Escolar Documentos
Profissional Documentos
Cultura Documentos
REVIEW ARTICLE
ABSTRACT
Worldwide, plant protein contributes substantially as a food resource because it contains essential amino acids for meeting
human physiological requirements. However, many versatile plant proteins are used as medicinal agents as they are produced
by using molecular tools of biotechnology. Proteins can be obtained from plants, animals and microorganism cells. The abundant
economical proteins can be obtained from plant seeds. These natural proteins are obtained by isolation procedures depending on
the physicochemical properties of proteins. Isolation and purification of single protein from cells containing mixtures of unrelated
proteins is achievable due to the physical and chemical attributes of proteins. The following characteristics are unique to each
protein: Amino acid composition, sequence, subunit structures, size, shape, net charge, isoelectric point, solubility, heat stability
and hydrophobicity. Based on these properties, various methods of isolation exist, like salting out and isoionic precipitation.
Purification of proteins is quiet challenging and, therefore, several approaches like sodium dodecyl sulfate gel electrophoresis and
chromatography are available. Characterization of proteins can be performed by mass spectrometry/liquid chromatographymass
spectrometry(LCMS). The amino acid sequence of a protein can be detected by using tandem mass spectrometry. In this article,
a review has been made on the sources, isolation, purification and characterization of natural proteins.
Key words: Amino acid, natural proteins, SDS gel electrophoresis, salting out
Table1: Essential amino acids and their roles Table2: Color reactions of proteins/amino acids
Amino acid Role Reaction Observations Specific group or
Isoleucine Formation of hemoglobin; prevents muscle amino acid
wasting in debilitated individuals Biuret reaction Violet or Two peptide linkages
Leucine Promotes healing of skin and broken bones; purple color
reduces muscle protein breakdown Ninhydrin reaction Blue color amino acids
Valine Influences brain uptake of other Xanthoproteic reaction Orange color Aromatic amino
neurotransmitter precursors(tryptophan, acids(phe, Tyr, Trp)
phenylalanine and tyrosine) Millions reaction Red color Phenolic group(Tyr)
Histidine Production of red and white blood cells; Hopkins-Cole reaction Violet ring Indole ring(Trp)
treatment of anemia Sakaguchi reaction Intense red Guanidio group(Arg)
Lysine Inhibits viruses; treatment of herpes simplex, color
Lysine and Vitamin C together form Lcarnitine, Nitroprusside reaction - Sulfhydryl groups(Cys)
a biochemical that enables muscle tissue to Sulfur test Black ppt Sulfhydryl groups(Cys)
use oxygen more efficiently, delaying fatigue Paulys test - Imidazole ring(His)
Methionine Increases the antioxidant levels(glutathione); Folin-Coicalteaus test - Phenolic groups
reduces blood cholesterol levels
Phenylalanine Production of collagen, precursor of tyrosine;
enhances learning, memory, mood and
E.coli).[7] Nend rule, which relates to the halflife of a
alertness protein, is used to identity its Nterminal residue.
Threonine Prevents fatty build up in the liver; amino
detoxifers Extinction coefficient
Tryptophan Prevents fatty buildup in the liver; precursor of
key neurotransmitter serotonin, which exerts a
It indicates at the amount of light absorbed by a protein at a
calming effect certain wavelength. This coefficient value helps in estimating and
identifying a protein when exposed to a spectrophotometer.[7]
2. Solubility depends on electrostatic charges; net The molar extinction coefficient of a protein can be estimated
charge depends on number, identity, location of by knowing its amino acid composition. By using the following
amino acids and pH of solvent equation, the native protein extinction coefficient in water
3. It depends upon isoelectric point(range 5-8.5): can be computed using the molar extinction coefficient values
Isoelectric point depends on sevencharge at a given wavelength of tyrosine, tryptophan and cystine(at
amino acids, viz. glutamate(carboxyl group), 280nm, the extinction value of Tyr is 1490, of Trp is 5500 and
aspartate(carboxyl group), cysteine(thiol group), of Cys is 125 in water).[8]
tyrosine(phenol group), histidine(imidazole
E1=no. of(Tyr) * Ext(Tyr) + no. of(Trp) * Ext(Trp) no.
