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Extraction and fractionation of wheat


flour proteins

Article in Journal of the Science of Food and Agriculture November 1982


DOI: 10.1002/jsfa.2740331109

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J . Sci. Food Agric. 1982,33, 1 117-1 128

Extraction and Fractionation of Wheat Flour Proteins


Aris Graveland, Pieter Bosveld, Willem J. Lichtendonk, Hans H. E. Moonen and
Auke Scheepstra
Institute for Cereals, Flour and Bread TNO, PO Box 1.5, 6700 AA Wageningen, The Netherlands
(Manuscript received I0 September 1981)

With the aid of a 1.5% sodium dodecyl sulphate solution (SDS) the proteins of two
Dutch wheat flours were separated into an SDS-soluble and an SDS-insoluble fraction.
The SDS-soluble fraction was fractionated with the aid of ethanol precipitation and
gel filtration chromatography into albumins, globulins, gliadins, glutenins I1 and 111,
and glutelins I, IT, I11 and IV. The SDS-insoluble proteinaceous material was separa-
ted into glutenins I and glycoproteins. The protein fractions were identified with the
aid of SDS-polyacrylamide gel electrophoresis and amino acid analysis. The glutenins
I and I1 consist of A, B and C subunits. Their molecular weights ranged from about
600000 to 10 million. The glutenins 111 consist only of B and C subunits and their
molecular weights ranged from 300 000 to 600 000. A hypothesis explains how the glu-
tenins I11 are the precursors for glutenins 1 and IT and how they contribute to gluten's
structure. The glutelins I consist of a, /3 and y subunits and their molecular weights
ranged from about 6OOOOO to several millions. The glutelins 11 consist also of
, and y subunits with a molecular weight of about 300000 to 600000, while
tc, B
the molecular weights of the glutelins I11 and IV ranged from 10000 to 200000.
All glutelins are insoluble in 70% aqueous ethanol and 5~ urea, but soluble in SDS
and 0 . 1 NaOH.
~ Only the glutelins 111 and 1V are soluble in 0 . 1 HAc.
~ The globulins
consist of a heterogeneous fraction of components, their molecular weights arranged
from 98000 to 10000. The gliadins form a heterogeneous but homologous group
of polypeptides with an average molecular weight of 35 OOO. The albumins consist of
components with molecular weights lower than 15 000.

1. Introduction
An extraction and fractionation method for wheat flour proteins was described in a previous
publication.' A 1.5 % SDS solution was chosen as the extraction solvent and for the fractionating
process ultracentrifugation, ethanol pecipitation, (NH4)z so4 precipitation and gel filtration
chromatography were used. A number of homogeneous protein fractions were obtained and charac-
terised by starch-gel electrophoresis. Most of these fractions appeared to contain sugars which could
not be removed with the techniques described and therefore were characterised as glycoproteins
at that time.
In this study the same extraction and fractionation methods have been used but more specific
methods for the characterisation of the protein fractions have been applied. This proved that the
fractions did indeed contain sizeable amounts of sugars, but that these are not chemically bonded
to the proteins. This has prompted a revision of the nomenclature used for the several protein
fractions.

2. Materials and methods


2.1. Wheat flours
Two Dutch wheat flours were used; one of the good breadmaking variety Sicco with a protein
0022-5142/82/1100-1117 $02.00 0 1982 Society of Chemical Industry
1117
1118 A. Graveland et al.

content of 11 % (N x 5.7) and one of the poor breadmaking variety Tundra with a Protein content
of 10.5 %. The flours were defatted with petroleum ether b.p. 40-60 by percolation.

2.2. Chemicals
Sodium dodecyl sulphate (SDS, specially purified for biochemical work) was purchased from
BDH Biochemicals Ltd, England.
Sephadex G-100 and Sepharose C L 4 B were obtained from Pharmacia Ltd, Sweden.
Acrylamide, N,N-methylenebisacrylamidewere obtained from UCB (Leuven), Belgium.

