2012 Abstract Effects of different solvents, ionic strength, and pH on Inca peanut seed protein solubility were assessed by quantitatively analyzing solubilized proteins using Lowry and
Trang 1ORIGINAL PAPER
Solubilization, Fractionation, and Electrophoretic Characterization
Shridhar K Sathe&Harshal H Kshirsagar&
Girdhari M Sharma
Published online: 12 August 2012
# Springer Science+Business Media, Inc 2012
Abstract Effects of different solvents, ionic strength, and
pH on Inca peanut seed protein solubility were assessed by
quantitatively analyzing solubilized proteins using Lowry
and Bradford methods Soluble proteins were fractionated
using Osborne procedure and the polypeptide composition
of solubilized proteins was determined by one dimensional
25 % monomer acrylamide linear gradient SDS-PAGE
Osborne protein fractions were analyzed by the 2D gel
electrophoresis Total seed proteins were efficiently
solubi-lized by 2 M NaCl among the tested solvents The soluble
seed proteins registered a minimum solubility at pH ~4.0
Osborne protein fractions, albumins, globulins, prolamins,
and glutelins accounted for 43.7, 27.3, 3.0, and 31.9 %,
respectively, of the total aqueous soluble proteins Soluble
seed flour proteins are mainly composed of polypeptides in
the MW range of 6–70 kDa of which the predominant
poly-peptides were in the 20–40 kDa range Prolamin fraction was
mainly composed of four polypeptides (MW < 15 kDa)
Glycoprotein staining indicated 32–35 and <14 kDa peptides
to be positive
Keywords Inca peanut Protein Solubility Protein fractions Electrophoresis
Abbreviations β-ME β-mercaptoethanol CBBR Coomassie brilliant blue R CHAPS
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate DTT Dithiothreitol EtOH Ethanol IEF Isoelectric focusing kDa Kilodalton
LSD Least significant difference MeOH Methanol
MW Molecular weight PAGE Polyacrylamide gel electrophoresis
pI Isoelectric pH
RT Room temperature SDS Sodium dodecyl sulfate Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol
Introduction Plant foods are important in human and animal nutrition According to FAO [1] statistical data, plant foods [cereals (46.4 %), roots/tubers (5.1 %), vegetable oils (9.6 %), sug-ars/sweeteners (8.1 %), fruits (5.9 %), and pulses (2.1 %)] together provided 77.2 % of the total food energy consumed during 2005–2007 Among plant foods, cereals typically provide the bulk of the calories (range 18.5 % for Burundi and Rwanda to 78.1 % for Bangladesh) Pulses, despite their high protein content, contribute in much smaller portion of global caloric intake (range 0 % for Armenia, Azerbaijan,
S K Sathe ( *):H H Kshirsagar:G M Sharma
Department of Nutrition, Food & Exercise Sciences,
College of Human Sciences, The Florida State University,
402 SAN,
Tallahassee, FL 32306-1493, USA
e-mail: ssathe@fsu.edu
Present Address:
H H Kshirsagar
Roquette America Inc.,
2000 South Batavia Avenue, Suite 400,
Geneva, IL 60134, USA
Present Address:
G M Sharma
Department of Health and Human Services, Public Health Service,
Food and Drug Administration,
Office of Applied Research and Safety Assessment,
Center for Food Safety and Applied Nutrition, U.S FDA,
8301 Muirkirk Road,
Laurel, MD 20708, USA
DOI 10.1007/s11130-012-0301-5
Trang 2Belarus, Georgia, Latvia, Samoa, Uzbekistan, and Vanuatu
to 18.7 % for Burundi) Legumes such as beans, peas, and
some oilseeds, make a significant contribution to human and
animal food protein supply [2–5] and therefore are
consid-ered to be valued crops Reviews on legume food and
nutrition [6–13] continue to periodically appear indicating
the global importance and interest in legume research and
utilization
Soybeans, peanuts, peas, and common dry beans are
some of the most widely investigated and utilized legume
crops However, there are many native legume species that