side chains), lysine(ammonium group) and
of(Cystine) * Ext(Cystine)
arginine(guanidinium group).[5]
E2=no. of(Tyr) * Ext(Tyr) + no. of(Trp) * Ext(Trp)
Molecular weight
Proteins molecular weight variation depends on the number Two values of the proteins produced in water at 280nm using
of amino acid residues. Each amino acid contributes 110 value the above equation indicate that the first value(E1) is due to
increases in a proteins molecular weight, e.g., Insulin5700; cysteine residues appearing as half cystines and that the second
Myoglobin1700; Hemoglobin64,450. value(E2) is due to no cysteine appearing as half cystine.
GRAVY=Sum of AA hydropathy values/residue numbers only single ions remain. Peptides are then fragmented and
in sequence the masstocharge ratio of this is measured. This process is
repeated with a different digestion enzyme, and overlaps in
An increasing positive score indicates greater hydrophobicity. sequences are used to construct a sequence for protein.[12]
2. Animal protein has a balanced combination of all peptide of four distinct fractions containing intramolecular
amino acids; hence, it is called complete protein. disulfide bonds.[40] These play a role in film formation,
In contrast, plant(vegetable) protein is incomplete strength and elasticity. Glutenin, a mixture of proteins, has
protein;soybean proteinis an exception to this. a molecular weight distribution between 100 and 1000kDa.
The disulfide bonds present in glutenin and gliadin help in
Plant proteins determining the strength of the protein matrix.[41]
Vegetables, legumes and fruits are good sources ofprotein.
Legumes have a higher content of protein than vegetables and Corn Zein
fruits.[14] Different parts of plants are sources of proteins as Zein contains high percentages of nonpolar amino acids,
given in Table3. Plants were studied for isolation of proteins namely glutamine(26%), leucine(20%) and proline(10%)
and basic and acidic amino acids in low proportions. It
that have applications in the pharmaceutical industry, as
is insoluble in water.[42] Two major fractions of zein are
shown in Table4.
zein, soluble in 95% ethanol, and zein, soluble in 60%
ethanol. Commercially, it is used for tablet coating and in
Some important proteins, their characteristics and uses are
biodegradable packaging.[41]
given below.[15]
Pea Proteins
Soybean proteins
It represents a 20-30% fraction extracted from pea seeds,
Soy proteins are a mixture of globular proteinsconglycinin
which includes mainly globulins(65-80%) and two
(140-170kDa with glycosylated three subunits) and Glycinin
minority fractions, albumins and glutelins. Globulins
(340-375kDa with six AB subunits comprised of an acidic
comprise of three different proteinslegumin, vicilin and
[A] and a basic[B] polypeptide linked via disulfide bonds) convicilin.[43] The 11S globulin fraction with a molar mass
and are obtained from the plant species Glycine max, family between 350 and 400kDa is represented by legumin, while
Fabaceae.[35] Based on the molecular weight and sedimentation the 7S globulin fraction with a molar mass of 150kDa is
coefficient, it separates into fractions 2S, 7S, 11S or 15S.[36] represented by vicilin and convicilin.[44]
The 7S globulin and 11S globulin comprise 37% and 31% of
the soy proteins, respectively. In combination with other Rice Proteins
filmforming proteins, glycinin is known as the gelling agent, During processing of white rice, rice bran is obtained, which
emulsifier and foaming agent.[37] conglycinin is less heat is a rich source of inexpensive highquality proteins obtained
stable than glycinin and gets denaturated at temperatures of from grain during the milling process.[45] Compared with