2.3. Extraction and fractionation of wheat flour proteins


The extraction and fractionation methods were used as previously describedl (see Figure l), but a
few modifications were made in the method of fractionation.

Extraction with 1.5% SDS ( 1 20)


Centrifuging ot 40 000 g

Glycoprolein (Light brown layer) Precipitation by adding


GluteninI (gel layer) ethanol to 7 0 % centrifuging
o t 2000 g

-
1 4
Rsrtdur Supernatant

1
Supernatant
Pentosans
-4 Residue
Extraction w i t h waler ( 2 x )
centrifuging at 4000 g

i
Evaporation of the
ethanol Sephodex G-100
column

Globulins E x t r x t i o n with 5 M urea and Glutenins m


20 % ethanol solution centrifuging Gliadins
a t 43 000 g Albumins

Supernatant Residue

I
Glutenins lT
Sepharose C L - 4 B
column

Glutelins I.E. m o n d m
Figure 1. Scheme for separating SDS-insoluble and SDS-soluble wheat flour proteins.

2.4. Separation of the SDS-insolubleprotein fractions


The gel layer was resuspended in 50 ml ethanol using a Polytron homogeniser for 15 s to remove
the SDS. After centrifuging the suspension at 2OOOg at 0C the residue was resuspended in 150 ml
H 2 0 using the Polytron homogeniser for 15 s. The proteinaceous material floated as water-insoluble
material (gel proteins or glutenin I) on the surface and were skimmed off with a spatula, the starch
and the residual material like hemicellulose fell to the bottom.
The light brown layer was also resuspended in SDS-solution using a Polytron homogeniser for
15 s and centrifuged at 40 OOOg for 20 min at 10C. Four layers were formed from the bottom to
the top; a starch layer, a dark brown layer, a white layer and a thin layer of gel. The latter was
identical with the gel layer named above (glutenin I). A complete separation between the brown
(SDS-insoluble glycoproteins) and white layer was obtained after resuspending them together in
water and centrifuging at 2000g. Starch particles could be removed by washing out over a sieve.

2.5. Fractionation of SDS soluble protein fractions


SDS-soluble proteins were fractionated as shown in Figure 1. Ethanol was added to the SDS
supernatant as described previously.' However, some details were changed. The ethanol was added
Extraction and fractionation of wheat flour proteins 1119

at 5'C. After standing for 15 min at O'C the mixture was centrifuged at 2000g for 5 min. The pre-
cipitate was suspended in 30 ml ethanol at 5C to remove the residual SDS. After that the precipitate
was resuspended in 100 ml He0 by stirring magnetically for 10 min at 5C. The suspension was
centrifuged at 4000g for 10 min. The supernatant (sugar complexes and globulins) was freeze dried
and the pellet was again suspended in 60 ml HzO at room temperature, by stirring magnetically for
15 min, and centrifuged. The supernatant (globulins and pentosans) was freeze dried. The resulting
pellet was resuspended in 50 ml of a solution containing 5~ urea and 20% ethanol, by stirring
magnetically for 15 min at room temperature, and centrifuged at 40 OOOg. The supernatant (glutenin
11) was dialysed and freeze dried. The residue was dissolved in SDS and separated into glutelins I,
11, 111 and IV with the aid of a Sepharose CL4B column. The ethanol-SDS supernatant (900 ml)
was reduced to about 40 ml under reduced pressure at about 50C and fractionated on Sephadex
G-100 with 1.5 % SDS solution as eluent. Three distinct peaks were obtained, which contained,
glutenins 111, gliadins and albumins respectively. The eluate was cleared of SDS using an AG2-
XI0 column, dialysed against distilled water and freeze dried.

2.6. Gel filtration


Gel filtration was carried out as described in the previous publication.'

