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Characterization of Sunflower Seed Proteins by
Electrophoretic Techniques
Reda Helmy Sammour*, M.N. EI-Shourbagy, A.M. Abo-Shady
and A.M. Abasary
Department of Botany, Faculty of Science, Tanta University,
Tanta, Egypt
ABSTRACT.The seed proteins of sunflower (Helianthus annuus) were
qualitatively and quantitatively investigated. Qualitative studies were
carried out using different electrophoretic techniques (SDS-PAGE,
Poro-PAGE, 2-D SOS-PAGE, lsoelectric focusing, Mapping gels).
Analysis of the water extracted flour on SOS-PAGE and
SDS-Poro-PAGE gave five major polypeptides with MWs of 63.5 KD,
60 KO, 58 KD, 55 KO and 51 KO. The pattern of the buffer extract
exhibited 8 major polypeptides with MWs of 65.5 KO, 63.5 KO, 60
KD, 58 KD, 55 KD, 54 KO, 51 KD and 42.5 KO. Second dimension
gel showed that the polypeptides with MWs
of
65.5 KO, 60 KO, 58
KO, 55 KD, 54 KD, 51 KD are legumin-like proteins. Isoelectric
points of the majority of the sunflower seed proteins were between 5
and 7. Mapping gels, however, showed that sunflower seed proteins
were highly heterogeneous, especially the major bands. A quantative
study indicated that the albumin, globulin, prolamin and glutelin
fractions amounted 38.32%, 39.04%, 5.53% and 17.09% respectively
of the extracted proteins.
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Oilcrops have a world importance. Some of them are directly used as food, while
most of them are utilized to obtain fats or oils and cake or flour (Lennerts 1984,
Hatje 1989).
Sunflower (Helianthus annuus) is an annual oilcrop (belonging to the family
Compositae (Lennerts 1984». The defatted flour of sunflower is used as a source of
highly digestible and nutritive protein for poultry (Smith 1968).
,Key words: Sunflower seed globulin,
Helianthus annuus;
molecular weights (MWs); two dimension-PAGE
(2-D SOS-PAGE); SDS (Sodium Dodecyl Sulphate); 2-ME (2-mercaptoethanol).
*
Correspondinb Author.
It has been reported that the major proteins (legumin-like proteins) of sunflower
have sedimentation constant of about lIS and that their MWs range from 300 KD-
350 KD (Joubert 1955, Gheyasuddin et al. 1970, Sabir et al. 1973, Young and
Huang 1981 and Hatje 1989). Heterogeneity and quaternary structure of the major
storage proteins have been investigated by Baudet and Mosse' (1977), Rahma et at.
(1981), Plietz et
at.
(1983), Dalgalarrondo et
at.
(1984) and Abasary (1992).
As far as is known, all the studies carried out on sunflower seed proteins were
done with the major seed proteins. The present work, therefore, is made to
investigate new major and minor components of the sunflower seed proteins.
Materials
Sunflower seeds were obtained from the Agriculture Research Center, Giza,
Egypt. The seeds were dehulled, and ground well in a mortar. The flour was defatted
by three hexane extractions (10 ml hexane/g flour), each for 2 hours with slow
stirring at 4°C. After the n-hexane layer was discarded, the flour was air-dried,
brushed through a sieve of 125 urn (115 mesh) and then stored at -10°C until used.
Water and Buffer Extracts
A portion (30 mg) of defatted flour was mixed with 0.5 ml distilled water
containing 0.02% (w/v) sodium azide in an Eppendorf tube for 30 min at 4°C, and
then centrifuged at 23 000 xg (Heraeus Christ Labofuge I-cooling centrifuged) for
20 minutes at 4°C. The residue was re-extracted another two times under the same
conditions. Another portion of the defatted flour was extracted with 0.125 M
Tris/borate buffer pH 8.9 containing 0.02% (w/v) sodium azide as described for
water extraction. Analogous extracts were made with 2-mercaptoethanol (2-ME).