70C and 80C.[38] rice bran, the protein content in rice grains is slightly lower,
varying from 6% to 15%.[46] It is generally prepared by alkali
Wheat proteins extraction followed by isoelectric precipitation[47,48] and by
Based on solubility, the wheat protein fractions are classified subcritical water treatment.[49,50] After sequential extraction
as albumins(water soluble), globulins(dilute salt solutions of the rice protein fractions, the following distribution
soluble), gliadins(soluble in 70-90% ethanol, comprise has been obtained: About 75% glutenin, 15% globulin, 6%
34% of the total protein) and glutenin(insoluble under all albumin and 3% prolamin.[51] The foaming properties of rice
of the previously mentioned conditions, comprise 47% of protein are similar to those of albumin from egg white; the
the total protein).[39,40] Gliadin(40kDa) is a singlechained emulsifying capacities of albumin from bovine serum(BSA)
are significantly higher than those of rice proteins; minimum acids),[61] lactalbumin(14.2kDa), BSA(66kDa, longest
protein solubility is close to isoelectric point at pH4 and singlechain protein) and immunoglobins(thermolabile
maximum at pH10; main amino acid content of rice proteins mixture of proteins).[62,63]
is similar to that of casein and soy proteins; and denaturation
temperature of the rice protein isolate is about 83.4C.[44] Meat proteins
Sarcoplasmic, stromal and myofibrillar are types of meat
Sunflower Proteins protein. Sarcoplasmic proteins contain enzymes myoglobulin
Proteins are majority constituents in sunflower oil cakes. and cytoplasmic. Collagen and elastin are the content of
Defatted sunflower flour contains a high quantity of proteins, stromal proteins while myosin, actin, tropomysin and
around 27% in dry weight.[52] The dehulled seed consists troponins are the content of myofibrillar proteins. Stromal
of about 20-40% crude protein. Four fractions of protein and myofibrillar proteins, soluble in salt solutions, are used
are present in the sunflower protein:[53] Globulins, 55-60%; for making edible films and coatings. Collagen, a fibrous
albumins, 17-23% of total proteins; and two minor fractions, stromal protein extracted from connective tissue, tendons,
glutelins and prolamins, comprising 11-17% and 1-4% of skin, bones and the vascular system, and is a waste products of
the total protein fractions, respectively. It shows two major meat processing. Collagen is a superhelical structure formed
fractions: 11S globulins(also named helianthinin) and 2S by a combination of three parallel alfachains, and forms
albumins. Helianthinin has been reported to be present as gelatine.[64] Collagen exposed to mild heat treatment under
a globular oligomeric protein with a molecular weight of acidic or alkaline conditions forms gelatin.[65]
300-350kDa,[54] and this protein mainly exists in the 11S
hexametric form. Egg albumin
Protein fractions: Ovalbumin(44.5kDa, contains free
Animal proteins sulfhydryl groups for crosslinking); ovotransferrin(77.7kDa,
Protein from animal sources contains essential amino acids ironbinding protein); ovomucoid, ovomucin and
needed for an adults diet. The important examples are as lysozyme, a gram negative antimicrobial present in
given below. albumin.[66] Ovalbumin, ovotransferrin and lysozyme protein
on denaturation form strong intermolecular betasheet
Casein structures as heatset gels due to their thermolabile nature.
There are four main subunits: s1 casein(23.6kDa, 4.94 a pI,
net charge21.9 at a pH of 6.6), s2 casein(net charge13.8, Silk proteins
5.37 pI, hydrophilic due to highcharge density), casein(polar The silkworm Bombyx mori produces silk to weave its cocoon.