2.7. Removal of sodium dodecyl sulphate (SDS)


SDS was removed from protein solution with the aid of the Bio-Rad cation exchange resin AG2-
XI0 column, as described in a previous publication.' Protein fractions which are insoluble in 70%
aqueous ethanol were cleaned of SDS by washing with 95% aqueous ethanol.

2.8. Electrophoresis
In this study we used the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
Protein samples were treated with sodium dodecyl sulphate and /3-mercaptoethanol and then
fractionated in 10% polyacrylamide gel slabs using a modification of the method of LaemmIi.2
Gels were loaded with 14 samples and subjected to electrophoresis at about 70 mA gel-l for about
4 h. The gel was stained with 2 mg ml-1 Coomassie brilliant blue R 250 in water-methanol-acetic
acid (45:45: 10) for 1 h at 50C and destained with water-methanol-acetic acid (175: 10: 15 v/v) at
50'C overnight.

2.9. Amino acid analysis


The protein samples were hydrolysed in evacuated sealed tubes in 6 M hydrochloric acid at 110C for
24 h. The determination of amino acids was performed using a Hitachi automatic amino acid
analyser, model KLA-3B. Tryptophan contents were determined by the method of Hugh and Moore.3

2.10. Sugar analysis in the SDS-PAGE gels


For the detection of glycoproteins the polyacrylamide gels were soaked in a thymol solution and
stained with sulphuric acid in ethanol using the method of Racusen.4

3. Results
3.1. Extraction
Starting from the point that no cleaving of disulphide bonds must take place, the best results were
obtained by extraction with a 1.5 % SDS solution, at a solvent, flour ratio of 20 : 1, at a temperature
of l O T , and with magnetic stirring.

3.2. Fractionation and characterisation of SDS-insoluble material


After centrifuging at 40000g of the SDS-flour suspension three SDS insoluble fractions were
obtained-a starch layer, a gel layer and a light brown layer.
1120 A. Graveland et af.

3.2.1. Glycoproteins and (hemi)cellulose


After resuspending the light brown layer in water and centrifugation a white and a brown layer
were obtained. The white layer consisted only of sugars. Table 1 summarises the sugar composition,
which was identical with the composition of hemicellulose B identified by Brillouet and Mercier.5
The brown layer on the other hand contained 5 % proteinaceous material. Resuspending followed
by centrifugation of the brown layer in water, 6h.1urea, 0 . l ~HAc did not change its chemical
composition. Reduction of the brown layer with 3 % 13-mercaptoethanol in a 1.5 94 SDS solution
did not change its composition either. Therefore we concluded that the proteinaceous material
was chemically bonded to the sugar complex and that the brown layer might therefore be indicated
as a glycoprotein. This fraction was classified as a SDS insoluble glycoprotein. Table 2 gives its
amino acid composition.

Table 1. Protein content and sugar composition of the sugar containing fractions

Neutral sugar composition


Neutral sugar ~-
Protein content content Not identified Ara Xyl Man Gal Gluc
(%) (%) ( Yo) (%) (%) (%) (%) (%)
~~ ~ ~~~ ~~

SDS-insoluble material
Brown layer (glycoprotein) 5 90 5 31 50 7 2 10
White layer (hemicellulose) 0 95 5 '11 54 5 1 9
SDS-soluble material
Pentosans 0 95 5 30 50 2 1 17

Table 2. Amino acid composition of protein fractions [g (100 g protein)-']

SDS A-compo- B and C


insoluble Glutenin I nents of components Glutenin Glutenin
glycoprotein (gelproteins) Glutenin I of Glutenin I II 111 Gliadins Glutelins
.~ ~~~~ ~ . ____ ~