For SDS-PAGE, the buffer and water extracts were treated with 2% (w/v) SDS by
boiling for 5 min. For complete proto mer formation, extracts were boiled for 3 min
with 2% SDS and 2% 2-ME.
SDS-Extract
For SDS-extraction, 30 mg of the defatted and dried flour were shaken with 0.5
ml of an aqueous solution of 5% or 15% (w/v) SDS respectively in an Eppendorf
centrifuge tube and centrifuged for at 23 000 xg for 20 minutes at room temperature.
The supernatant was used for electrophoresis. Analogous extracts were made with
2-ME.
Urea-Extract
Samples of 30 mg defatted flour were stirred for 30 minutes in an Eppendorf
tube with 0.5 ml of an aqueous solution of 9 M urea, 2% (v/v) ampholyte pH 3-10
(Pharmacia (Ampholine) and 2% 2-ME. The mixture was centrifuged (20 min, 22°C,
23 000 xg), and the supernatant was used for electrophoresis. Analogous extraction
was done without 2-ME or 2-ME/ampholyte (Shah and Stegemann 1983). All the
extracts were stored at -10°C.
Protein determination
Albumin, globulin, prolamin and glutelin were extracted using the protocol used
by Shah and Stegemann (1983). Total protein was determined by the method of
Bradford (1976) using bovine serum albumin as standard protein.
Electrophoresis
Protein separation was carried out in vertical slabs using the LKB-2201 Vertical
Electrophoresis Unit. The polymerization mixture for PAGE contained 22.5 ml of 1
M Tris pH 8.8, 19.5 ml of a mixture of 30% acrylamide and 0.43% bisacrylamide,
14.5 ml distilled water, 20 ug ammonium persulfate and 30 ul TEMED.
Two-dimensional SDS-PAGE was carried out according to Sammour (1985). In
this protocol the extracted sample was analyzed in the first dimension on 12%
SDS-PAGE. The gels were stained overnight with 0.05% (w/v) Coomassie Blue- R-
250 in methanol, acetic acid and distilled water (50:7:43, by v/v) and destained in
methanol, acetic acid water (Laemmli 1970). After destaining, the track was cut with
a sharp razor-blade and left in sample buffer containing 5% SDS and 2% 2-ME for
20 minutes. The gel strip was then inserted onto a 17% SDS-PAGE and developed
with a constant current of 25 mA.
Electrophoresis was performed in 17% SDS-PAGE following the same protocol
as that used by Abasery (1992). For the determination of the protomer MW s a
mixture of the following marker proteins, treated. with SDS, were used: human
transferrin (76.7 KD), bovine serum albumin (68 KD), albumin egg (43 KD),
oc-chymotrypsinogen-A (25.7 KD), and cytochrome-C (12.7 KD).
Poro-PAGE was carried out in a 6-26% gradient polyacrylamide in 0.125 M
Tris/borate buffer. For determination of proto mer MW s the same protein markers
used in SDS-PAGE were applied to SDS-Poro- PAGE.
Characterization of Sunflower Seed Proteins by ...
Isolectric focusing was carried out as described by Stegmann et ai. (1988) using
6% polyacrylamide tube gels containing 6M urea.
Mapping: Isoelectric focusing in the first dimension and SDS in the second
dimension was run as described by Stegmann et al. (1981) and Laemmli (1970).
The gels were stained with Coomassie Blue- R-250 as described above.
Sunflower seed flour was successively extracted with distilled water (albumin),
salt (NaCl) (globulin), alcohol (prolamin) and alkaline solution (glutelin) using the
method of Bradford (1976). Protein contents of 134.43
±
13.62 mg/g seed flour and
137.39 ±6.34 mg/g seed flour were found respectively in the water and salt extract
(Table
I).