Nterminal amphipathic protein with large hydrophobic Biocompatibility, slow biodegradability, selfassembly,
domain, Ca 2 + sensitive, at 4C solubility increases) and excellent mechanical properties, controllable structure and
casein(not Ca 2 + sensitive), which make up 38%, 10%, morphology make it promising materials for drug delivery
36% and 13% of the casein composition, respectively, and and tissue engineering.[67] Fibrous protein fibroin, a core of
has a unique property to form films.[5557] s1 casein is silk and a gluelike protein sericin that envelop fibroin in
amphipathic due to the charge between the hydrophobic cocoon formation, are major components of silk.[68]
Nand Cterminals. It has 8 phosphorylated serine clustered
with glutamine residues possessing calciumbinding sites and Isolation of proteins[69]
hence is Ca 2+sensitive, 17 proline, 25 glutamine residues Selective precipitation of proteins can be used as:
and no cysteine residues. Here, the Ca 2+sensitivity means 1. Bulk method to recover majority of the proteins
aggregation and precipitation in low ion concentrations. It does from a crude lysate
not participate in disulfide bond formation and crosslinking
2. Selective method to fractionate a subset of proteins
due to the absence of free cysteine. Caseins are heat stable
from a protein solution
because they are prolinerich, which interrupt alfahelix and
3. Specific method to recover a single protein of
beta strands, resulting in the absence of disulfide bridges in the
structure. It has relatively little secondary or tertiary structure. interest from a purification step.
These undergo proteolytic cleavage due their open structure
Selective precipitation methods
imparted due by the high proline content. This characteristic,
along with acidsoluble calciumphosphate bridging, makes 1. Salting out
an excellent targetactivated release mechanism for unloading 2. Isoionic precipitation
drug in the stomach.[58,59] 3. Organic cosolvent precipitation
4. Two carbon(C2) organic cosolvent precipitation
Whey protein of proteins
Technically, whey proteins are those that remain in milk 5. C4 and C5 organic cosolvent precipitation, phase
serum after coagulating caseins at 4.6 pH and 20C.[60] It partitioning and extraction of proteins
contain lactoglobulin(18.3kDa, containing 160 amino 6. Protein exclusion and crowding agents(neutral
The principal sample applied to the gel has been treated with Table5: Separation mechanism and technique
detergent SDS and mecaptoethanol. This will denature Property of proteins Technique
the proteins and the SDS binding tightly to the uncoiled
Diameter of molecule Size exclusion chromatography
molecule makes it negatively charged. SDSpolyacrylamide Electric charge of molecule Ionexchange chromatography
gel electrophoresis(PAGE) gel separates proteins primarily Presence of hydrophobic Reversed phase chromatography
according to size because SDScoated proteins have a uniform domains
charge: Mass ratio. Presence of hydrophobic Hydrophobic interaction
groups chromatography
Biospecific interaction Affinity chromatography
Onedimensional gel electrophoresis
This type of electrophoresis can provide information about
the molecular size and purity of the proteins as well as can be detected using Coomassie Blue staining. Because
the number and molecular size of its subunits. In PAGE, Coomassie Blue is predominantly nonpolar, it is usually
proteins migrate in response to an electrical field through used in methanolic solution and excess dye is removed from
pores in the gel matrix; the pore size decreases with higher the gel later by distaining. Stacking gels are usually discarded
acrylamide concentrations. The combination of gel pore size before staining the resolving gels. Gel slabs are placed in
and protein charge, size and shape determines the migration staining solution and are stained fully within 4-6h at room
rate of the protein. temperature if only 1.5mm thick.
