Agricultural Research Review. 67(4): 611 -618 (1989)
Printed in Egypt.
61 1
RELATION BETWEEN AMINO ACIDS CONTENT OF GLIADIN,
GLUTENIN, GLUTEN, AND ISOELECTRIC POINT OF
THESE PROTEINS IN SOME CEREAL CROPS
N.A. EL-HAWWARY1,
A.I. ABDEL-GAWWAD2,
AND
M.T. SHALABY2
' Central Laboratory for Agricultural Industries,
Agricultural Research Centre, Giza, Egypt.
2 Faculty of Agriculture, Mansoura University, Mansoura, Egypt.
[Manuscript accepted 13 April 1987]
-----------------------------------------------------------------------------------------------
Abstract
Amino acids content and isoelectric point (IEP) of gliadin and glutenin of four
cereal plant proteins, i.e. rice, sorghum, wheat, and corn, as well as gluten of wheat,
were determined. Data obtained from chemical analysis, as well as other data obtained
from literature, were used to establish a mathematical equation to reduce the IEP of
gliadin, glutenin,andgluten (the major important proteins from the stand-point of
(bread making) with information about glutamic + aspartic (A) and lysine + arginine
(B) amino acids content as follows:
IEP = 2.553 + 3.000 log A/B, where :
IEP = the isoelectric point of gliadin, glutenin, or gluten.
A = glutamic + aspartic amino acids.
B = lysine + arginine amino acids.
2.533 and 3.000 = constants.
Differences between values calculated from the above suggested equation and
those obtained from chemical analysis are not significant. With information about
amino acids content, one can calculate the IEP of gliadin, glutenin, and gluten by the
use of such equation as a substitute! it of titration curves. Consequently, we can get
information about the adequate IEP for precipitating gliadin, glutenin, and gluten
fraction protein.
INTRODUCTION
It is well known that amino acids migrate in alkaline solution to the anode and in acid
solution to the cathode, and there is a pH value at which no migration occurs. This pH
value has been called the isoelectric point (IEP), which is determined by a titration curve
612
N.A. EL-HAWWARY. A.I. ABDEL-GAWWAD. AND M.T. SHALABV
relation and characterization of gliadin-like sub-unit from glutenin and reported that the
most precipitate of gliadin occurred between pH 4.6 to 5.5 and 5.0 to 7.4 above which no
further precipitation occurred.
El-Hawwary and Abdel-Gawwad (1983) suggested mathematical equation to deduce
the IEP of true plant protein with information about glutamic + aspartic (A) and lysine +
arginine (B) amino acids as follows: IEP = 3.37 + 1.79 log A/B, where 3.37 and 1.79 are
constants. Differences between values calculated from the equation and those obtained
from chemical analysis were not significant up to 2.88 A/B ratio.
Since the two major important proteins in gluten from the standpoint of bread making
are gliadin and glutenin (Bietz and Rothfus 1970), and the nondializable water soluble
compounds (glycoproteins) has been found to be responsible for gas retention and gluten
extensibility (Pomeranz et a/. 1970), even the wheat gluten is producing and marketing
for the use of the food industry (AACC 1978), it was' decided to make an attempt to use
the results of the chemical analysis of the more acidic amino acids: glutamic and aspartic,
the more basic amino acids: lysine and arginine, and pH of IEP of precipitated proteins
(gliadin, glutenin, and gluten) with other data obtained from literature in order to
establish mathematical equation to facilitate calculation of IEP of these proteins with
information about the above mentioned amino acids.
MATERIALS AND METHODS
Four cereal seed varieties, namely rice (Giza 172), sorghum (Giza 114), corn
(double hybrid), and wheat (Giza 155) constituted the materials used in this study to
determine amino acids content in gliadin, glutenin, and gluten. Seeds of samples were
milled through a Willey mill with a 60 mesh screen. Flour was defatted in a soxhlet
extractor with n-hexane for 20 h. The deffated flour was air dried and stored at 5°C.
Protein of seed varieties was extracted by using alkaline solution. Ten grams of
ground seeds (particle size of 1.0 and 0.4 mm) were used to extract protein for every
sample by using 300 mL of 2% sodium hydroxide for 30 min extraction time. The supernatant was considered to have 100% soluble nitrogen. Aliquots of 20 mL for every
sample were adjusted to pH values ranging from 1.0 to 13 and left for 3 h under
refrigeration for equilibration. Volumes of suspensions were brought to 50 mL with
distilled water and centrifuged at 3000 rpm for 15 min. Supernatants were analyzed for
their nitrogen content and results were expressed as percent of total extracted nitrogen in
the initially prepared alkaline extract.
Separation of the extractable proteins was similar to the procedure adopted by Chon
and Bushuk (1970). Analysis of amino acids content was carried out according to Block
RELATION BETWEEN AMINO ACIDS CONTENT AND ISOELECTRIC POINTS
613
et al. (1958). The isoelectric point was determined by titration curves of protein isolated according to the methods of Smith et al. (1959).