The protein extracted with the alkaline solution represented nearly half of
the protein content found after salt extraction. Protein analysis cannot be carried out
in extracts containing ampholyte, since the ampholytes complex with copper ions
(Shah and Stegemann 1983). A protein content in sunflower of 16.6 to 20.3% has
been reported by Hatje (1989) which contradicts our findings (35%). Youle and
Huang (1981) reported that 33% of sunflower seed protein was albumin. However
the variation between their data and the data reported in the present work could be
due to methodological or varietal differences.
Table 1. Protein contents of the albumin. globulin, prolamin and glutelin fractions of sunflower
seed flour
Protein species Quantity Percent to the
rnglg Seed flour total protein content
Albumin 134.43 ±13.2* 38.33
Globulin 137.39 ±10.7* 39.04
Prolamin 019.39 ±05.4* 05.54
Glutelin 059.95 ±07.3* 17.09
Total protein content 35
J.J
6
In SOS-PAGE the seed proteins extracted with distilled water showed five
major polypeptides with MWs of 63.5 KD (kiloOalton), 60 KO, 58 KD, 55 KD and
5] KO (Fig. 1, lane 1). The pattern of the buffer exract (lane. 2) exhibied 8 major
polypeptides with molecular weights of 65.5 KD, 63.5 KO, 60 KD, 58 KD, 55 KD,
54 KO, 51 KO and 42.5 KD. The extraction with SOS (Fig. 1, lane 3) extracts gave
electrophoretic patterns similar to that of the buffer extract, but with higher intensity.
There was also an additional strong band at 15 KO. The greater amount of seed
proteins extracted with SOS extracts could be due to the ability of SOS to
dissociated cell membrane-binding proteins (Gennis and Jonas 1977). The pattern of
the urea extract showed an increase in the numbers and intensity of the bands (Fig. 1,
lane 5). However, the electrophoretic pattern of the urea extract in the presence of
ampholyte shwoed an electrophoretic pattern similar to those of the extracts
analyzed under reducing conditions (Fig. 1, lanes 6-14). This shows the reducing
nature of the ampholyte. The extracts with urea, water, buffer or SOS in the presence
of 2-ME showed similar electrophoretic patterns (Fig. 1, lanes 7-14). However there
was a variation in the quantity of the protein extracted. Urea extracts in the presence
of 2-ME showed high protein quantity and a new protein band denoted with arrow in
Fig.
I,
lanes 7-9.
Two- dimensional SOS-PAGE gel was done for further resolution, first by
SOS-PAGE, and in the second dimension by SOS-PAGE under reducing conditions
(Fig. 2). The polypeptides which dissociated under reducing conditions (65.5 KD, 60
KD, 58 KD, 55 KD, 54 KD, 51 KD) are legumin-like proteins. These polypeptides
are separated into acidic subunits (designated
I
a, 2a, 3a, 4a, 5a, 6a) and basic
subunits (designated 1b, 2b, 3b, 4b, 5b, 6b) (see Table 2). These polypeptides can be
defined as falling into three groups corresponding to bands G 1, G2 and G3 in Fig. 1.
