2-6758690. Fax: 972-2-5410740. E-mail: gorin@cc.huji.ac.il. This author is affiliated with the David R. BloomCenter for Pharmacy.

The Hebrew University-Hadassah Medical School. Institute of Agricultural Chemistry. Kaplan Medical Center.

5595J. Agric. Food Chem. 2001, 49, 55955601

10.1021/jf010627g CCC: $20.00 2001 American Chemical SocietyPublished on Web 10/27/2001

sequence of the used solvents was the following: (a) water-albumins (Alb-1.Os), 0.5 M NaCl (Glo.Os), then water (Alb-2.Os), and (b) 0.5 M NaCl, then separated by dialysis againstwater [albumins-1 (Alb-1.K) and globulins (Glo.K)]. Anotherfraction (Alb-2.K) was extracted with water after removingAlb-1.K and Glo.K. Then all fractions Glo, Alb-1, and Alb-2were dialyzed and freeze-dried. The nitrogen content in eachfraction was determined by the micro-Kjeldahl method com-bined with a colorimetric determination (12).

Amino Acid Analysis. Samples of extracted proteins werehydrolyzed with 6 M HCl and 3% phenol solution in an MLS-MEGA-Microwave system for 20 min at 160 C. The energywas 1000 W the first 5 min and then 500 W for 15 min. Thesamples were then dissolved in 100 L of HCl (20 mM) andfiltered through a 0.45 m filter. Derivatization was done with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (13). Thesample was injected into a Multi-Pump Gradient Water HPLCsystem with a vertex knauer column B1184742 (Knauer,Berlin), length XID 150 4, 6 mm ID, spherimage-80 ) DS2-5m. The Millenium chromatography manager system fromWaters (Waters, Milford, MA) was used to evaluate the aminoacids. Scanning fluorescence detector was used at an excitationof 250 nm and emission of 395 nm. Gradient program consistedof 40% acetate phosphate buffer and 60% acetonitrile.

The values of leucine and lysine were added and presentedas a total of both. TYR + CYS eluted together and arepresented as TYR. The results are given as g/100 g of protein.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electro-phoresis (SDS-PAGE). SDS-PAGE was performed accord-ing to Laemmli (14) using gels of T ) 10% and C ) 2.7% withsome modifications (15). The gels were 1.5-mm thick andconsisted of a 2-cm stacking gel and a 8-cm running gel. Atotal of 25 g of protein was applied to sample slots. Electro-phoresis was carried out at 75 mA for 95 min. Gels werestained with 0.25% Coomassie Brilliant Blue R in methanol/water/acetic acid (5:5:1 v/v) and destained in the same solvent.MW standards IV (Merck, Darmstadt, Germany) were usedto estimate protein subunit molecular masses in kDa: carbonicanhydrase (30); ovalbumin (42.7); bovine serum albumin(66.25); ovotransferin (78). Amaranth albumin 1 (A.Alb-1),amaranth albumin 2 (A.Alb-2), amaranth globulin (A.Glo),soybean albumin 1 (S.Alb-1), soybean albumin 2 (S.Alb-2), andsoybean globulin (S.Glo) were dissolved in sample buffer thatcontained 10% glycerol, 5% 2-ME, and 2% SDS in 0.0625 M(Tris-HCl), pH 6.8. Then the extracts were boiled for 5 minbefore being loaded. Similar bands of Alb-2 from amaranthand soybean at 34.2 and 36.4 kDa, respectively, were cut fromthe electrophoresis gels. Dialysis against GLY after electro-phoretic separation to avoid the artifacts from the glycinebuffer and mercaptoethanol was done. Hydrolyzed gel wasanalyzed as a blank for comparison. The entire protein bandswere hydrolyzed with 6 M HCl containing 3% phenol solutionin an MLS-MEGA II microwave (MLS GmbH, Leutkirch,Germany) for 20 min at 160 C. The energy supply was 1000W for the first 5 min and then 500 W for 15 min (16). Theamino acid concentration was determined as described byCohen and Michaud (13).

Fluorescence Spectra. Fluorescence measurements weredone using a model FP-770 Jasco spectrofluorometer. Thetemperature of the samples was maintained at 30 C with athermostatically controlled circulating water bath. Fluores-cence emission spectra measurements for all native anddenatured Glo, Alb-1, and Alb-2 samples were taken atexcitation wavelengths (nm) of 274 and 295 and recorded overthe frequency range from the excitation wavelength to awavelength of 500 nm. Fluorescence intensity (FI) was mea-sured according to Arntfield et al. (4) and Zemser et al. (17).

Hydrophobicity (So). So was determined by 1-anilino-8-naphthalenesulfonate (ANS)-fluorescent probe measurementswith 0.01 M phosphate buffer, pH 7.0, from 0.001 to 0.02%protein concentration at ex ) 357 nm. The fluorescenceintensity was measured at 513 nm (18). Index of proteinhydrophobicity was calculated as the initial slope of fluores-cence intensity versus protein concentration (%) plot.

Differential Scanning Calorimetry (DSC). The thermaldenaturation of proteins was assessed with a Perkin-Elmer

DSC system 4. Lyophilized samples of about 1 mg were sealedin aluminum pans (19). Denatured samples were prepared byhomogeneous mixture of native protein and denaturant in thedry state: protein sample + urea (1:1). Then the mixed sampleof 1 mg was sealed in an aluminum pan in the same way asthe native one. As a reference, an empty pan was used. Thescanning temperature was 30-120 C at a heating rate of 10C/min. Indium standards were for temperature and energycalibrations. Td and H were calculated from the thermograms(20).

Circular Dichroism (CD) Spectra. CD spectra weremeasured with a Jasco J-600 spectropolarimeter (JapanSpectroscopic Co., Ltd., Japan) at room temperature underconstant nitrogen purge. Solutions (0.03 mg/mL) of proteinswere prepared by dissolving the lyophilized powder in 0.01 Mphosphate buffer, pH 7.2. The absorbancies of all solutionswere kept below 1.0 (17, 21). Denaturation of proteins wasperformed with 8 M urea. CD spectra represent an average ofeight scans collected in 0.2-nm steps at average rate of 20 nm/min over the wavelength range 180-250 nm of far-UV (FUV).CD spectra were baseline-corrected. The data are presentedas the mean residue ellipticities (). Secondary structurecontent was calculated using nonlinear least-squares curve-fitting program and the results of CD measurements (22),allowing the comparison of secondary structures from differentplants.

Statistics. To verify the statistical significance of studiedparameters means (M), their 95% confidence intervals of 3times analyzed samples ( SD were defined. The p values of