Lysine 4.34 I .03 1.15 0.43 I .oo 0.42 0.45 5.50


Histidine 2.61 I .60 I .30 I .65 1.62 I .60 1.95 2.90
Arginine 6.30 2.98 3.15 2.45 2.90 2.47 2.25 8.30
Aspartic acid f 8.46 2.48 2.60 I .75 2.50 1.75 2.75 7.95
Asparagine
Threonine 4.32 2.42 2.85 I .90 2.40 1.87 1.80 3.65
Serine 5.31 5.10 5.20 5.55 5.20 5.50 4.50 4.70
Glutamine acid 11.07 38.12 39.45 38.60 38.10 38.70 44.30 12.65
Glutamine
Proline 5.49 11.20 10.50 11.60 11.10 11.55 15.20 3.95
Glycine 7.47 2.80 3.00 I .40 3.00 1.35 1.45 4.60
Alanine 5.31 2.05 2.30 1.45 2.05 I .45 2.10 5.10
Valine 5.31 3.25 2.40 3.25 3.25 3.25 3.70 5.35
Methionine 0.31 1.35 0.92 1.40 I .40 I .40 1.15 1.82
lsoleucine 3.60 2.84 2.10 3.70 2.80 3.68 4.35 3.75
Leucine 6.75 5.56 4.95 6.20 5.56 6.24 7.05 6.85
Tyrosine 4.05 3.49 5.90 I .75 3.50 1.76 2.70 1.30
Phenylalanine 4.32 3.45 2.80 5.15 3.36 5.10 6.35 4.00
Tryptophan 0.80 0.70 1.10 0.35 0.80 0.40 0.55 0.90
Cysteine 3.56 2.00 1.10 2.30 2.10 2.18 2.00 1.55

3.2.2. Glutenins Z
After resuspending the SDS-insoluble gel layer in alcohol and water consecutive with the aid of a
Polytron the proteinaceous material could be separated from the residual starch, (hemi)cellulose
and SDS-insoluble glycoproteins. The protein content of this fraction was 97%. Table 2 (column 3)
Extraction and fractionation of wheat flour proteins 1121

shows the amino acid composition and Figure 2a,b the SDS-PAGE composition of the subunits
after reduction with 8-mercaptoethanol. The subunits of these gel proteins can be divided into three
groups on the basis of their molecular weights: Group A with subunits of a molecular weight higher
than 70 000; group B with subunits of a molecular weight between 40 000 and 50 000; group C
with subunits of a molecular weight between 30 000 and 40 000. The gel proteins also contained a
few minor bands with a molecular weight of about 68 000.

Figure 2. SDS-polyacrylamide-gel electrophoresis of protein samples from Sicco (S) and Tundra (T). Left io
right: a, glutenins I (S); b, glutenins I (T). c, A-components (S); d, A-components (T); e, B f C-components (S);
f, B f C components (T); g, gliadins (S); h, gliadins (T); i, glutinens 111 (S); j, glutenins 111 (T); k, glutenins I1 (S);
I , glutenins I1 (T); m, standards. The following standards were used; Phosphorylase b (94 000 d), Albumin (67 000 d),
Ovalbumin (43 000 d), Carbonic Anhydrase (30 000 d), Trypsin Inhibitor (20 I00 d) and oi-Lactalbumin (14 400 d).

After reduction with P-mercaptoethanol in a SDS solution the high molecular weight subunits
(group A) of the gelproteins could be easily separated from the subunits B and C by adding alcohol
to 70%. The A subunits were alcohol insoluble in contrast to the B and C subunits, which were soluble
in 70% ethanol. Figure 2c, d and e, f respectively shows the electrophoretic pattern and Table 2
the amino acid composition of both these fractions.
The electrophoretic results show that the fraction of A subunits also contained two subunits of
68 000 and three subunits of approximately 45 000. These subunits belonged to the glutelins I fraction
(see below). The hydrated A subunits had particularly strong elastic properties, while the hydrated
B and C subunits had viscous properties. On the basis of these results we classified the gel proteins,
in contrast to the previous publication,' as SDS insoluble glutenins or glutenins I. This classifi-
cation is comparable with those of other workers.6-10 They formed 35 and 22% of the total N of
Sicco and Tundra wheat flour respectively (Table 3). The SDS-insoluble glutenins I are also in-
soluble in AUC ( 0 . 1 acetic
~ acid, 3~ urea and 0 . 0 1 cetyltrimethyl
~ ammonium bromide). Therefore
we conclude that the molecular weights of the glutenins I were at least several millions, and they
contained quite a number of intermolecular disulphide bonds.
72
1122 A. Graveland ef al.

