Proteomic Quantitative UV Absorption Spectrum Analysis of Effect of Heat Stress on Protein Extract from Cowpea Seed (Vigna unguiculata (L) Walp)

Aim: Proteomic quantitative UV absorption spectrum analysis was used to study the effect of heat stress on protein extract from cowpea seeds (Vigna unguiculata (L) Walp). Study Design: Protein extracts were obtained from 9 cowpea accessions obtained from GeneBank of International Institute of Tropical Agriculture in January 2014. Each protein extract was divided into four batches out of which three batches were subjected to different temperature treatments and incubation at 37°C, 60°C and 100°C for 1 hour and the remaining one batch served as control. Protein content in each control protein extract and 37°C, 60°C and 100°C treated protein extracts Original Research Article Onasanya et al.; BBJ, 8(1): 1-18, 2015; Article no.BBJ.18429 2 from each of the 9 cowpea samples were determined at 280 nm using bovine serum albumin standard curve. Place and Duration of Study: Biochemistry Unit, Department of Chemical Sciences, Afe Babalola University Ado Ekiti, Nigeria between January 2014 and June 2014. Methodology: A200-A960 UV wavelengths absorption spectrum analysis was carried out on control protein extract and 37°C, 60°C and 100°C treated protein extracts respectively from each of the 9 cowpea samples. In order to establish the relationship between protein extracts (control) and protein extracts heat treated (37°C, 60°C, and 100°C), cluster analysis of optical density (OD) data was carried out using numerical taxonomy and multivariate analysis system. Results: The protein content (control) in Vigna unguiculata (L) Walp was between 8.4 and 10.8 mg/ml (10.5-13.5%) in seed, while protein content (heat treated) in Vigna unguiculata (L) Walp was between 8.9 and 9.5 mg/ml (11.2-11.9%), 8.7-9.5 mg/ml (10.9-11.9%), 9.0 and 11.8 mg/ml (11.314.7 %) in heat treatments of 37°C, 60°C, and 100°C respectively. The protein UV absorption spectra of control protein extract and 37°C, 60°C and 100°C treated protein extracts from each cowpea accession were generally different due to differential UV wavelength protein absorption. Cluster analysis of absorbance spectra optical density values revealed five clusters (cluster 1, cluster 2, cluster 3, cluster 4, and cluster 5) among control protein extracts and protein extracts heated at 37°C, 60°C, and 100°C. Cluster1 was made up of protein extracts heated at 37°C and 60°C, while cluster2 and cluster3 constituted closely related protein extracts heated at 37°C and 100°C respectively. Cluster4 was typical of control protein extracts, while cluster5 was made up of distinct protein extracts heated at 100°C. Conclusion: Heating protein extracts at 37°C, 60°C, and 100oC has altered proteomic diversity in different cowpea accessions and this could make protein extraction more difficult with implications on protein properties.