remain under-utilized (e.g., lablab, moth, and Tepary beans)
An extensive report on tropical legumes compiled by the US
National Academy of Sciences over 30 years ago [14] is
particularly informative and useful in this regard In addition
to their use in foods, many legumes are also utilized for
non-food uses (e.g., ornamental uses of several Acacia spp and
production of luxury timbers from Pterocarpus spp.) [14]
Plukenetia L (Euphorbiaceae) is a pantropical genus that
reportedly consists of 19 species belonging to Plukenetieae
(subfamily Acalyphoideae) [15,16] Plukenetia species are
twining vines found in tropical rain forests to seasonally dry
forest or scrub Plukenetia penninervia Muell Arg., a
spe-cies found in rainforests of Mexico is used for basket
weav-ing and other craft activities for several centuries datweav-ing
back to the Mayan period [17] Inca peanut (Plukenetia
volubilis L.), also known as Sacha inchi, is an underutilized
plant that grows wild in the rain forests of the Andean
region This plant belongs to the Euphorbiaceae family
[18] High in oil (54 %, w/w) and protein (27 %) [19], the
plant seeds are typically flat, <2 cm across, and slightly
dome shaped in the middle (Fig.1) The seeds are protected
by a tough, dark outer shell with soft thin tissue layer on the
inside Since the seeds are bitter, they are typically eaten
after suitable processing (e.g., roasting) and are valued as a
significant source of dietary protein by the native people
Unlike soybean, peanut, cottonseed and sunflower oils that
are high in linoleic but low in linolenic acid, Inca peanut oil
contains approximately equal proportions of the essential fatty acids, linoleic- the C18:2, ω-6 (36.8 % w/w) and linolenic-C18:3,ω-3 (45.2 %, w/w) A recent report indi-cates that these two fatty acids accounted for 35.34 and 46.92 % respectively with reported average of the published data of 33.74 and 44.79 %, respectively The quantity of monounsaturated oleic acid (9.6 %, w/w) in Inca peanut lipids is less compared to the corresponding amounts in soybean (22.3 %), peanut (41.3 %), cottonseed (18.7 %), and sunflower (29.3 %) seed oils [19] The Inca peanut seed proteins are soluble in aqueous buffers and a water soluble albumin has been reported to constitute ~25 % (w/w) of the defatted seed flour weight or 31 % of total proteins in the seed This albumin protein is a dimeric, glycosylated [con-tains 4.8 % (w/w) sugar], basic protein (pI~9.4) with an ultracentrifuge sedimentation value of 3S [20] The results
of amino acid composition analyses indicate high levels of cysteine, tyrosine, threonine, and tryptophan and low amounts of phenylalanine in the seed proteins [19, 21] Legume proteins are known to be deficient in sulfur amino acids [6, 22] Inca peanut seed proteins contain high amounts of sulfur amino acids (methionine + cysteine),
37 mg/100 g seed meal protein, as compared to the FAO/ WHO/UNU recommended scoring pattern for these sulfur amino acids—25 mg/100 g protein [1,19] Taken together, Inca peanut seed flour proteins are a valuable source of dietary proteins However, except for some of the reported studies, little is known about the Inca peanut seed proteins Here we report our findings on protein solubility and elec-trophoretic properties of Inca peanut seed proteins
Materials and Methods Materials
The source of seeds used for Fig.1has been reported earlier [19] Hexane defatted seed flours (two separate batches, ~5 kg each) used for the current investigations were a gift from Cristina Thibaut (Cobiosa, Industrias Asociada SL, Madrid, Spain) The defatted flour batches were stored at−20 °C until further use Sources of the reagents and chemicals used have been reported earlier [21,23]
Methods All experiments were conducted at least in duplicate using separate batches of defatted flours
