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6. ESTIMATION OF PROTEIN BY HIGH PERFORMANCE

LIQUID CHROMATOGRAPHY (HPLC)

6.1 INTRODUCTION

Groundnuts make an important contribution to the diet in many countries.

Groundnut seeds are a good source of protein, lipid and fatty acids for human

nutrition (Grosso et al., 1999). Protein is a major macromolecular constituent of

cells, consisting of carbon, hydrogen, oxygen, nitrogen and usually sulphur and

phosphorous. A linear polymer of amino acids linked together by peptide bonds in

a specific sequence. In groundnut protein content was higher among valencias

(Nagaraj, 1990). The groundnut crop has high potential of protein built up through

subjected to variable conditions such as roots growth, factors and ontogenic

development. The protein contents are modified by photosynthetic surfaces, the

product of photosynthetic assimilatory reactions and biosynthetic reactions during

nitrogen fixations (Deshmukh and Dev, 2005).

Nitrogen being the constituent of protein, enhances the protein content in

grain which is the most important parameter of making quality (Tiwari et al.,

2008). The protein and starch ratio often influenced by moisture and temperature

(Deshmukh and Dev, 2005). Ghallab and Salem (2001) stated that in wheat plant,

growth characters and nutrients, sugar, amino acids and plant regulators as well as

crude protein contents of the tested plant increased by using biofertilizers.

High performance liquid chromatography (or) high pressure liquid

chromatography (HPLC) is a chromatographic technique that can separate a

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mixture of compounds and is used in biochemistry and analytical chemistry to

identify and purify the individual components of the mixture. Chromatographic

process can be defined as separation technique involving mass-transfer between

stationary and mobile phase. HPLC utilizes a liquid mobile phase to separate the

components of a mixture. The stationary phase can be a liquid or a solid phase.

These components are first dissolved in a solvent, and then forced to flow through a

chromatographic column under a high pressure. In the column, the mixture

separates into its components. The amount of resolution is important, and is

dependent upon the extent of defined as the immobile packing material in the

column. The interaction of the solute with mobile and stationary phases can be

manipulated through different choices of both solvents and stationary phases. As a

result, HPLC acquires a high degree of versality not found in other

chromatographic systems and it has the ability to easily separate a wide variety of

chemical mixtures.

Reversed HPLC is the most commonly used form of HPLC. Reversed-

phase high-performance liquid chromatography (RP-HPLC) involves the separation

of molecules on the basis of hydrophobicity. The separation depends on the

hydrophobic binding of the solute molecule from the mobile phase to the

immobilized hydrophobic ligands attached to the stationary phase, i.e., the sorbent.

The solute mixture is initially applied to the sorbent in the presence of aqueous

buffers, and the solutes are eluted by the addition of organic solvent to the mobile

phase. Elution can proceed either by isocratic conditions where the concentration

of organic solvent is constant, or by gradient elution whereby the amount of

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organic solvent is increased over a period of time. The solutes are, therefore, eluted

in order of increasing molecular hydrophobicity. RP-HPLC is a very powerful

technique for the analysis of peptides and proteins because of a number of factors

that include the excellent resolution that can be achieved under a wide range of

chromatographic conditions for very closely related molecules as well as

structurally quite distinct molecules. The experimental case with which

chromatographic selectivity can be manipulated through changes in mobile phase

characteristics and the excellent reproducibility of repetitive separations carried out

over a long period of time, which is caused partly by the stability of the absorbent

materials under a wide range of mobile phase conditions (Aguilar et al., 1996).

However, RP-HPLC can cause the irreversible denaturation of protein samples

thereby reducing the potential recovery of material in a biologically active form.

The RP-HPLC experimental system for the analysis of peptides and

proteins usually consists of an n-alkylsilica-based absorbent from which the solutes

are eluted with gradients of increasing concentrations of organic solvent such as

acetonitrile containing an ionic modifier such as trifluoroacetic and (TFA) (Mant

and Hodges, 1996). Complex mixtures of peptides and proteins can be routinely

separated and low picomolar-femtomolar amounts of material can be collected for

further characterization. Separations can be easily manipulated by changing the

gradient slope, the operating temperature, the ionic modifier, or the organic solvent

composition.