Table 3. Distribution of nitrogen in the protein fractions (per-


centages of total nitrogen)

Fraction Sicco Tundra

SDS-insoluble
Glycoprotein 3 4
Glutenin I 35 22

SDS-soluble and 70 % alcohol insoluble


Glutenin I1 4 8
Glutelins I, 11, 111 and IV 11 18
Globulins 1 I

SDS-soluble and 70% alcohol soluble


Glutenin 111 6 8
Gliadins 31 30
Albumins 9 9

3.3. Fractionation and characterisation of SDS-soluble and 70 % ethanol-insolublematerial


On addition of ethanol (up to 70%) to the supernatant, which had been obtained after ultracentri-
fuging of SDS-flour mixture, a precipitate was produced.

3.3.1. Sugar complexes


By carefully washing the precipitate with an excess of cold water (5C) nearly all the sugar com-
plexes could be extracted from the precipitate. Table 1 gives the sugar composition. This fraction
is usually denoted in the literature as water soluble pentosans. The freeze dried material contained

Figure 3. SDS-polyacrylamide-gel electrophoresis of protein samples from Sicco (S) and Tundra (T). Left to
right: a, globulins ( S ) (1st water extract); b, globulins (T)(1st water extract); c, globulins (S) (2nd water extract);
d, globulins (T) (2nd water extract); e, albumins (S); f, albumins (T); g, gliadins (S); h, gliadins (T), i, standards.
Extraction and fractionation of wheat flour proteins 1123

3 proteins. With the aid of SDS-PAGE we have characterised them as globulins (Figure 3a and b)
see below.

3.3.2. Globulins
By washing the precipitate a second time with an excess of water at room temperature with the aid
of magnetic stirring the rest of the globulins could be isolated from the precipitate. and were
separated into 15 components by starch gel electrophoresis.1 With the aid of SDS-PAGE the
globulins could also be separated into many bands (see Figure 3c and d). They formed 1 % of the
total N.

3.3.3. SDS-soluble gluferiins 1I


The residual precipitate after two water extractions could be separated by an extraction with a
solution containing 5M urea and 20% ethanol. The soluble material had the same SDS-PAGE
pattern (see Figure 2k and 1) and the same amino acid composition (Table 2, column 3 and 6) as
glutenin 1. Accordingly it may be concluded that this fraction consists of SDS-soluble glutenins,
indicated as glutenins 11. A typical separation of this fraction using Sepharose CL-4B as the gel
filtration medium is shown in Figure 5. The glutenins I1 eluted as a very broad fraction, indica-
ting a wide molecular weight distribution (600 000 to 10 million) in SDS solvent. All the protein
fractions that were eluted from the Sepharose gel had the same PAGE-electrophoretic pattern. How-
ever, we found that the proportions of the subunits changed progressively; as the molecular weight
of the glutenins decreased, the proportion of group A subunits declined compared with the B and
C subunits. This observation is comparable with the results of Payne and Corfield.6The main differ-
ences between glutenins I and II on the basis of the SDS-extraction method was the molecular
weight. Glutenins I are a multiple of glutenins IT. Table 3 gives the percentage of the total N that
is represented by this fraction in both wheat flours.