Protein Solubilization and Extraction Effect of Buffer Proteins were solubilized in the selected solvent and for the indicated extraction time using defatted Fig 1 Inca peanut whole seed and kernel The kernel is packed inside
a dark brown hard shell with an inner soft white tissue lining
Trang 3seed flour to solvent ratio of 1:10 (w/v) at room temperature
(RT, ~25 °C) with constant mechanical shaking Aqueous
solvents used for protein solubilization were: A) distilled
deionized water, B) 2 M NaCl, C) 0.1 M Tris-HCl (pH 8.1),
D) 0.05 M Na3PO4buffer (pH 7.5), E) 0.1 M borate saline
buffer (BSB, 0.1 M H3BO3, 0.025 M Na2B4O7, 0.75 M
NaCl, pH 8.45), F) 0.1 M NaOH, G) 0.1 M HCl, H) 2 %
(w/v) sodium dodecyl sulfate (SDS), I) 70 % (v/v) aqueous
ethanol (EtOH), J) 10 % (v/v) acetic acid, K) 0 1 M
NaHCO3, L) 10 % (w/v) Na2SO4. The pH of the slurry
was adjusted using 1 M HCl and 1 M NaOH (pH range
1–12) At the end of the extraction period, the slurry was
centrifuged (12,300g, 15 min, RT), the supernatant collected
and used for further analysis
Effect of Extraction Conditions on Protein Solubility Effects
of ionic strength (0–4 M NaCl), time of extraction (1–5 h,
1 M NaCl), pH (pH range 1–13, 1 M NaCl extraction), and
repeated (five) consecutive extractions in 1 M NaCl on the
protein solubility were determined by extracting 100 mg
defatted flour with the appropriate solvent (1 ml) [e.g., flour
to solvent ratio01:10 (w/v)] at RT with continuous vortexing
at room temperature (RT, ~25 °C) followed by
centrifuga-tion at 16,000 g for 15 min at RT To investigate the effects
of pH on seed flour protein solubility, defatted flours (2 g
each) were first dispersed in either distilled deionized water or
aqueous 1 M NaCl and the pH of the dispersion was adjusted
to the desired value with diluted (0 1 M) NaOH and/or HCl
and continued stirring for 30 min until pH was stabilized
Then, the final flour to solvent ratio 1:10 (w/v) was adjusted
and the soluble proteins were extracted for 1 h with constant
magnetic stirring At the end of extraction, the slurry was
centrifuged (15,000 g, 30 min, 4 °C), the supernatant
collected and analyzed for soluble protein within 48 h
Osborne Fractionation Protein fractionation of the defatted
seed flour proteins was done by the Osborne method [24]
Briefly, the defatted seed flour (100 g) each was sequentially
extracted with 2 M NaCl (albumins + globulins), 70 %
aqueous ethanol (prolamins), and 0.1 M NaOH (glutelins)
at flour to solvent ratio 1:10 (w/v) at room temperature (RT)
for 1 h with constant magnetic stirring The pH of flour
suspensions in distilled water, 2 M NaCl, and 70 % aqueous
EtOh was approximately 6.6–6.7 while in 1 N NaOH the pH
was ~12.6 At the end of each extraction the slurry was
centrifuged (15,000 g, 30 min, 4 °C), the supernatant
col-lected, and the residue was used for next extraction The
residue was discarded after the 0.1 M NaOH extraction step
Each supernatant was filtered through Whatman filter paper
number 4 and dialyzed (molecular weight cut-off of the
dialysis tubing06–8 kDa, Spectra-Por Dialysis, Spectrum
Laboratories, Inc., Rancho Dominguez, CA) against
dis-tilled deionized water for 48 h (4 °C, 10 lit each, six
changes); each dialyzed extract, except for the 2 M NaCl extract, was separately lyophilized True albumins were separated from the true globulins by subjecting the dialyzed 2 M NaCl extract to centrifugation (15,000 g,
30 min, 4 °C) The separated true albumins (superna-tant) and the true globulins (residue) were separately lyophilized The lyophilized protein fractions were stored at −20 °C in plastic screw-capped bottles until further use
Gel Electrophoresis SDS-PAGE Sodium dodecyl sulfate polyacrilamide gel elec-trophoresis (SDS-PAGE) was run by the method of Fling and Gregerson [25] as described in Sharma et al [26] Appropriate protein samples were loaded on 8–25 % linear acrylamide gradient separating gel and 4 % monomer acryl-amide stacking gel The gel was typically run at 10 mA/gel until the tracking dye migrated to the gel edge (typically