1 g/l of the stock sample was prepared in 0.1%(v/v) soybean seeds accumulating novokinin[84]
TFA in water (buffer A) and filtered through a 0.22 m 3. As solubility enhancer of curcumin in the
filter if any undissolved material remained. Baseline was food industry due to proteinmicelle
obtained using 100% buffers with a flow rate of 1mL/min structure(betacasein), acting as a nano vehicle[85]
at 215nm. Ten microliters of the sample was injected using 4. As vehicles for bioactives, like milk proteins
a linear gradient from 0% to 100% buffer B: 100% CH3CN 5. As a source of bioactive peptides, e.g.,caseinderived
containing 0.1%(v/v) TFA over30min to elute the sample. four main bioactive peptides act on the
Ten microliters of MilliQ water was used as the blank. cardiovascular system, nervous system, immune
In the literature, the HPLC system with a reversedphase
system and nutrition system[85]
octadecylsilica(C18) column(4.6mm id[internal diameter]
6. As a novel antifungal, e.g.,Pisumin proteins
250mm length, 5 m particle size, 300 pore size, C18
guard column) had been used.[74]
obtained from Pisum sativum. Sugar snap pea
legumes.[86]
Mass spectrometry of proteins 7. In microencapsulation, e.g.,vegetable proteinssoy
Large and polar biomolecules are not easily ionized and proteins, pea proteins, wheat proteins, rice proteins,
transferred into the gas phase; hence, electrospray(ES) oat proteins and sunflower proteins[83]
and matrixassisted laser desorption ionization(MALDI) 8. As pest control: Proteinaceous cysteine proteinase
techniques are used. Mass spectrometry in proteomics inhibitor, an insecticidal protein found in pulses used
is used in three major areas.[78] to control the proteolytic activity of endogenous
1. R e c o m b i n a n t p r o t e i n s , m a c r o m o l e c u l e digestive cystein proteinase in the midgut of some
characterization and quality control in the field of insects.
biotechnology
2. Protein identification, either in classical biochemical REFERENCES
projects or in largescale proteomic ones
3. Detection and characterization of posttranslational 1. HermannJR.Protein and the Body.Oklahoma Cooperative
modifications or any method like any covalent Extension Service, Division of Agricultural Sciences and Natural
Resources. Oklahoma State University: T31631T31634.
modification that alters mass of a protein using
2. McArdleWD, KatchFI, KatchVL. Vitamins, minerals, and
versatility of it to detect the molecular weight of water, In: Exercise physiology: Energy, Nutrition, and Human
the protein.[79] Performance, 5thed. Philadelphia, PA: Lippincott Williams and
Wilkins; 2001. p.4781.
Liquid chromatographymass spectrometry and 3. MillwardDJ. The nutritional value of plant based diets in relation
tandem mass spectrometry of peptides and proteins to human amino acid and protein requirements. Proc Nutr Soc
1999a; 58:24960.
Protein and peptide tandem mass spectrometry involves
4. StreitwieserA, Jr. HeathcockCH. Introduction to organic
application of two separate stages: LCMS and LCMS/
chemistry 3rded., 1985.
MS, to solve problems that require protein identification
5. FurstP, StehleP. "What are the essential elements needed for
and software tools to identify proteins by matching mass the determination of amino acid requirements in humans? J
spectrometric data to protein sequence databases. [80,81] Nutr 2004;134(6Suppl):155865S.
Additionally, LCMS provides highquality data for peptide 6. SatyanarayanaU, ChakrapaniU. Books of Biochemistry. 3ed.,
mass as compared with MALDI mass spectrometry in 2006.
fingerprinting searches. Tessier etal. reported the prediction 7. BachmairA, FinleyD, VarshavskyA. Invivo halflife of a
of plant protein hydrolysate containing small peptide amino protein is a function of its aminoterminal residue. Science
1986;234:17986.
acids composition performed by LCMS and capillary
8. PaceCN, VajdosF, FeeL, GrimsleyG, GrayT. How to measure
electrophoresis-mass spectrometry.[82]
and predict the molar absorption coefficient of a protein. Protein
Sci 1995;11:241123.
Applications of proteins 9. EdelhochH. Spectroscopic determination of tryptophan and
Proteins are used in drug and gene delivery systems tyrosine in proteins. Biochemistry 1967;6:194854.
as proteinbased nanocarriers. Extended applications 10. GillSC, von HippelPH. Calculation of protein extinction
include use in controlled delivery, as a film coater, coefficients from amino acid sequence data. Anal Biochem
1989;182:31926.
as hydrogels, as composites, as albuminbased
11. IkaiA. Thermostability and aliphatic index of globular proteins.
nanoparticles, as microparticles and as beads. Some
JBiochem 1980;88:18958.
examples are:
12. KyteJ. DoolittleRF. Asimple method for displaying the
1. Whey proteins used as hydrogels, nanoparticle hydropathic character of a protein. JMol Biol 1982;157:10532.
systems for encapsulation and controlled delivery 13. AltermanMA, HunzikerP. Methods in Molecular Biology, Amino
of bioactive compounds[83] Acid Analysis, Methods and Protocols, 2012.