RESULTS AND DISCUSSION
Gliadin and glutenin were separated from rice, sorghum, corn, and wheat flours, while
gluten was separated from wheat .Values of amino acids content in these proteins are
presented in Table 1. Four amino acid had been chosen to establish the relation between
the amino acids and the isoelectric point of gliadin, glutanin, and gluten fraction protein.
These amino acids were the more acidic amino acids: glutamic (pK n = 4.00) and
aspartic(pKa = 3.50), and the more basic amino acids: lysine (pKa = 12) and arginine (pKa
= 10.64). Values and total of above mentioned four amino acids are presented in Table 2,
which shows that wheat seeds contained the highest value of aspartic + glutamic amino
acids (A) for both gliadin and glutenin compared with other cereal seeds, while rice and
corn contained the lowest values of aspartic + glutamic amino acids for gliadin and
glutenin, respectively. On the other hand, rice seeds contained the highest values of lysine
+ arginine (B) for both gliadin and glutenin compared with other seeds.
From Table 2, it is apparent that wheat gliadin and glutenin contained the highest total
values of aspartic + glutamic and lysines- arginine amino acids (52.60 and 48.99%,
respectively) while gliadin of sorghum and glutenin of corn contained the lowest A-f-B
values (32.59 and 33.66% respectively). It can be also seen that ratios between the more
acidic amino acids (A) and the more basic amino acids (B) showed the highest value in
sorghum for gliadin and in wheat for glutenin, while wheat and rice showed the lowest A/
B values for gliadin and glutenin, respectively.
Semi-logarithmic relation between log A/B ratios on the abscissa axis and isoelectric
point on the ordinate axis was proved. Hence, log A/B
ratios and IEP of proteins obtained from chemical analysis, as well as data obtained from
literature, were used to establish mathematical equation to calculate the IEP of above
mentioned cereal proteins with information about aspartic + glutamic (A) and lysine +
arginine (B) amino acids.
Relation between log A/B ratios and IEP of gliadin, glutenin and gluten fraction protein was linear. Therefore, a straight line equation was used to establish the suggested
equation as follows:
IEP = 2.553 + 3.000 log A/B, where
IEP = isoelectric point of gliadin, glutenin, or gluten.
A = aspartic -t- glutamic amino acids.
B = lysine + arginine amino acids.
2.553 and 3.000 = constants obtained by a straight line equation.
Lysine
Histidine
Arginine
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Cysteine
Valine
Methionine
Leucine + isoleucine
Tyrosine
Rice
Sorghum
Corn
Wheat
Gliadin Glutenin Gliadin Glutenin Gliadin Glutenin Gliadin Glutenin Gluten
0.51
3.47
0.14
3.12
0.14
2.48
0.73
1.75
1.00
0.91
2.27
0.67
3.12
1.39
4.34
2.32
2.01
2.16
5.92
6.60
0.66
5.91
1.89
4.82
3.58
3.58
5.00
7.78
9.92
6.72
9.07
5.85
5.32
2.65
3.06
8.00
2.86
3.92
4.58
4.88
2.97
5.95
2.14
3.09
2.56
5.53
5.10
3.32
5.38
5.04
5.35
3.99
5.25
4.67
21.40
18.80
25.07
29.48
26.48
21.04
46.64
40.90
10.40
4.08
6.41
11.63
14.86
11.62
13.23
17.04
13.12
15.80
3.22
4.60
1.28
5.33
1.35
4.35
1.87
5.85
3.36
6.58
6.52
13.96
9.40
10.68
7.39
2.22
3.02
2.10
0.30
2.68
Trace
1.21
2.27
0.78
2.27
2.27
6.97
7.31
5.88
5.50
4.21
5.15
5.03
4.80
4.92
0.50
2.61
1.33
Trace
0.44
2.23
1.78
1.79
1.23
15.98
13.46
20.37
16.56
25.44
15.60
12.98
11.14
11.64
8.70
3.82
5.17
3.23
5.61
5.43
2.89
4.16
3.46
N.A.EL-HAWWARY, A.I.ABDEL-GAWWAD, AND M.T.SHALABY
Amino acids
614
Table 1. Amino acids content in gliadin and glutenin of rice, sorghum, corn, wheat, and gluten (g/100 protein)
RELATION BETWEEN AMINO ACIDS CONTENT AND ISOELECTRIC POINTS
615
Table 2. Aspartic-t-glutamic, lysine+arginine amino acids, and isoelectric points of
gliadin and glutenin of rice, sorghum, corn, wheat, and gluten of wheat.