20 h) The gels were stained with Coomassie Brilliant Blue
R (CBBR) or silver stain followed by appropriate destaining
as described earlier [23]
2D Gel Electrophoresis were conducted as described below (a) SDS-PAGE gels without the reducing agent [2 %v/v mercaptoethanol (β-ME)] were run in the first dimen-sion followed by SDS-PAGE in the presence of the reducing agent Gels were typically run at a constant current (10 mA/gel) with tap water cooling provided during the gel run The gels were stained with Coo-massie Brilliant Blue R and appropriate lanes were excised for 2D analysis The excised gel strips were soaked in SDS-PAGE sample buffer [0.05MTris-HCl,
pH 6.8; 0.1 % (w/v) SDS; 0.01 % (w/v) bromophenol blue; 30 % (v/v) glycerol] containing 2 % (v/v)β-ME and heat-denatured (~100 °C) in a microwave oven (Kenmore, model 565.68301790, Sears, Hoffman Estates, IL) for 30 s at 1,000 W The strips were then cooled to RT, turned 90º counterclockwise, laid on top
of the 4 % stacking gel with a 8–25 % linear monomer acrylamide gradient SDS-PAGE separating gel, and electrophoresed as indicated above The gels were then stained (CBBR) and destained as described by Sathe et
al [23] Briefly, the gels were stained overnight with 0
25 % Brilliant blue R (B0149-100 G, Sigma-Aldrich)
in 50 % methanol and 10 % acetic acid The gels were destained with destain solution (50 % methanol and
10 % acetic acid) once followed by diluted destaining solution (~1:1 with water) until the background was clear
(b) IEF-SDS-PAGE Fractionated proteins were subjected
to 2D gel separations using isoelectric focusing (IEF)
in the first dimension followed by SDS-PAGE as de-scribed by Sharma et al [26] About 1.1–1.4 mg of
Trang 4freeze-dried protein fractions were suspended in 500μl
rehydration buffer [8 M urea, 2 %w/v
3-[(3-Cholami-dopropyl)dimethylammonio]-1-propanesulfonate
(CHAPS), 0.002 % bromophenol blue, 0.5 % carrier
ampholyte, and 20 mM dithiothreitol (DTT)] for 1 h at
room temperature followed by centrifugation at
16,000 g for 15 min An appropriate amount (30–
50μl) of the supernatant was mixed with the
rehydra-tion buffer to a final volume of 250μl and was used to
rehydrate pH 3-10NL 13 cm IPG strips (17-6001-15,
GE healthcare) overnight (thus loading the protein
sample on the strips) The strips were covered with
mineral oil to prevent evaporation The rehydrated
strips (containing the protein samples) were used for
IEF as per manufacturer’s recommendation (IEF100,
Hoefer Scientific Co., CA) using following conditions:
1- gradient volt, 1,000 V, 1 h; 2- gradient volt,
12,000 V, 1 h; 3- constant volt, 12,000 V, 25,000 V,
1 h; 4- constant volt, 1,000 V, 1 h For the 2nd
dimension, the focused strips were incubated
15 min in equilibration buffer (6 M urea, 75 mM
Tris-HCl pH 8.8, 29.3 % glycerol, 2 % SDS,
0.002 % bromophenol blue) containing 10 mg/ml
DTT, followed by 15 min in equilibration buffer
containing 25 mg/ml iodoacetamide The IPG strips
were then horizontally placed on a 8–25 % gradient
monomer linear acrylamide gel and sealed with 1 %
(w/v) agarose An appropriate amount (10 μl) of
same supernatants and protein markers were loaded
on the either side of the gel, and run overnight at
constant current of 10 mA/gel followed by 20 mA/
gel next day, until the tracking dye reached gel
edge The gels were then stained (CBBR) and
destained as described by Sathe et al [23])
Glycoprotein Staining
Glycoprotein staining was done on SDS-PAGE gels using the
Gelcode glycoprotein staining (Pierce Chemical Co.,
Rock-ford, IL) procedure following manufacturer’s instructions
Proximate Composition
Moisture An accurately weighed sample (0.1 g) was placed in