2. As antihypertensive use, like genetically modified 14. CreightonTE.Proteins: Structures and molecular
29. DucelV, RichardJ, PopineauY, BouryF. Rheological interfacial 50. ChandiGK, SogiDS. Functional properties of rice bran protein
properties of plant protein Arabic gum coacervates at the concentrate. JFood Eng 2007;79:5927.
oilwater interface. Biomacromolecules 2005;6:7906. 51. AgboolaS, NgD, MillsD. Characterisation and functional
30. EzpeletaI, IracheJM, StainmesseS, ChabenatC, GueguenJ, properties of Australian rice protein isolates. JCereal Sci
PopineauY, etal. Gliadin nanoparticles for the controlled release 2005;41:28390.
of alltransretinoic acid. Int J Pharm 1996b; 131:191200. 52. OrdonezC, AsenjoMG, BenitezJL, GonzalezJL. Obtaining
31. IwamiK, HattoriM, NakataniS, IbukiF. Spray dried gliadin a protein concentrate from integral deffated sunflower flour.
powders inclusive of linoleic acid(microcapsules): Their Bioresour Technol 2001;78:18790.
preservability, digestibility and application to bread making. Agric 53. GonzalezPerezS, VereijkenJM. Sunflower proteins: Overview
Biol Chem 1987;51:33017. of their physicochemical, structural and functional properties.
32. Mauguet MC, Legrand J, Brujes L, Carnelle G, Larre C, JSci Food Agric 2007;87:217391.
Popineau Y. Gliadin Matrices for microencapsulation processes 54. LindenGL. Biochimie agroindustrielle: Valorisation alimentaire
by simple coacervation method. JMicroencapsul 2002;19:37784. de la production agricole. Masson, Paris. 1994.
33. YuJY, LeeWC. Microencapsulation of pyrrolnitrin from 55. Audic JL, Chaufer B, Daufin G. Nonfood applications of
Pseudomonas cepacia using gluten and casein. JFerment milk components and dairy coproducts: Areview. Lair
Bioeng 1997;84:4448. 2003;83:41738.
34. The Wealth of India Raw Material Series, National institute of 56. SwaisgoodHE. Review and update of casein chemistry. JDairy
Science Communication and Information Resources, CSIR, Sci 1993;76:305461.
57. SwaisgoodHE. Characteristics of milk. In: FennemaO, editor. 74. AguilarMI, HearnMT. High resolution reversed phase high
Food chemistry. NewYork: Marcel Dekker; 1996. p.1067. performance liquid chromatography of peptides and proteins.
58. RosembergM, YoungSL. Whey proteins as microencapsulating Methods Enzymol 1996;270:326.
agents. Microencapsulation of anhydrous milk fat structure 75. MantCT, HodgesRS. Analysis of peptides by high performance
evaluation. Food Struct 1993;12:3141. liquid chromatography. Methods Enzymol 1996;271:350.
59. LivneyYD. Milk proteins as vehicles for bioactives. Curr Opin 76. PurcellAW, AguilarMI, HearnMT. Conformational effects in the
Colloid Interface Sci 2010;15:7383. RPHPLC of polypeptides. II: The role of insulin A and B chains in the
60. MorrCV, HaEY. Whey protein concentrates and isolates: chromatographic behavior of insulin. JChromatogr 1995;711:719.
Processing and functional properties. Crit Rev Food Sci Nutr 77. LinS, KargerBL. Reversed phase chromatographic behavior of
1993;33:43176. proteins in different unfolded states. JChromatogr 1990;499:89102.