INTRODUCTION
Cowpea [Vigna unguiculata (L.) Walp.] is an important crop in many countries of tropical Africa, Asia and South America. It is one of the most important food legume crop grown in semiarid tropics covering Africa, Latin America, West Indies, India, South-east Asia and the southern United States [1] with an annual production of more than five million metric tons world-wide [2]. West Africa produces about 80% of the world's total cowpea production, with Nigeria, Niger and Senegal as the principal producers [3]. In Nigeria, the grain legume is mostly grown in the drier climate of the North than in the humid climate of the South, where high humidity causes diseases and drying problems [4].
Cowpea grains and leaves are edible products that are rich and cheap sources of high-quality protein. They supplement to the lower quality cereal or root and tuber protein commonly consumed in tropical Africa [5,6]. Cowpeas are mainly consumed as a favorite food-stuff in the form of dried seeds, either as flour or split [7,8]. They are a good source of carbohydrates, vitamins, and protein, providing more than half of plant protein in human diets in some areas of the semi-arid tropics [9,10]. On average cowpea grains contain 23-25% protein and 50-67% starch in dry bases [11]. The seeds make up the largest contributor to the overall protein intake of several rural and urban families hence [12] regarded cowpea as the poor man's major source of protein. Their amino acid complements those of cereals [13][14][15]. Cowpea is a most versatile African crop, it feeds people, their livestock and because of its ability in nitrogen fixation, it improves soil fertility, and consequently helps to increase the yields of cereal crops when grown in rotation and contributes to the sustainability of cropping systems [16].
Cowpea [Vigna unguiculata (L) Walp.] is one of the important leguminous crops, which belongs to the family Leguminosae, subfamily Faboideae, tribe Phaseoleae, genus Vigna, subgenus Ceratotropis and species unguiculata [17]. It is subdivided into four cultivar groups (cv. gr.): unguiculata (the common cowpea), biflora (the catjang), sesquipedalis (the yard-long bean) and textilis (used for fibers) [18,9]. It is primarily originated in West Africa [19]. It has a number of common names which includes crowder pea, black eyed pea and southern pea [20]. It is generally called beans in Nigeria.
Apart from its protein content (about 25%), cowpea also contains some vitamins and minerals and it's also an excellent source of fibre, calcium, folic acid and vitamin A [21]. As with most legumes, they are poor in some of the sulphur-containing amino acids needed for a complete protein. Several methods are commonly used for the determination of protein concentration. Each of these methods depends on the source of the protein, amount of protein present, the specificity of the method, the presence of interfering substances, and the amino acid composition of the protein. Most of these methods rely either on colorimetric assays or the use of UV absorbance spectroscopy [22]. A number of methods have been devised to measure protein concentration, which are based on UV-visible spectroscopy. These methods use either the natural ability of proteins to absorb (or scatter) light in the UV-visible region of the electromagnetic spectrum, or they chemically or physically modify proteins to make them absorb (or scatter) light in this region. The basic principle behind each of these tests is similar. First of all a calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve. The main difference between the tests are the chemical groups which are responsible for the absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and aggregated proteins.
Temperature treatments include the use of freeze-thaw and heat treatments. Freezethawing uses the effect of ice crystal formation in the tissue during the freezing process. Lysis of the cells or tissues is usually achieved by flashfreezing the cells in liquid nitrogen and homogenizing in a mortar with a pestle. Examples of this process are found in the analysis of leaves [23], fruits [24], and seeds [25][26][27]. [28] developed a very efficient cell disruption method for grape berry clusters, which were pulverized frozen with dry ice using a stainless steel blender. The use of heat is common in protein processing. Heating protein solutions usually improves their solubility, emulsifying, and foaming properties, but it makes protein extraction more difficult and alter proteomic diversity in grains [29,30,27]. The aim of the present study was to compare the effect of heat stress at various temperatures on protein extract from seeds of Vigna unguiculata (L) Walp accessions using UV absorption spectrum analysis.

Vigna unguiculata (L) Walp Seed
The Vigna unguiculata (L) Walp seeds used in this study were obtained from International Institute of Tropical Agriculture (IITA), Ibadan in January 2014.

Protein Extraction
A total of 9 seed samples (Table 1) were used for protein extraction. The protein extraction procedure used was according to Barbarino and Lourenco [31] and Onasanya et al. [27]. 1 g of seed was grinded in 5 ml of 0.1 M NaOH using mortar and pestle. The homogenate was transferred into 2.5 ml Eppendorf tubes and incubated at room temperature for 12 hours. After incubation, the homogenate was centrifuged at 4ºC at 12,000 rpm for 20 min using refrigerated centrifuge. The supernatant was collected as crude protein extract. The collected protein extract was stored at 4ºC for use in heat treatment and UV absorbance spectrum analyses.

Heat Treatment of Protein Extract
The procedure used for heat treatment was according to Hiller et al. [32] and Rees and Robertson [33] with slight modification. The protein extracts were divided into four batches out of which three batches were was subjected to different temperature treatments and incubation at 37ºC, 60ºC and 100ºC respectively for 1 hour and the remaining one batch served as control ( Table 2).

UV Absorption Spectrum Analysis of Protein Extract
The procedure used for UV absorption spectrum analysis of protein extracts was according to Onasanya et al. [27] and Grimsley and Pace [34]. 0.4 ml of crude protein extract was added to 1.6 ml of 0.1 M NaOH, mixed well, and the optical density (OD) absorbance value was taken from A 200 -A 960 UV wavelengths using Spectronic 20 spectrophotometer. All the 36 protein samples were analyzed (Table 2).

Graphical Analysis of Optical Density (OD) Data
The procedure used for the graphical analysis of optical density (OD) data was according to Mattley and Garcia-Rubio [35] and Pavokovic et al. [36]. A Standard Curve was prepared by plotting the optical density (OD) absorbance valuesatA 280 UV wavelengths against the concentrations of protein standard (bovine serum albumin). The concentration of each protein extracts was obtained using the linear equation generated from the protein Standard Curve. A plot of optical density (OD) absorbance values against A 200 -A 960 UV wavelengths was also carried out to generate a combined UV absorbance spectra profile for protein extracts (control) and protein extracts heat treatments (37ºC, 60ºC, and 100ºC).

Cluster Analysis of Optical Density (OD) Data
In order to establish the relationship between protein extracts (control) and protein extracts heat treated (37ºC, 60ºC, and 100ºC), cluster analysis of optical density (OD) data was carried out using numerical taxonomy and multivariate analysis system (NTSYS-PC), version 2.1 [37]. OD data were first converted to pairwise distance matrices using the Jaccard coefficient of similarity (Jaccard, 1908) present in NTSYS-PC 2.1 and dendrogram cluster was created by Unweighted Pair Group Method with Arithmetic mean (UPGMA) cluster analysis [38].