an aluminum pan and was dried in a previously heated
vacu-um oven (Barnstead Lab-Line, Melrose Park, IL; model
3608-5; 95-100 °C, 25 in of Hg) to a constant weight AOAC
Official Method 925.40 [27]
Protein AOAC Official Method 950.48 [27] The
micro-Kjeldahl method was used to determine total proteins using
0.1 g sample Sample nitrogen content was calculated using
the formula: Protein %ð Þ ¼ total N %ð Þ 5:7
Soluble Protein Soluble protein was determined using the methods of Lowry et al [28] and Bradford [29] Each sam-ple was typically suitably diluted and analyzed in duplicate Tannins A known weight of the sample (0.1 g) was extracted for 1 h in absolute methanol (MeOH) and acidified (1 % HCl, v/v) MeOH with continuous vortexing followed
by centrifugation (15,000 g, 10 min, RT) Aliquots of the supernatant were immediately analyzed for tannin using a
4 % (w/v) vanillin assay [30] A catechin standard curve (0–
1 mg/ml) was prepared simultaneously Tannin content was expressed as catechin equivalents
Statistical Analysis All statistical analyses were performed using SPSS statistical software (version 15; Chicago, IL) All experiments were conducted at least in duplicate, and data are expressed as the mean ± standard deviation (SD) One-way ANOVA and Fish-er’s least significant difference (LSD) test as described by Ott [31] were used to determine statistical significance, and results were considered to be significant if the differences between the two means exceeded the LSD value (p00.05)
Results and Discussion Defatted Flour
Three samples from each of the two defatted flours batches (n06) were analyzed for proximate composition The average percent protein (%N x 5.7), moisture, ash, and soluble carbo-hydrate content of the defatted flours were 59.1±3.3, 8.32± 0.11, 6.46±0.05 and 1.03±0.06, respectively The average total soluble phenolics in the defatted flours were 0.117± 0.021 and 0.112±0.016 g/100 g flour when extracted with
Fig 2 Seed flour protein solubility in a Distilled deionized water, b
2 M NaCl, c 0 1 M Tris –HCl (pH 8.5), d 0.05 M Sodium phosphate buffer (pH 7.5), e 0.1 M BSB (pH 8.45), f 0.1 M NaOH, g 0.1 M HCl,
h 2 % (w/v) SDS, i 70 % (v/v) EtOH, j 10 % (v/v) acetic acid, k 0.1 M NaHCO 3 , l 10 % (w/v) Na 2 SO 4 LSD (p 00.05) is 7.76 (Lowry) and 0.97 (Bradford), n 06 for both
Trang 5methanol and acidified methanol, respectively, indicating the
phenolics both solvents The chemical composition of the
defatted flour found in the current investigation is consistent
with soybean [32], peanut [33], sesame [34], and lupin [35,36]
seed flours The protein content of the defatted flour found in
the current investigation is comparable to the reported
protein content (53 %) for the defatted flour from inca
peanut grown in Peru [19]
Protein Solubility
Among the tested solvents, 0.1 N NaOH and 2 M NaCl were
the most effective solvents for solubilizing the flour proteins
(Fig.2) Since the albumin fraction constitutes a significant
portion of the total seed proteins, a good solubility was
expected in aqueous solvents [21] Although 0.1 M NaOH
was the most effective protein solubilizer, the exposure to
alkali is known to alter proteins through amino acid
deamida-tion and may therefore alter protein properties [37,38] Alkali
exposure may also cause destruction of some of the essential
amino acids, such as lysine, thereby decreasing the nutritive
value of the solubilized proteins For these reasons, 2 M NaCl
was considered to be the best solvent to solubilize the Inca
seed flour proteins in this investigation When soluble proteins
were analyzed by Lowry et al [28] and Bradford [29]
meth-ods, the latter method consistently registered lower protein
content (Fig 2), regardless of the solvent used Bradford
method is reportedly ~4× more sensitive than the Lowry