Figure 4. SDS-polyacrylamide-gel electrophoresis of protein samples from Sicco (S) and Tundra (T). Left to
right: a, total glutelins (S); b, glutelin 1 (S); c, glutelins I1 (S); d, glutelins I11 (S); e, glutelins IV ( S ) ; f, standards:
g. total glutelins (T); h, glutelins I (T); i, glutelins 11 (T), j, glutelins IT1 (T); k, glutelins IV (T); 1, standards.
1124 A. Graveland ef a/.

3.3.4. Glutenins I, II, III and I V


The residue insoluble in a solution containing 5~ urea and 209; ethanol could be separated after
reduction with p-mercaptoethanol into many subunits Figure 4a and g. Their molecular weights
ranged from about 10 000 to 95 OOO. This fraction was not only insoluble in 70% aqueous ethanol
but also in 5M urea. As this fraction was comparable to the glutelins of barley, oat, maize, rice and
sorghum it was classified as glutelins (see Discussion).
On a Sepharose CL4B column the glutelins could be separated into four peaks (Figure 5) with a
maximum at 2 to 3 million, 400 OOO, 160 OOO and 40 000 respectively. The proteins present in the
column fractions were shown by electrophoresis (Figure 4).

a GLUTENINS II

-
46x104D 16x104D
t 1
E
C d

0
m b GLUTENINS Ill
N

w
V
z
6
m
[L
I
2-
m G L U T E L I N S l.II,mand Izz
6

20 40 60 80 100
Fraction no.

Figure 5. Gel-filtration profiles of a, glutenins 11, b, glutenins 111 and c, glutelins I, 11, 111 and IV on Sepharose
CL-4B, 2 3 x 5 0 cm, solvent; 1.5% SDS; sample 10 mg. Internal standards were: Thyroglohulin (800 000). Fer-
ritin (460 000) and Lactate dehydrogenase (36 000 d).

The first peak I contained subunits of a molecular weight of approximately 95 OOO (a- subunits),
subunits of 68 000 ( jsubunits) and subunits of 45 000 ( y subunits). We classified this fraction as
glutelins I. The second peak I1 contained the same subunits as glutelins I.
This fraction was classified as glutelins 11. The third and fourth peak consisted of many compo-
nents of relative low molecular weight (glutelins 111). Comparison of Figures 2 and 4 shows that
the subunits of glutelins I and I1 were present in the glutelins I. Table 2 gives the amino acid composi-
tion of the combined glutelins. They formed 11 and 18% of the total wheat flour N of Sicco and
Tundra respectively (Table 3).

3.4. Characterisation of the SDS-soluble and 70 % ethanol-soluble material


The supernatant obtained after ethanol precipitation (see Figure 1) could be separated into three
fractions by means of a Sephadex G-100 column after evaporating the ethanol. Figure 6 shows the
elution pattern.

3.4.1. Glutenins III


The first peak could not be analysed on SDS-PAGE without reduction. However, after reduction
with 8-mercaptoethanol the B and C subunits of glutelins I and I1 were obtained (Figure 2i and j).
The amino acid composition was exactly the same as that of the B and C subunits of glutelins
I (Table 2 columns 5 and 7). These results caused us to classify this fraction as glutenins 111. On a
Extraction and fractionation of wheat flour proteins 1125

I , GLUTENINS (m)
E, GLIADINS
m, ALBUMINS
Figure 6. Gel-filtration profile of SDS-soluble and 70%
ethanol-soluble material on Sephadex G-100, 2.5 x 50 cm,
solvent; I .5 7; SDS; sample 100 mg. 60
20 40
Fraction no.

Sepharose CL4B column (Figure 5), the molecular weights of this fraction varied from 200 OOO co
600 000 with a peak at 420 000. The glutelins I11 distinguished themselves from glutenins I and I1 by
the absence of A subunits. Besides, their molecular weights were relatively low and, therefore
this fraction was soluble in 70 %aqueous ethanol. The fraction also contained sugars. After resuspend-
ing in water the sugars could be separated from the glutenins. Table 3 gives the percentages of total
N occurring in this fraction.

