Agricultural Research Review, 66(3): 433-439(1988) 433
Print in Egypt
RELATION BETWEEN ISO-ELECTRIC POINT AND SOLUBLE PROTEIN EXTRACTED FROM PALNT SEEDS AT pH 1.0
N. A. El-HAWWARY
Field Crops Research Institute ,Agricultural Research Centere , Giza,Egypt
______
Abstract
Amino acids contents and iso –electric point of six plant protein namely wheat ,
horsebean , barely , sorghum , lupine and fenugreek were determined . Highest
protein extraction fromseed samples occurred at 1:30 solid : solvent ratio except
that from sorghum where protein occurred at 1:40.
Data obtained from literaturey curves of protein as well as other data obtained
from literature were used to establish mathematical equation to predict the IEP of
protein withinformation about soluble protein percent of total extracted protein in
the initially prepared alkaline extract at pH 1.0 as follows :
IEP = 4.9895 – 1.1000 Log . R/10.
Where : IEP is the iso –electric point of protein .
R is soluble protein percent of total extracted protein in the initially prepared
alkaline extracted at pH 1.0 . while 10 is a factor.
4.9895 and 1.1000 are constants .
Differencesbetween values calculated from equation and those obtained from titr- ation curves are not significant .
Information obtained from the above mentioned equation are valid from 30 to 100% of total extracted protein at pH 1.0 .
INTRODUCTION
Recently , iso-electric point is determined by titration curves of isolated protein after precipitation at various pH .Clusky et al (1973) , reported that the minimum solubility ofoat concentrated protein was about pH 5 . Rhee et al (1973) , studied the effect of pro-cessing pH on the properties of protein isolates during precipitation . They found that maximum recovery was obtained at precipitation of pH 4.5, while subsequent studies showed that optimum precipitation pH would be 4.0 ±0.25. Patel and Jahnson (1974) , reported that the protein in the clarifed liqour was precipitated after adjustment to 4.25 with 6 N Hcl . El – Iraqi (1976) , found that the maximum precipitation was at pH 4.0 for sesame seeds protein .
434 N.A.EL-Hawwary
A mathematical equation to deduce the iso- electric point of true protein with information about amino acids glutamic + aspartic (A) and lysine + arginine (B) was suggested by El- Hawwary and Abdel –Gawwad (1982) . The equation was as follows :
IEP = 3.73 + 1.79 Log A/B
Where : 3.73 and 1.79 are constants .
Differences between values calculated from equation and those obtained from chemical analysis are not significant .
Information obtained from above mention equation are valid up to 2.88 of A/B ratios .
The aim of the present work was to find out a new formula related to the iso –electric point , measured by titration curve , and the percentage of soluble protein extracted pH 1.0.
MATERIALS AND METHODS
This study was carried out during 1986 at soil tests and plant Analysis Laboratory , Faculty of Agriculture ,MansouraUniversity.
Seeds of Sixvarieties , namely , wheat (Giza 155) , horsebean ( Giza 1) , barley (Giza 119) , sorghum (Giza 114) , Lupine (Giza 2) and fenugreek (Giza 2) constituted the materials used in this work to determine amino acids content and protein extraction . Analysis of amino acids was carried out according to Block et al (1958).
Different ratios of solid : solvent (1:20 , 1:25 , 1:30 , 1:35 and 1: 40) , 2% sodium hydroxide as extractant and 30 mintues length of agitation were used for obtaining maximum protein extract from milled seeds ( particles . size of 0.4 mm ).
Ten gm of milled seeds were used to extract protein by using 300 ml . of sodium hydroxide for 30 minutes 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 hours under refrigeration for equilibration . Volumes of suspensions were brought to 50 mL with distilled water and centrifuged at 300 rpm for 15 mintues.Supernatants were analysed for their nitrogen content and results were expressed as percent of total extracted nitrogen in the initially prepared alkaline extract.
The iso-electric point was determined by titration curves of protein extracted according to the methods of Smith et al ,(1959) .
All the statistical analysis were carried out according to Snedecor &Cochran (1967).
RELATION BETWEEN ISO-ELECTRIC POINT AND SEED PROTRIN 435
RESULTS AND DISCUSSION
Amino acids of wheat , horsebean , barley , sorghum , lupine and fenugreek seeds were determined . Values of amino acids are presented in Table 1 . From table 1 , it can be seen that essential amino acids were found in different ratios in the six types of seeds . Lupine and fenugreek contained the highest values of total essential amino acids (53.91 and 52.02%) of total protein , respectively, while sorghum seeds showed the lowest value of total essential amino acids ( 37.09%) compared with other seeds .On the other hand , it can be seen also , from data presented in Table 1 . that wheat and fenugreek seeds con-tained the highest values of asparatic and glutamic non- essential amino acids, respecti- vely, compared with other seeds .