method and susceptible to interference by 1 % SDS, 1 %
Triton X-100, and to a lesser extent by 1 % hemosol, 2 M
Tris, acetone, and 5 % phenol Lowry protein assay is
susceptible to a number of reagents and chemicals commonly found in biological samples Among these, amino acid these, glycine (at 0.5%) is known to decrease color with protein by
50 % Glycine, at 118 mg/g protein, is the second most abundant amino acid, behind glutamic acid (133 mg/g pro-tein), in Inca peanut flour proteins [19] A lower color yield and lower protein content would be expected, all provided glycine was in free amino acid form when Lowry method was used It is unlikely that all glycine residues in Inca peanut seeds would be in the free amino acid form Solvents used for protein solubilization are also unlikely to contain significant amounts of glycine The lower protein content (Bradford method) of 0.1 M Tris-HCl (pH 8.1) extracted protein samples although possible, is not likely due to the interference by Tris
as the final Tris concentration in the assay (≤0.1 mM) was far less than the reported 2 M Tris interfering in the Bradford assay The consistent lower protein estimation by Bradford
Fig 4 Seed flour protein solubility in distilled deionized water at the indicated pH (Lowry method) Note the minimum protein solubility at
pH 4 LSD (p 00.05)01.26, n06
Fig 3 Effects of a indicated
NaCl concentration, b
extraction time, c consecutive
extraction (1 h each) in aqueous
2 M NaCl, and d pH on seed
flour protein solubility For b
and d, aqueous 1 M NaCl was
used for protein extractions.
The LSD (p = 0.05) values for
Lowry method were 2.81, 6.8,
4.46, and 8.18 respectively for
a, b, c, and d LSD (p = 0.05)
values for the Bradford method
were 1.52, 1.76, 0.82, and 1.26,
respectively for a, b, c, and d,
n = 6 for both
Trang 6assay [28] as compared to Lowry procedure [27], in all tested
samples, remains unresolved and therefore warrants further
investigations to determine the cause(s) for this difference
At NaCl concentration≥0.5 M protein solubility increased
significantly≥ 0.05NaCl concentration (Fig.3a) Extraction
time of 15–30 min was considered adequate to maximally
solubilize the flour proteins in 2 M NaCl (Fig.3b) and two
consecutive extractions were able to solubilize the majority
of the flour proteins (Fig 3c) Bradford assay once again
registered lower protein readings than the Lowry assay when
procedure the NaCl solubilized samples were analyzed
(Fig.3a, b, and c) In the presence of 1 M NaCl the influence
of pH on protein solubility (Fig.3d), as determined by Lowry
method [27], was statistically not significant as indicated by
the narrow range (40.93 to 47.00 g solubilized protein/100 g
deffated flour at pH 1 and 12, respectively) The difference in
protein solubility was less than the LSD08.18 (p00.05) for
this tested pH range (1-13) Protein solubility of these
sam-ples, determined by Bradford method, exhibited a narrow
range [8.42 to 13.38 g solubilized protein/100 g deffated flour
at pH 4;] With the exception of solubility at pH 3, protein
solubility as determined by Bradford method, significantly
decreased at pH 4 The low protein solubility in the pH range
3–5 was further confirmed when the influence of pH on protein
solubility in distilled water, i.e in the absence of 2 M NaCl,
was determined (Fig 4) The observed pH dependence of
protein solubility is consistent with the reported dominance
of acidic amino acids in the make-up of the seed flour
proteins [19] Taken together, protein solubility results
in-dicate the importance of ionic strength in solubilizing Inca
peanut seed proteins
Osborne Protein Fractionation and Electrophoresis
With the exception of 70 % aqueous EtOH, solubilized
proteins exhibited a consistent polypeptide pattern,
regard-less of the solvent used (Fig.5) The soluble seed proteins