3.4.2. Gliadins
The second peak of the Sephadex G-100 column contained the gliadins (1). The gliadins could
be separated on starch gel by electrophoresis and gave a very characteristic pattern.11
With SDS-PAGE the pattern was more simple but characteristic for gliadins (Figure 2, slot g and h).
The separation by electrophoresis was possible without previous reduction. This means that they do
not contain intermolecular disulphide bonds. Table 2, presents the amino acid composition and
Table 3 the percentage of total N occurring in this fraction. After separation in a polyacrylamide
gel no sugars could be detected.

3.4.3. Albumiris
The third peak of the Sephadex G-100 column contained the albumins.' The albumins were separa-
ted on starch gel by electrophoresis into eight components.l Figure 3 (slot e and f ) shows the analysis
by SDS-PAGE. From these results it is concluded that the molecular weights of the albumins are
lower than 15 000.

4. Discussion
It is known that wheat flour protein, which is insoluble in water, saline solution and 70% alcohol,
differs greatly with respect to the physical properties and chemical composition from flour protein
from other cereals. Only insoluble protein from wheat flour has elastic properties and is partially
soluble in 0 . 1 HAc.
~ The amino acid composition is also different. It contains a large amount of
glutamine (40%) and little lysine (1.0%). Insoluble protein from other cereals, like barley,lz oat,13
maize,l* riceI5 and sorghum16 contains c. 15% glutamine and c. 6% lysine. These differences can
be attributed to the fact that wheat flour contains a great many acetic acid-insoluble glutenins with a
high molecular weight,l7 which are only found in much smaller quantities in barley and rye and
not at all in rice, maize and sorghum (unpublished results). In the presence of phenol acetic acid,
SDS or 70% 2-chlorethanol the glutenins form a gel, hence the reason why they are often called
gel proteins.18- 2 0
This study shows that in addition to the glutenins, wheat flour protein also contains a protein
fraction which is insoluble in 70% alcohol, HAc and 5M urea but soluble in 1.5% SDS solution.
As regards the amino acid pattern and electrophoretic composition (SDS-PAGE) the protein is
similar to the residual proteins (glutelins) from other cereals (unpublished results). This frac-
tion was termed glutelins in common with other cereals.

4.1. Glutenins
The glutenins I from both flour samples have the same chemical composition as the glutenins
which were isolated by Payne and Corfield6 and Khan and Bushukqg
1126 A. Graveland et al.

It was also found that a fraction of the glutenins is soluble in a 1.5% SDS solution and insoluble
in 70% aqueous ethanol. This fraction, designated glutenins IT, consists of the same subunits
A, B and C as glutenins I, but differs from the latter in that it has a lower molecular weight. A
small fraction of the glutenins is both soluble in SDS and in 70% aqueous ethanol. This fraction,
designated glutenins 111, contains no A subunits.
The difference in the solubility of the various glutenin fractions therefore lies in a difference in
molecular weights (Table 4). Glutenins I are so large that they do not dissolve in an SDS solution.
After kneading dough in air or oxygen, causing SS bonds to reduce using a radical mechanism,

Table 4. Molecular weights of the protein fractions

Intermolecular
Protein fraction Molecular weight disulphide bonds

Glutenins I 10 million
Glutenins I1 800 000-10 million
Glutenins 111 200 000-600 000

Glutelins 1 800 000-10 million


Glutelins I1 300 000-800 000
Glutelins 111 and IV < 300 000
Globulins 20 000-90 000
Gliadins 30 000-40 000
Albumins < 20 000

glutenins I (gel proteins) become soluble in SDS.lQIt is reasonable to suppose that glutenins 111,
consisting of B and C subunits and with an average molecular weight of 420 000, are the precursors
for glutenins I and 11. The A subunits probably act as crosslinks between the B-C units. The number
and type (reactivity) of intermolecular SS-bonds undoubtedly play a role in the build-up of the
glutenins to units with a high molecular weight.
Glutenins I1 are probably identical to the AUC-extracted glutenins of Payne and Corfield.6
Glutenins III are identical to the ethanol-soluble fraction isolated by Payne and Corfield6 and
the h.m.w.g. (high molecular weight gliadins) isolated by Bietz and

