Characterization of Seed Storage Protein Pattern of Amaranthus viridis: SDS-PAGE Study

Amaranthus viridis (AV) belongs to Amaranthaceous family. AV is commonly known as green amaranth or karund, is a common gene, found in many parts of the world. Greek people called it as “never fading flower”. In many parts of the world, Amaranthus has been rediscovered as leafy vegetables, cereals etc. [1,2]. It is characterized by a high degree of diversity and a wide spectrum of adaptability to different agroecological conditions [3]. Being very cheap and widely distributed Amaranth vegetable is very popular in rural areas because it is very nutritious and provides essential nutrition to them and also prevention of diseases associated with nutritional deficiencies, such as blindness due to vitamin A deficiency [4]. Amaranthus has received considerable attention in many countries because of the high nutritional value of some species [5]. The protein of AV includes albumins, globulins, prolamines and glutelins; are of good digestibility nature [6,7]. Amaranthus species have different centers of domestication and origin, being widely distributed in North America, Central America and South American Andes, where the greatest genetic diversity is found [8,9]. It is estimated that there are 87 species of Amaranthus, 17 in Europe, 14 in Australia and 56 in America [10].


Introduction
Amaranthus viridis (AV) belongs to Amaranthaceous family. AV is commonly known as green amaranth or karund, is a common gene, found in many parts of the world. Greek people called it as "never fading flower". In many parts of the world, Amaranthus has been rediscovered as leafy vegetables, cereals etc. [1,2]. It is characterized by a high degree of diversity and a wide spectrum of adaptability to different agroecological conditions [3]. Being very cheap and widely distributed Amaranth vegetable is very popular in rural areas because it is very nutritious and provides essential nutrition to them and also prevention of diseases associated with nutritional deficiencies, such as blindness due to vitamin A deficiency [4]. Amaranthus has received considerable attention in many countries because of the high nutritional value of some species [5]. The protein of AV includes albumins, globulins, prolamines and glutelins; are of good digestibility nature [6,7]. Amaranthus species have different centers of domestication and origin, being widely distributed in North America, Central America and South American Andes, where the greatest genetic diversity is found [8,9]. It is estimated that there are 87 species of Amaranthus, 17 in Europe, 14 in Australia and 56 in America [10].
Proteins of amaranth have better balanced amino acid composition and thus higher biological values than those of most cere als. They can safely be consumed by people suffering from celiac disease and more sustainable than those from animal sources [11][12][13][14]. There is currently much interest in the use of pseudo cereals for developing nutritious food products. They contain high levels of starch, protein, dietary fibers, minerals, vitamins and other bioactive. Their proteins have well balanced amino acid composition. Pseudo cereal protein mainly consists of albumins and globulins, the predominant cereal proteins are prolamins and glutelin's [15]. Amaranthus is classified as pseudo cereals which in turn good food crop too because it has exceptionally good nutritive values owing to its protein, lipids and essential amino acids like lysine contents [16]. Understanding of the function of biological systems requires knowledge of their chemical composition. The state of life of a cell at any given time is defined by its protein composition, i.e. its proteome.
That is why it is necessary to have a rapid and an efficient tool for characterization to the proteome [17]. There are several ways of protein identification, however majority of them are very time-consuming procedures. Separation techniques became important tools for proteins and proteome analysis [18]. These methods have been successfully applied for the identification of proteins in numerous biological system [19,20]. The principal energy sources within the grains are protected from infestation by outer coverings. The largest morphological component of all grains is the endosperm and approximately 80% of this is starch. Other important compound classes are the storage proteins, which makes the next largest contribution to endosperm dry weight. Proteins are important both as nutrients and as active chemicals [17].
There are four categories of proteins occurring in seeds: a.
Albumins-soluble in water and comprise mostly enzymatic proteins. b.
Globulins-soluble in dilute salt solutions and occur in protein bodies. c.
Prolamins-soluble in aqueous ethanol solution and are also found in protein bodies as true storage proteins. d.
Glutelin's-soluble in alkaline or acid solutions or in detergents and are probably mainly structural proteins. Thus, the purpose of this study was to determine the amounts of albumins, globulins, prolamins and glutelin present in the proteins of AV grains.

Protein Extraction
The seeds of AV were collected and grounded for flour. The defatted flour was separated by centrifugation and then air dried for 2 days at room temperature and finally stored at 4° C until used. Total protein was estimated according to the Lowry's method [21]. Sequential extraction was performed according to the method of Sammour [22] with minor modifications. Amaranthus flour (1g) was extracted consecutively in four different solvents. First, the flour sample was extracted with distilled water (10ml, w/v). The suspension was stirred at room temperature for 20 minutes and then centrifuged at 8,000 rpm for 20 minutes. The supernatant was used as the extract 1 (Albumin fraction). The remaining insoluble sample was mixed with aqueous 5% (w/v) NaCl (10ml) solution, with the repetition of extraction procedure and the extract 2 was collected as globulin fraction. Subsequently extractions were followed with aqueous 70% (v/v) ethanol and aqueous 0.2% NaOH solution, the extract 3 as prolamin fraction and the extract 4 as glutelin fraction were obtained.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE]
All gels were run in minilabs; (Bangalore Genie Vertical mini gel system). SDS-PAGE was carried out according to the method of Laemmle [23]. The runs were carried out in the following discontinuous buffer system: 0.5 M Tris-HCl pH 6.8 (4x stacking gel buffer), 1.5 M Tris-HCl at pH 8.8 (4x separating or resolving gel buffer) and 0.025 M Tris-HCl, 0.192 M glycine/1% (w/v) SDS, pH 8.3 for the running buffer. The protein samples (100 μl) were dissolved in the 100 μl of loading buffer (0.5 M Tris-HCl pH 6.8, 20% (v/v) glycerol, 1% (w/v) SDS/0.05 % (w/v) bromophenol blue and centrifuged at 5,000 rpm for 5 minutes: 25-30 μl/lane supernatants were loaded in the gel. The electrophoretic runs were conducted for about 3-4 hours at a constant voltage of 80 V. The gels were stained with 0.5% R-250 Coomassie Brilliant Blue in water/methanol/acetic acid (4:5:1) for overnight and detained with water: methanol: acetic acid (9:9:1). The molecular weights of polypeptides were calculated by using the protein standard molecular marker.

Results & Discussion
Amaranth seeds flour was successively extracted with distilled water (albumin), NaCl (glutelin), ethanol (prolamin) and an alkaline solution (glutelin). The concentration of proteins extract was determined by the method of Lowry et al. [21]. According to the results, the amount of total protein was 14.9% of seed flour and the content of albumin, globulin, prolamin and glutelin were 3.61, 3.56, 0.64 and 6.62 g 100g-1seed flour respectively. The content of glutelin was highest in comparison to all the protein fractions and it was approximately 10 folds higher than prolamin, which was lowest in total protein. In SDS-PAGE, the amaranth seed flour protein extracted with distilled water (albumin), showed the dominant bands of low molecular weight of <32KDa (Figure 1 Lane 1) and some components of intermediate molecular mass between 43-70KDa in agreement with our previous studies [5,7]. In globulins (

Conclusion
Storage proteins accounts for about 50% of the total protein in mature cereal grains and have important impact on their nutritional quality for humans and livestock and on their functional properties in food processing [24]. The storage proteins of grains are of immense importance in determining the quality and end use properties of the grain. Understanding the structures of these proteins is important to improve the quality of grains. Though plant has been extensively studied for its nutritional properties, but more experimental and clinical trials are warranted.