Comparison of Absorbance Spectra of Protein with Different Heat Treatments in TVu -8775
Difference between the absorbance value of protein with different heat treatments in TVu-8775 was compared. TVu-8775 protein extract heated at 100ºC (V2-100C) has more protein concentration at the wavelength of 280 nm than in the control (V2), protein extract heated at 37ºC (V2-37C), and protein extract heated at 60ºC (V2-60C) respectively (Fig. 3). Other protein molecules are found along other wavelengths (300-960) at even higher concentrations in TVu-8775 protein extract heated at 100ºC (V2-100C) than in the control (V2), protein extract heated at 37ºC (V2-37C), and protein extract heated at 60ºC (V2-60C) respectively (Fig. 3). Comparing absorbance spectra of control and heat treated protein extracts, TVu-8775 protein extract heated at 100ºC (V2-100C) has two peaks at 300 nm and 460 nm respectively with maximum absorbance peak at 300 nm, both the control (V2) and protein extracts heated at 37ºC (V2-37C) have two peaks at 300 nm and 440 nm respectively with maximum absorbance peak at 300 nm, while protein extract heated at 60ºC (V2-60C) has two peaks at 320 nm and 440 nm respectively with maximum absorbance peak at 440 nm (Fig. 3).

Comparison of Absorbance spectra of Protein with Different Heat Treatments in TVu-7846
Comparative studies revealed that TVu-7846 protein with different heat treatments showed difference in protein concentration across the same wavelength of 280 nm and at different wavelengths, other protein molecules with even higher protein concentration are present (Fig. 4). The protein concentration in TVu-7846 protein extract heated at 100ºC (V3-100C) at the wavelength of 280 nm was higher than in the control (V3), protein extract heated at 37ºC (V3-37C), and protein extract heated at 60ºC (V3-60C) respectively (Fig.  4). Comparing absorbance spectra of control and heat treated protein extracts, both the control (V3) and TVu-7846 protein extract heated at 100ºC (V3-100C) have two peaks at 300 nm and 460 nm respectively with maximum absorbance peak at 300 nm, while both protein extracts heated at 37ºC (V3-37C) and at 60ºC (V3-60C) have two peaks at 300 nm and 440 nm respectively with maximum absorbance peak at 300 nm (Fig. 4).

Comparison of Absorbance Spectra of Protein with Different Heat Treatments in TVu-586
Given the comparison of absorbance spectra of TVu-586 protein with different heat treatments, it was observed that the absorbance value of the protein extract heated at 37ºC (V6-37C) has it maximum absorbance at 460 nm unlike the protein absorbance in the protein extract control (V6), protein extract heated at 60ºC (V6-60C), and protein extract heated at 100ºC (V6-100C) respectively (Fig. 7). Comparing absorbance spectra of TVu-586 protein with different heat treatments, protein extract control (V6) has three peaks at 320 nm, 440 nm and 480 nm, while protein extract heated at 37ºC (V6-37C) produced two peaks at 320 nm and 460 nm. However, protein extract heated at 60ºC (V6-60C) has three peaks at 320 nm, 440 nm and 480 nm, while protein extract heated at 100ºC (V6-100C) produced four peaks at 320 nm, 440 nm, 480 nm and 560 nm respectively (Fig. 7).

Comparison of Absorbance Spectra of Protein with Different Heat Treatments in TVu -3947
Given the comparison of absorbance spectra of TVu-3947 protein with different heat treatments, it was observed that the absorbance value of the protein extract heated at 37ºC (V8-37C) has it maximum absorbance at 420 nm unlike the protein absorbance in the protein extract control (V8), protein extract heated at 60ºC (V8-60C), and protein extract heated at 100ºC (V8-100C) respectively (Fig. 9). Comparing absorbance spectra of TVu-3947 protein with different heat treatments, protein extract control (V8) has one peak at 320 nm, while protein extract heated at 37ºC (V8-37C) produced two peaks at 320 nm and 420 nm. However, protein extract heated at 60ºC (V8-60C) has two peaks at 320 nm and 440 nm, while protein extract heated at 100ºC (V8-100C) produced four peaks at 320 nm, 440 nm, 480 nm and 560 nm respectively (Fig. 9).