4.2. Glutelins
Like glutenins, glutelins also consist of different subunits by means of intermolecular SS bonds,
and they too can be subdivided, according to the difference in molecular weight and molecular
composition, into glutelins I, 11,111 and IV. The glutelins differ from the glutenins in their solubility
properties. Even after reduction with P-mercaptoethanol, they remain insoluble in 70'4 aqueous
ethanol and 5h.1urea. The difference between glutenins subunits and glutelins subunits is also shown
by the SDS-PAGE patterns.
Glutelins I have a molecular weight of 800 000 to c. 10 million. Glutelins 11, which consist of the
same subunits, have a spread in molecular weight ranging from 300 000 to 800 000. We suppose
that glutelins I1 are the precursors for glutelins I. Glutelins 111 and 1V differ from glutelins I and 11
in that they consist of other subunits. They have a lower molecular weight, i.e. lower than 300 OOO.
It is not yet clear what the relationship there is between glutelins TI1 and IV and glutelins I and 11.
Figure 2, slot c and d shows that glutenins I have glutelins I as an impurity. It would seem that
wheat flour protein contains a small fraction of glutelins I with a very high molecular weight, the
result being that they are insoluble in SDS and that they remain behind in the gel layer during
fractionation.
In comparing gel tracks 2a,k with 4b,c and tracks 2c,l with 4h,i it is clear that some of the ic-
glutelin subunits of Figure 4 are much the same as the A-glutenin subunits of Figure 2. It would also
seem that the glutelin I and I1 contain a small fraction of glutenin I.
Extraction and fractionation of wheat flour proteins 1127

4.3. Gliadins
The gliadins form an extremely heterogeneous but homologous group of polypeptides with an
average molecular weight of 35 000 (Figure 3, slot k and I). The majority of gliadins are classified
as or-and /3-gliadins on the basis of their electrophoretic mobility at pH 3.1. We were unable to detect
any sugar on the SDS-PAGE gel using specific sugar colour reagent. Unlike the glutelins and glu-
tenins they contain no intermolecular disulphide bonds.

4.4. Glycoproteins
In contrast with the previous study, we Were only able to isolate And partly characterise one fraction
as SDS-insoluble glycoproteins. The amino acid composition of the protein from this fraction
strongly resembles that of the glutelins.

4.5. Functional properties of the different protein fractions


Table 3 shows that Sicco contains more glutenins than Tundra, viz. 45 and 38 respectively of the
total protein. Sicco is especially rich in glutenins I and Tundra in glutenins I1 and 111. These figures
demonstrate that in the case of Sicco a greater build-up of glutenins into more, larger units has
taken place than in the case of Tundra. This also explains why more sediment was obtained with
Sicco, using the SDS-sedimentary method, than with Tundra. It should also be noted that the gel
layer which was obtained after centrifuging of the SDS-flour suspension was much firmer with
Sicco than with Tundra.
Our research and the results of other research workers17 have shown that the glutenins constitute
the most significant protein fraction for the creation of a continuously visco-elastic protein network
in dough. Table 3 also shows that the total glutelin content in Sicco is 11% and in Tundra 18%.
The addition of glutelins to dough clearly showed that the glutelins have negative functional
properties (unpublished results). Since Tundra contains more glutelins than Sicco, it can be reason-
ably assumed that the negative effect of the glutelins will be greater with Tundra than with Sicco.
The difference in bread volume between the good breadmaking variety, Sicco, and the poor
breadmaking variety, Tundra, can largely be explained by the difference in the number and mole-
cular composition of the glutenins on the one hand and of the amount of glutelins on the other.

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