were characterized by polypeptides in the 10–70 kDa range The soluble seed flour proteins are mainly composed of two molecular species (32–35 kDa and ~60–62 kDa) Electro-phoretic profiles in the absence of the reducing agentβ-ME (Fig 5a) also indicated that seed proteins contained disul-fide bond-linked polypeptides Upon reduction, the electro-phoresis profile (Fig 5b) indicated that the 60–62 kDa polypeptide is composed of a disulfide bond linked dimer
of 32–35 kDa and a 60–62 kDa monomeric polypeptide Although disulfide bond reduction facilitated a significant disappearance of the 60–62 kDa polypeptides, it did not allow their elimination (e.g., compare the band width and intensity of these bands in lanes J, K, and L in Fig 5a versus
Fig 6 Two dimensional SDS-PAGE of BSB (0.1 M, pH 8.45) soluble proteins First dimension (top gel) was run in the absence of a reducing agent (2 % (v/v) β-ME) Protein (100 μg load) migration was from left to right Second dimension gel was run by the presence of a reducing agent (2 % (v/v) β-ME) using soluble protein (50 μg) in the left reference lane and molecular weight markers in the right reference lane Protein migra-tion was from top to bottom
Fig 5 SDS-PAGE (8 –25 % linear monomer acrylamide gradient)
analysis of total seed flour soluble proteins using silver a, b and
glycoprotein c staining in indicated solvents (A-L, same as in Fig 2 )
in the absence a and presence b, c of a reducing agent (2 %v/v β ME).
Protein load in each lane was 10 μg a, b, and 40 μg c This figure is a composite of 3 different gels (15 wells each) Protein (S) and polypep-tide (P) standards used are indicated by their molecular weights (kDa)
on the left
Trang 75b) Comparison of the corresponding lanes of Fig.5a and b
demonstrate the increased concentration and number of
poly-peptides with MW<30 kDa indicating the importance of
disulfide bonds in the seed protein organization These results
indicate the presence of two different types of 60–62 kDa
proteins A 2D analysis of the aqueous 2 M NaCl soluble
proteins in SDS-PAGE (Fig.6) further confirmed the presence
of two different types of 60–62 kDa proteins The polypep-tides solubilized by 70 % (v/v) aqueous EtOH stained poorly usnig silver staining (lane I, Fig 5a and b) Glycoprotein staining (Fig.5c) of the solubilized proteins demonstrated that the 32–35 kDa polypeptides are glycosylated The glycopro-tein staining also revealed that the 70 % (v/v) aqueous EtOH soluble polypeptides (range 10–30 kDa) that were poorly stained in silver staining were readily recognized (Fig 5c, track I)
Of the total soluble proteins, true albumin, globulin, and glutelin were the major fractions in the seed flour soluble proteins with prolamin contributing in a small proportion (Fig 7b) Typically, over 90 % of the seed flour proteins could be solubilized by the aqueous solvents used for pro-tein fractionation One dimensional SDS-PAGE analysis of the Osborne fractions (Fig.7) indicates (judged qualitatively based on band width and intensity) that the major polypep-tides (30–40 kDa range) that constitute the albumin fraction also appear to be the major polypeptides in globulin and glutelin fractions The main difference between the albumin and globulin fraction was the distinctly different polypep-tides present in the globulin fraction (indicated by an * next
to the polypeptide on the right of the globulin track) Glob-ulin and glutelin fractions had comparable polypeptide com-position with the glutelin fraction exhibiting higher proportion of polypeptide marked by the arrow (right side
of the glutelin track) Prolamin fraction was mostly
Fig 7 SDS-PAGE (8-25% linear monomer acrylamide gradient) in the
presence of a reducing agent ( 2 % (v/v) β-ME) for Osborne protein
fractions Protein load, except for the standards, was 75 μg each Note