Subunits
No. Polpeptides Acidic Subunits
MWs Basic Subunits
Code MWs Code MWs
165.5 al 50.5 bl 15.0
260.0 a2 43.0 b2 17.0
358.0 a3 40.0 b3 18.0
455.0 a4 40.0 b4 15.0
554.0 a5 37.0 b5 17.0
651.0 a6 36.0 b6 15.0
The first group includes subunit 1a, 2a, 3a, 4a; the second a5 and a6; and the third
1b, 2b, 3b, 4b, 5b, 6b. These analysis are not in a good agreement with the work of
Dalgalarrondo et al. (1984) and Allen et at. (1985) who reported that legumin-like
proteins of sunflower seeds consists of four polypeptides wit MWs of 60 KD, 54
KD, 48 KD, 40 KD. The appearance of the band with molecular weight 130 KD
(denoted with an arrow in Fig. 1) was found to consist of disulphide linked pairs of
76.7
68.0
Fig. 1. SOS-Polyacrylamide gel electrophoresis (17% SOS-PAGE) of different extracts of
sunflower seed proteins. 1) water extract, 2) Trislborate buffer (pH 8.9) extract, 3) 5%
SOS extract, 4) 15% SOS extract,'5) Urea extract, 6) Urea/ampholyte extract, 7) Urea/2%
ampholytel2% 2-ME extract, 8) Urea/ 2% 2-ME extract, 9) Urea/ 2% 2-ME extract (after
boiling for 3 min. 2% 2-ME is added), 10) water! 2% 2-ME extract (after 30 min. 2%
2-ME is added), 11) water! 2% 2-ME extract, 12) Trislborate buffer (pH 8.9) 12% 2-ME
extract, 13) 5% SOS! 2% 2-ME extract, 14) 15% SOS! 2% 2-ME extract, M. Protein
standards (Human transferrin. Bovine Serum Albumin, Ovaalbumin
oc:-Chymotrypsinogen-A,and Cyochrome-C).
large acidic and small basic subunits. However these linked pairs of acidic and basic
subunits were dissociated into their components in Fig. 2.
SOS/PAGE
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Fig. 2. Two dimensional SDS-PAGE of the total sunflower seed protein extract. Ist-D:
SDS-PAGE under non-reducing conditions, and 2nd-D: SDS-PAGE under reducing
conditions.
Poro-PAGE revealed a variation in the electrophoretic patterns of the various
unreduced sunflower extracts (Fig. 3, lanes 1-4). After reduction with 2-ME,
however, the pattern was strikingly similar; fewer bands in the upper part of the gel
and more bands in the lower part were observed (lanes 5-7). As expected, meals
extracted with 2-ME showed more bands than did extracts with 2-ME. However urea
extraction out in the presence of ampholyte increased the dissociation of the proteins
(Fig. 3, lane 4). The close similarity of the protomers of the extracts in lanes 5-7 and
10 does not mean that these proteins are the same classes of the proteins, since the
separation in the Poro-gel is dependent partly on the shape and charge and partly on
the molecular, weight of the polypeptides (Derbyshire et ai. 1976 and Sammour
1985).
Isoelectric focusing was carried out between pH 3-10 in cylindrical tubes
containing 8 M urea (Fig. 4). As with Vida faba and potato tuber, the patterns for
the urea extracts did not allow for good differentiation or clear separation of the
-
-
.
-
-
.
.
-
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-
.
-
--
-
-
-
-
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-
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-
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-
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--
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-
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--
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Fig. 3. Porosity gradient polyacrylamide gel electrophoresis (6-26% Poro-PAGE) of different
extracts of sunflower seed proteins. 1) water extract, 2) Tris/borate buffer (pH 8.9)
extract, 3) urea extract, 4) ureal 2% ampholyte extract, 5) ureal 2% ampholyte/ 2% 2-ME
extract, 6) ureal 2% 2-ME extract, 7) urea/2% 2-ME extract (after boiling for 3 min. 2%
2-ME is added), 8) water/ 2% 2-ME extract (2% 2-ME is added after 30 min), 9) water/
2% 2-ME extract 10) Tris/borate buffer (pH 8.9)/2% 2-ME extract.
-
-
-
-
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-
.
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-
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-
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---
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.-
--.
--.
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.
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---
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Fig. 4. PAGIF in 6% polyacrylamide, 2% ampholyte pH 3-10. Samples of sunflower flour extraced
with water (1), Trislborate buffer (2), waterl2-ME (3) and Trislborate buffer/2-ME.
bands. This poor differentiation may be attributed to the presence of phytic acid in
the extracts (Stegemann et al. 1980). The patterns for the water or buffer extracts
showed a number of band with pI-values between 9 and 5. The patterns for the water
and buffer extracts analyzed in the presence of 2-ME showed little variation.