The highest extraction of protein occurred at 1:30 solid : solvent ratio , where protein extracted were 95.6 , 92.0 , 91.0 , 93.0 and 92.3 % of total protein for wheat , horsebean , barley , lupine and fenugreek seeds, respectively . While maximum protein extracted from sorghum ( 93.3 % of total protein )occurred at 1.40 solid :solvent ratio (Table 2)..
Table 1 .Amino acids contents of wheat , horsebean , barley , sorghum , lupine, and
fenugreek seeds ( g / 100 g protein ) .
Amino acids / Wheat / Horsbean / Barely / Sorghum / Lupine / FenugreekLysine / 3.50 / 5.20 / 2.61 / 1.81. / 4.45 / 7.10
Histidine / 4.40 / 3.00 / 2.17 / 2.10 / 1.81 / 1.75
Arginine / 8.80 / 8.90 / 7.90 / 7.80 / 11.10 / 10.50
Asparatic / 5.10 / 9.50 / 6.70 / 6.80 / 9.69 / 11.20
Threonine / 3.20 / 2.50 / 3.62 / 2.10 / 3.12 / 3.61
Serine / 4.10 / 6.78 / 3.57 / 4.20 / 7.48 / 6.56
Glutamic / 34.10 / 15.80 / 20.10 / 14.10 / 16.90 / 21.10
Proline / 11.80 / 6.78 / 11.30 / 10.40 / 4.76 / 2.50
Glycen / 4.60 / 2.69 / 3.40 / 2.88 / 3.20 / 1.26
Alanine / 4.10 / 4.50 / 3.65 / 1.31 / .2.50 / 5.71
Cystine / 3.00 / 0.75 / 1.83 / 1.78 / 0.68 / 0.86
Valine / 5.00 / 6.00 / 4.00 / 5.10 / 4.90 / 5.20
Methinine / 1.25 / 0.80 / 2.40 / 2.22 / 0.50 / 0.45
Leuc+isoleu / 14.10 / 13.31 / 11.10 / 13.70 / 20.90 / 19.60
Tyrosine / 2.70 / 3.70 / 3.40 / 3.50 / 3.50 / 3.30
Tryptophan / 0.90 / 1.10 / 0.90 / 1.01 / 1.25 / 1.20
Total / 45.25 / 45.31 / 38.79 / 37.09 / 52.02 / 53.91
436 N.A.EL-HAWWARY.
Table 2.Effect of different solid : solvent ratios on the protein extraction efficiency from wheat , horsebean, barley , sorghum , lupine , and fenugreek , milled seeds .
Solid: solvent ratios / Protein extracted (%)wheat / Horsbean / Barely / Sorghum / Lupine / Fenugreek
1:20 / 62.0 / 75.0 / 50.0 / 36.0 / 70.5 / 55.0
1:25 / 85.0 / 83.0 / 75.2 / 43.0 / 76.0 / 85.0
1:30 / 94.5 / 93.0 / 95.0 / 55.0 / 92.0 / 91.1
1:35 / - / - / - / 70.0 / - / -
1:40 / - / - / - / 92.2 / - / -
Soluble protein at pH values ranging from 1.0 to 13.0 were determined , and the data summarized in Table 3 . At pH 1.0 , soluble nitrogen ( R ) ranged from 50.75 to 100.00 of
Table 3.Protein solubility of seeds at various pH values .
Soluble protein (%)pH / Wheat / Horsebean / Barely / Sorghum / Lupine / Fenugreek
1.0 / 50.75 / 85.00 / 80.00 / 70.00 / 70.00 / 100.00
2.0 / 46.80 / 76.00 / 25.00 / 70.00 / 63.00 / 80.00
3.0 / 42.00 / 15.00 / 16.00 / 57.00 / 50.00 / 80.00
3.5 / 25.00 / 15.00 / 16.00 / 30.00 / 27.00 / 65.00
4.0 / 22.00 / 14.00 / 15.00 / 17.00 / 18.00 / 50.00
4.5 / 26.00 / 17.00 / 32.50 / 31.00 / 27.00 / 48.50
5.0 / 40.00 / 17.00 / 33.00 / 57.00 / 50.00 / 50.00
6.0 / 59.00. / 55.00 / 33.00 / 63.00 / 70.00 / 70.00
7.0 / 76.00 / 72.00 / 33.00 / 70.00 / 77.00 / 89.00
8.0 / 82.00 / 75.00 / 40.00 / 70.00 / 83.00 / 90.00
9.0 / 92.00 / 90.00 / 40.00 / 77.00 / 92.00 / 90.00
10.0 / 100.00 / 100.00 / 44.00 / 83.00 / 92.00 / 90.00
11.0 / - / - / 44.00 / 97.50 / 98.00 / 99.00
12.0 / - / - / 85.00 / 100.00 / 100.00 / 100.00
13.0 / - / - / 90.00 / - / - / -
RELATION BETWEEN ISO-ELECTRIC POINT AND SEED PROTRIN 437
total extracted nitrogen in the initially prepared alkaline extract of wheat and fenugreek seeds , respectively , while nitrogen soluble at pH 1.0 ( R ) was 70.0%of total extracted nitrogen for both lupine and sorghum seeds . On the other hand , soluble nitrogen of bar- ley and horsebean at pH 1.0 were 85.0 and 85.0%of total extracted nitrogen in the initially prepared alkaline extract .