the different polypeptides in globulin (*) compared to the albumin
fraction profile The polypeptide marked ( ←) in the alkali glutelin
fraction profile was distinctly different when compared with globulin
fraction polypeptide profile
Fig 8 2D (IEF top gel left to
right- pH range 3 –10 followed
by SDS-PAGE- top to bottom)
gel electrophoretic analysis of
the fractionated proteins
pre-pared from the defatted seed
flour Protein load was ~60 μg
each in both directions
Molec-ular weights (kDa) of the
stan-dard proteins are indicated in
each gel on the left The protein
fractions in the second
dimen-sion gel are in the
corresponding gel (right lane)
indicated as A albumin, Gb
Glubulin, P Prolamin, and Gn
Alkali Glutelin
Trang 8composed of four polypeptides in the molecular weight
range 6–12 kDa Two dimensional gel electrophoresis of
the protein solubility fractions (IEF in the first dimension
followed by the SDS-PAGE in the presence of β-ME
(Fig 8) further confirmed that the seed flour proteins
were mainly composed of two types of polypeptides
with estimated molecular weights in the range 32–
35 kDa that were linked together by disulfide bonds
These results also demonstrated that the glutelin fraction
is different than the globulin fraction (compare the
globulin profile to that of the profile for glutelin
frac-tion in Fig 8), mainly with respect to the isoelectric pH
of individual polypeptides A closer examination
indi-cates that the globulin fraction contained polypeptides
that are slightly more basic (judged based on their
mobility in the first dimension) than those in the
glu-telin fraction although the molecular weight, indicated
by their mobility in the second dimension, appear to be
similar Although alkali exposure of the insoluble
glob-ulin polypeptides during glutelin fraction preparation
may possibly cause deamidation resulting in the
forma-tion of acidic polypeptides, it does not explain why the
globulin polypeptides were not solubilized during 2 M
NaCl extraction (the first step) during seed flour protein
fractionation especially since the flour to solvent ratio
of 1:10 (w/v) provided more than sufficient amount of
solvent It is possible that when albumins and globulins
are present together, their collective behavior, as
op-posed to their individual behavior, may differ
signifi-cantly, an observation similar to the one reported earlier
in the Great Northern bean (Phaseolus vulgaris L.)
proteins [39] One possible reason of the different
be-havior between albumins and globulins, when present
together, is, the protein-protein interactions These
protein-protein interactions are facilitated by dfferent
bonding forces (e.g., ionic, hydrogen, hydrophobic, and
disulfide) depending on the environmental conditions
permitted by the experimental conditions used (e.g.,
pH, ionic strength, and temperature) Such protein-protein
interactions may lead to formation of large protein complexes
(soluble and insoluble) thereby affecting their solubility If
globulin solubility is limited due to the presence of albumins,
the insoluble globulins would then be extracted as the
glute-lins solubilized by alkali solution Note the appearance of new
polypeptides [>56 kDa and <72 kDa and several polypeptides
in >26 and ~34 kDa range)] in the second dimension that were
not part of the globulin fraction The >26 kDa and ~34 kDa
polypeptides have more acidic pIs compared to pIs of
the original globulin fraction Since alkali solubilization
would possibly deamidate susceptible globulin
polypep-tides, they may appear as part of the glutelin fraction
(Fig 8) Further investigations in understanding the
molecular, nutritional, and functional properties of Inca
peanut seed proteins would be helpful in utilizing this protein source for a variety of purposes and are there-fore underway
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