Mapping was done for further resolution, first by isoelectric focusing and in the
second dimension by SDS-PAGE (Fig. 5). The polypeptide distribution of the buffer
extracts consists of three groups matching the three groups of polypeptides reported
in Fig. 1. It is very interesting to notice that the acidic subunits have pIs between 5-7,
whole the basic subunits have pIs between 9.5-7. The gel also show that the basic
and acidic subunits are highly heterogeneous. However, the acidic subunits are
distributed in a narrow pH range, while the basic subunits are distributed over a
wider pH range. The heterogenity can be explained as follows: 1) the subunits could
be composed of several nearly identical polypeptides; 2) the proteolytic
modifications could produce charge variants and in this event the differences
between molecules in each preparations would be evident only at the ends of the
molecules; 3) glutamine and asparagine could be diamidated in some peptides.
In conclusion, sunflower seed proteins contain approximatly equal amounts of
albumin and globulin proteins. These proteins represent about 77% of the total seed
proteins. The total seed proteins in turn represent about 35% of the seed meal. This
percentage makes sunflower seed proteins a good resource for feeding animals. The
richness of the legumin-like proteins in sulphur amino acids makes them highly
nutritional. On reduction with 2-ME, the legumin-like proteins were cleavaged into
acidic and basic subunits. The pI-values of sunflower seed proteins range between
9.5 and 5.0. Urea and ampholyte (which are denaturating agent) gave electrophoretic
patterns similar to those of the proteins extracted with Tris/borate buffer and
analyzed under reducing conditions. The extraction with SDS appears to release the
proteins binding to membrane.
Abasery, A.W. (1992) Electrophoretic and biochemical studies on oil seed proteins. M.Sc. Thesis,
Tanta University, Tanta, Egypt.
Allen, R.D., Craig, C.L. and Thomas, T.L. (1985) Developmental expression of sunflower 11S
storage protein genes. Plant Mol. Biol. 5: 165-173.
Baudet, J. and Mosse, J. (1977) Fractionation of sunflower seed. J. Amer. Oil Chem. Soc. 54:
82A-86A.
Bradford, M.M. (1976) A rapid and sensitive method, method for quantitation of microgram
quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72:
248-254.
Dalgalarrondo, M., Raymond, J. and Azanza, J.L. (1984) Sunflower seed proteins:
Characterization and subunit composition of the globulin fraction. J. Exper. Bot. 35:
1618-1628.
Derbyshire, E.D., Wright, D.J. and Boulter, D. (1976) Legumin and vicilin, storage proteins and
of legumin seeds. Phytochemistry (Oxf.) 15: 3-24.
Gheyasuddin, S., Cater, C.M. and Mattil, K.F. (1970) Effect of several variables on the
extractibility of sunflower seed proteins.
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McGraw-Hill Publishing Company, New York.
Joubert, F.J. (1955) Sunflower seed proteins. Biochemica et Biophysica Acta 16: 520-523.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature (London) 227: 680685.
Lennerts, Dr. L. (1983) Oelschrote, Oelkuchen, pflanzliche. Oele und Fette, Herkunft,
Gewinnung, Verwendung. Bonn (1983) Verlag Alfred Strothe, Hannover.
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fraction of sunflower seeds. J. Agric. Foods Chem. 29: 518-521.
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proteins. J. Agric. Chern. 21: 988-993.
Sammour, R.H. (1985) The use of protein character in the taxonomy of peas and beans. Ph.D.
Thesis, Faculty of Science, Tanta University, Tanta, Egypt.
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and characterization by electrophoresis. J. Agron. and Crop Sci. 152: 39-47.
Smith, J.K. (1968) A review of the nutritional value of sunflower meal. Feedstuffs 40: 20-25.
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Electrophoretic studies on seeds of some German and Egyptian cultivars. Z. Acker- und
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pp. Inst. Biochem Messeweg 11, 0-3300 Braunschweig.
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Amer..
J.
Bot.
68(1): 44-48.
(Received 01/05/1993;
in revisedform 15/102/1995)
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