Amino acids
(g/100g protein)
Samples
Kinds of protein
Rice
Gliadin
Aspartic
Lysine
+ glutamic + arginine
(A)
(B)
Total
A/B ratio
IEP
29.18
6.43
36.61
4.53
4.50
Sorghum
31.79
0.80
32.59
39.74
7.50
Corn
32.33
2.03
34.36
15.93
6.00
Wheat
49.92
3.31
52.60
14.89
6.00
28.72
10.07
38.79
2.85
4.00
Sorghum
38.15
9.03
47.18
4.22
4.50
Corn
26.36
7.30
33.66
3.61
4.50
Wheat
43.96
5.03
48.99
8.74
5.50
112.00
6.00
118.00
18.66
6.25
Rice
Wheat
Glutenin
Gluten
Values of IEP of gliadin, glutenin, and gluten calculated from te above suggested
equation at various A/B ratios and IEP of the same proteins obtained from chemical analysis are presented in Table 3. Differences between calculated IEP of proteins and IEP
obtained from chemical analysis are not significant, and confidence limits were ± 0.711.
The minimum and maximum of A/B ratios ranged form 2.85 (min.) to 39.74
(max.) and IEP of protein ranged from 3.92 (min.) to 7.35 (max.). It can be seen that as
the range of acidic to basic amino acids is low, the isoelectric range is also low,
otherwise, increasing of IEP of protein is due to increasing the range of acidic to basic
amino acids.
The relation between A/B ratio and IEP was also tested for gliadin, glutenin,
and gluten fraction protein, i.e. 39.74-2.85/7.35-3.92 = 10.75. From obtained results it
can be seen that every single pH affected the A/B ratio by 10.75, where IEP was
expressed as pH value.
Summarizing the results obtained in this study the isoelectric point of protein
could be mathematically calculated by use of the proposed equation if the aspartic,
glutamic, lysine, and arginine amino acids content is available
Summarizing the results obtained in this study the isoelectric point of protein
could be mathematically calculated by use of the proposed equation if the aspartic,
glutamic, lysine, and arginine amino acids content is available
61 6
N.A. EL-HAWWARY. A.I. ABDEL-GAWWAD. AND M.T. SHALABY
Table 3. Comparison between experimental and calculated IEP ofgliadin, glutenin,
and gluten at different A/B ratios (confidence limits « ±0.711).
IEP
IEP
experime
experimental
ntal
pH
pH
A/B ratio
IEP
calculated
pH
2.85
3.92
4.00
3.61
4.22
4.53
8.74
14.89
15.93
18.66
39.74
5.75
6.53
7.50
4.30
4.43
4.53
5.37
6.07
6.16
6.37
7.35
5.39
5.50
5.62
4.50
4.50
4.50
5.50
6.00
6.00
6.25
7.50
5.50
5.50-5.80
5.30
7.80
5.10
. 9.08
5.68
9.85
5.86
10.06
5.88
10.10
10.20
10.80
10.87
11.00
11.38'
12.27
12.80
13.60
14.02
16.76
5.87
5.89
5.94
5.94
5.94
5.88
6.05
6.10
6.17
6,32
6.30
4.00
References
Experimental data
4.50
Experimental data
4.50
Experimental data
4.50
Experimental data
5.50
Experimental data
6.00
Experimental data
6.00
Experimental data
6.25
Experimental data
7.50
Experimental data
5.50
Experimental data
5.50-5.80 Dexter and Matsuo (1978)
5.30
Bietz and Wall (1973)
around
around 4.50
Dexter and Matsuo (1978)
4.50
Domah and Mohamed
5.60 .
5.60 .
(1974)
5.50-5.80 5.50-5.80 Dexter and Matsuo (1978)
around
around 6.40
Bietz and Wall (1973)
6.40
5.60
5.60
Orth and Bushuk (1973)
5.40
5.40
Dexter and Matsuo (1978)
5.50-5.80 5.50-5.80 Dexter and Matsuo (1978)
5.50-5.80 5.50-5.80 Bietz and Wall (1973)
5.80
5.80
Bietz and Wall (1973)
5.80
5.80
Dexter and Matsuo (1978)
6.40
6.40
Dexter and Matsuo (1970)
5.80
5.80
Dexter and Matsuo (1978)
5.80
5.80
Dexter and Matsuo (1978)
6.40
6.40
Dexter and Matsuo (1978)
5.80-7.00 5.80-7.00 Bietz and Wall (1973)
Therefore, by use of such equation we can get information about the adequate
IEP for precipitating gliadin, glutenin, or gluten as a substituent of titration curves.
Differences between calculated values of IEP of protein and values of IEP obtained by
chemical analysis are not significant.
RELATION BETWEEN AMINO ACIDS CONTENT AND ISOELECTRIC POINTS
61 7
REFERENCES
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glu-tenin. Cereal Chem., 47: 381.
3. Bietz, J.A. and J.S. Wall. 1973. Isolation and characterization of gliadin like
subunits from glutenin. Cereal Chem., 50 (5): 541.
4. Block, R.J., E.L. Durrum, and G. Zwerg. 1958. A manual of paper chromatography
and paper electrophoresis, 2nd ed. Academic Press Inc. Publishers, New York.
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species. 1-Solubility characteristics and amino composition of endosperm
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pasta dough rheolgy and spaghetti making quality. Cereal Chem., 55 (1): 44-47.
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8. El-Hawwary, N.A. and A.I. Abdel-Gawwad. 1983. Relation between some amino
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