Cluster Analysis Comparison of Absorbance Spectra Optical Density Values of Protein with Different Heat Treatments
Cluster analysis of absorbance spectra optical density values revealed remarkable differences in the form of clusters among control protein extracts and protein extracts heated at 37ºC, 60ºC, and 100ºC respectively (Fig. 11). Five clusters (cluster1, cluster2, cluster3, cluster4, cluster5) have been identified among control protein extracts and protein extracts heated at 37ºC, 60ºC, and 100ºC. Cluster1 was made up of protein extracts heated at 37ºC and 60ºC, while cluster2 and cluster3 constituted closely related protein extracts heated at 37ºC and 100ºC respectively. Cluster4 was typical of control protein extracts, while cluster5 was made up of distinct protein extracts heated at 100ºC (Fig. 11).

DISCUSSION
Quantitative UV absorption spectrum proteomic study is widely used in biochemistry and molecular biology for quantification of protein extracts from different sources including prokaryotic and eukaryotic cells [39]. The present study used proteomic analysis to study the effect of heat stress on protein extract from cowpea seeds (Vigna unguiculata (L) Walp). However, the present results showed that protein content in Vigna unguiculata (L) Walp seed was between 10.5-13.5% as compared to 6.6-16.9% previously reported [27]. Protein variability in cowpea population could be as a result of exposures of cowpea seeds to different temperature [29,30]. Previous studies have grouped cowpea varieties on the basis of protein content as high protein lines (above 30%), medium protein lines (20 to 30%), and low protein lines (less than 20%) [40,41]. In the present study, protein extracts exposed to different temperatures (37ºC, 60ºC, 100ºC) gave different protein contents (11.2-11.9%, 10.9-11.9%, 11.3-14.7%) which revealed some levels of protein variability in different cowpea accessions used. In addition, protein content of different accessions of Vigna unguiculata (L) Walp seeds tested in this study varied greatly and could possibly depending on different levels of heat tolerance among different chemical composition of the seeds and, in theory, this may lead to differences in the protein content and adsorption spectra [42, 29,30].
Large amounts of information are usually generated from single multiwavelength measurement which consequently makes UV absorption spectrum proteomic assay a powerful characterization tool that has several applications [39,43]. The sample information contained in a typical multiwavelength UV spectrum includes cell size, chemical composition and shape [39]. This information is obtained from the spectroscopic analysis of a sample measured over a broad range of wavelengths (200 -900 nm) [43]. In the present study, absorbance spectra between 200-960 nm UV wavelengths have been used to compare protein content from protein extracts from Vigna unguiculata (L) Walp heated at 37ºC, 60ºC, and 100ºC. Within the same Vigna unguiculata (L) Walp accession difference protein absorbance spectra were obtained from control protein extract and protein extract heated at 37ºC, 60ºC, and 100ºC. For examples, protein extract from TVu-702 heated at 37ºC have two peaks at 320 nm and 420 nm, another heated at 60ºC has two peaks at 320 nm and 440 nm, while those heated at 100ºC produced five peaks at 320 nm, 440 nm, 480 nm, 540 nm and 700 nm respectively. The difference in protein absorbance spectra could possibly be due to different levels of heat tolerance among protein chemical composition and shape [42,43,29,30] Cluster analysis comparison of protein multiwavelength absorbance spectra for the characterization of Vigna unguiculata (L) Walp revealed the sensitivity and discriminating power of the spectroscopic approach. Cluster analysis of absorbance spectra optical density values revealed five clusters (cluster1, cluster2, cluster3, cluster4, cluster5) among control protein extracts and protein extracts heated at 37ºC, 60ºC, and 100ºC. Heating protein extracts at 37ºC, 60ºC and 100ºC have altered proteomic diversity in different cowpea accessions and this could make protein extraction more difficult with implications on protein properties [29,30].

CONCLUSION
The present study used UV absorption spectrum proteomic assay to study the effect of heat stress of protein extract from cowpea seeds (Vigna unguiculata (L) Walp). The assay is widely used in biochemistry and molecular biology for quantification of protein extracts from different sources including prokaryotic and eukaryotic cells. Large amounts of information are usually generated from single multiwavelength measurement which consequently makes UV absorption spectrum proteomic assay a powerful characterization tool that has several applications. As shown in this study, cluster analysis comparison of protein multiwavelength absorbance spectra for the characterization of Vigna unguiculata (L) Walp revealed five clusters among control and heated protein extracts which further revealed the sensitivity and discriminating power of the spectroscopic approach. The ability to discriminate between crude protein content with different heat-treatment levels from cowpea have been demonstrated with the light scattering and absorption interpretation model and optical properties developed in our laboratory. Heating protein extracts at 37ºC, 60ºC, and 100ºC have altered proteomic diversity in different cowpea accessions and this could make protein extraction more difficult with implications on protein properties. With this level of detail in the analysis, the multiwavelength UV proteomic assay has the potential to provide a sensitive biosensor for the detection, identification and enumeration of various biomolecules. Future studies are highly recommended in the areas of developing the necessary interpretation models for more cowpea varieties and investigating the sensitivity of the approach.