Minimum protein solubility and iso – electric point were occurred at around pH 4.0 for wheat , horsebean , barely , sorghum and lupine and around pH 4.5 for fenugreek prot-eins.
Semi logarithmic relation between log R/10 ratios on the abscissa axis and iso-electric points on the ordinate axis was proved . Accordingly , log R/10 ratios and iso-electric points ( IEP) of proteins obtained from titration curves as well as other data from literature were used to establish mathematical equation to calculate theIEP of protein .
The relation between log R/10 ratios and IEP proteins was linear . The correlation coefficient was negative and significant at the level of 0.01 ( r = -0.9969) . therefore , a straight line equation was used to establish the suggested equation as follows :
IEP = 4.9895 – 1.1000 log . R/10 . where:
IEP is iso- electric point of protein .
R is soluble nitrogen at pH 1.0 and 10 is a factor
4.9895 and 1.1000 are constants obtained by a straight line equation .
Values of IEPof proteins calculated from above suggested equation at various R/10 ratios and IEP of proteins obtained fromtitration curves are presented in Table 4 .
Table 4.Comparison between experimental and calculated IEP of proteins at different R ratios .
Rratios / Log. R/10 / IEP
Experimental
pH / IEP
Calculated
pH / Source of protein
30 / 0.4770 / Around / 4.5 / 4.46 / Rice*
40 / 0.6020 / Around / 4.0 / 4.32 / Sesama seeds**
50 / 0.6989 / Around / 4.0 / 4.22 / Wheat
70 / 0.8450 / Around / 4.0 / 4.06 / Sorghum
70 / 0.8450 / Around / 4.0 / 4.06 / Lupine
70 / 0.8450 / Around / 4.0 / 4.06 / Barley
85 / 0.9294 / Around / 4.0 / 3.97 / Horsbean
100 / 1.0000 / Around / 4.0 / 3.89 / Fenugreek
* After Domah and Mohamed (1974)
** After el-iraqi (1976)
438 N.A.EL-HAWWARY
It is evident from data presented in Table 4 . that differences between calculate IEP of protein and IEP obtained from titration curves are not significant .
Summarizing the results obtained in this study , it can be supposed that iso-electric point of protein could be mathematically calculated by the use of the proposed equa- tion if soluble nitrogen percent at pH 1.0 of protein extracted by alkaline solution is availa- ble . Thus , by the use of such equation we can get information about the adequate IEP for precipitating protein as substituent of titration curves . Calculated values of IEP are valid from 30 to 100 % of soluble protein at pH 1.0 .
REFERENCES
1.Block , R.J ; E. L .Durrum and G. Zwerg .1958 . A manual of paper chromatography and paper electrophoresis , 2nd Ed . , AcademicPress Inc . Publishers , New York .
2.Cluskey , Y.V. ; J .S . Wall and G.E. Inglett . 1973 . Oat protein concentrates from a Wet-milling process , composition and properties . Cereal Chem . 50 ( 4 ) P. 481.
3.Domah , M . B. and M. S. Mohamed . 1974 . Studies on rice starch industry in Egypt ;nitrogen fraction in rice its starch manufacture wastes . Alex . J . Agric . Res . Vol .,22(1):45-54
4-El-Hawwary , N. A and A. I . Abdel – Gawwed .1983 . Relation between some amino cids content in plant seed proteins and iso-electric point of protein.
5-El- Iraqi , S.M .1976. Biochemical studies on sesame seed protein as influenced by processing . Ph . D . Thesis , Faculty of Agric . Alex . Univ .
6-Hassan , S . A . 1980. Separation and evaluation of cereal and legume protein . Ph . D . Thesis , Faculty of Agric . Ain Shams Univ.
7-Patel , K . A and J, A. Johnson .1974. Horsebean as protein supplement in bread making 1- Isolation of horsebean protein and its amino acids composition .Cereal Chem .Vol 51, p . 435.
8-Rhee , K . C ; C .M Cater and R. E . Mattil .1973. Effect of processing pH on the prop- erties of peanut protein isolates and Oil . Cereal Chem .50 ( 4 ) P . 395.
9-Smith , G . R . and I . A . Wolf .1959. Comparison of solubility characteristies of selected seed proteins. J. Agric . Food Chem .Vol .1, ( 2) .
10-Snedecor , G. W . and W.C . Cochran . 1967. Statistical methods . IowaState Univ . Press . Ames, Iowa .