61. DeWitJN, KlarenbeekG. Effects of various heat treatments 78. MannM, HendricksonRC, PandeyA. Analysis of proteins
on structure and solubility of whey proteins. JDairy Sci and proteomes by mass spectrometry. Annu Rev Biochem
1983;67:270110. 2001;70:43773.
62. KinsellaJE. Milk proteins: Physicochemical and functional 79. YatesJR. Database searching using mass spectrometry data.
properties. Crit Rev Food Sci Nutr 1984;21:197262. Electrophoresis 1998;19:893900.
63. SawyerL, KontopidisG, WuSY. Betalactoglobulin: 80. TessierB, SchweizerM, FournierF, FramboisierI, ChevalotR,
A threedimensional perspective. Int J Food Sci Tech VanderesseC. Harscoat,I. Marc, Prediction of the amino
1999;34:40918. acid composition of small peptides contained in aplant
64. HaugIJ, DragetKI, SmidsrodO. Physical and rheological proteinhydrolysate byLCMSand CEMS. Food Res Int
properties of fish gelatin compared to mammalian gelatin. Food 2005;38:57784.
Hydrocoll 2004;18:20313. 81. ElzoghbyAO, Abo ElFotohWS, ElgindyNA. Caseinbased
65. BadiiF, HowellNK. Fish gelatine: Structure, gelling properties formulations as promising controlled release drug delivery
and interaction with egg albumen proteins. Food Hydrocoll systems. JControl Release 2011;153:20616.
2006;20:63040. 82. ElzoghbyAO, SamyWM, ElgindyNA. Albuminbased
66. MineY. Recent advances in the understanding of egg white nanoparticles as potential controlled release drug delivery
protein functionality. Trends Food Sci Technol 1995;6:22532. systems. JControl Release 2012;157:16882.
67. NumataK, KaplanDL. Silkbased delivery systems of bioactive 83. YukoY, KeitoN, MegumiY, HuiZ, KunihikoO, MasayoshiT,
molecules, Adv Drug Deliv Rev 2010;62:1497508. etal. Antihypertensive activity of genetically modified soybean
seeds accumulating novokinin. Peptides 2008;29:3317.
68. AltmanGH, DiazF, JakubaC, CalabroT, HoranRL, ChenJ,
etal. Silkbased biomaterials. Biomaterials 2003;24:40116. 84. EsmailiM, GhaffariMS, MoosaviMovahediZ, AtriMS,
SharizadehA, FarhadiM, etal. Beta caseinmicelle as a nano
69. RothsteinF. Differential precipitation of proteins. In protein vehicle for solubility enhancement of curcumin: Food industry
purification process Engineering, HarrisonRG, editor. NewYork: application in LWT. Food Sci Technol 2011;44:216672.
Marcel Dekker; 1994. p.115208.
85. SilvaSV, Malcata FX. Caseins as source of bioactive peptides.
70. TanfordC. Multiple equilibria. In Physical Chemistry of Int Dairy J 2005;15:115.
Macromolecules. NewYork: John Wiley and Sons; 1996. p.5614.
86. YeXY, NgTB. Isolation of pisumin, a novel antifungal protein
71. EdsallJT, WymanI. Biophysical Chemistry. NewYork: Academic from legumes of the sugar snap pea Pisum sativum. Comp
Press; 1958. p.591662. Biochem Physiol PartC Toxicol Pharmacol 2003;134:23540.
72. HamesBD, RickwoodD. Gel electrophoresis of proteins:
Apractical approach. 2nded. NewYork: Oxford University Press; How to cite this Article: Nehete JY, Bhambar RS, Narkhede MR,
1990. Gawali SR. Natural proteins: Sources, isolation, characterization
73. AguilarMI. Methods in Molecular Biology. Vol.251, HPLC of and applications. Phcog Rev 2013;7:107-16.
Peptides and Proteins: Methods and Protocols. Totowa, NJ:
Source of Support: Nil, Conflict of Interest: None declared
Humana Press Inc.; 1996.