The extensive use of RP-HPLC for the purification of peptides, small

polypeptides with molecular weights up to 10,000 and related compounds of

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pharmaceutical interest has not been replicated to the same extent for larger

polypeptides (molecular mass > 10 KDa) and globular proteins. The combination

of the traditionally used acidic buffering systems and the hydrophobicity of the

n-alkylsilica supports which can result in low mass yield or the loss of biological

activity of larger polypeptides and proteins have often discouraged practitioners

from using RP-HPLC methods for large-scale protein separations. The loss of

enzymatic activity, the formation of multiple peaks for compositionally pure

samples, and poor yields of protein can all be attributed to the denaturation of

protein solutes during the separation process using RP-HPLC (Purcell et al., 1995).

RP-HPLC is extremely versatile for the isolation of peptides and proteins

from a wide variety of synthetic or biological sources and is used for both

analytical and preparative applications. Analytical applications range from the

assessment of purity of peptides following solid-phase peptide synthesis to the

analysis of tryptic maps of proteins. Preparative RP-HPLC is also used for the

micropurification of protein fragments for sequencing to large-scale purification of

synthetic peptides and recombinant proteins. The complexity of the mixture to be

chromatographed will depend on the nature of the source and the degree of

preliminary clean-up that can be performed. In the case of synthetic peptides,

RP-HPLC is generally employed both for the initial analysis and the final large-

scale purification. The purification of synthetic peptides usually involves an initial

separation on an analytical scale to assess the complexity of the mixture followed

by large-scale purification and collection of the target product. A sample of the

purified material can then be subjected to RP-HPLC analysis under the same or
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different elution conditions to check for purity. The isolation of proteins from a

biological cocktail derived from a tissue extract or biological fluid for example,

often requires a combination of techniques to produce a homogenous sample.

HPLC techniques are then introduced at the later stages following initial

precipitation, clarification, and preliminary separations using soft gels.

The challenge facing the scientist who wishes to analyse and/or purify their

peptide or protein sample by RP-HPLC is the selection of the initial separation

conditions and subsequent optimization of the appropriate experimental parameters.

This study was aims to determine the effects of biofertilizers using high

performance liquid chromatography and characteristics of seed protein patterns in

Arachis hypogaea L. var TMV-7 groundnut were observed through HPLC.

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6.2 MATERIALS AND METHODS

6.2.1. Estimation of protein (Dong, 2006)

Sample preparation

Desired samples are dissolved in specific solvent (HPLC Grade) and

filtered through the 0.22 micron filter. Collect the filtrate and degassed by using

sonicator for 50 times at 4ºC.

Solvent preparation

Solvent are prepared by desired concentration of Acetonitrile,water (65 :

35) respectively. Every solvents should be in HPLC grade. Otherwise use 0.22

micron filter.

Solvent are degassed by using Sonicator for 50 times at 4ºC.

Column equilibration.

Column equilibration using 65% Acetonitrile water until zero base line.

Sample injection

20 microlitre of sample injected to the injection head using injection needle.

Then set required time, wave length.

Purification profiles are seen on the screen, contains each compound with

its retention time.

Column specification

Reserve Phase HPLC (Cyberlab,USA)

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Column: C 18 (250 x 4.6 mm) Varian (Lake forest, CA, USDA)

Elution: Isocratic elution (65% acetonitrile water)

Absorbance at 280 nm

Reserve phase HPLC (Cyberlab, USA) analysis was carried out in a C 18

column (250 mm x 4.6 mm) Varian (Lake forest, CA, USA) equipped with a C18

guard column. The compounds were eluted with a Isocratic elution of Acetonitrile

Vs water at the flow rate 1 ml/min. The effluents were monitored by recording the

absorbance at 280 nm.

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6.3 RESULTS

The results of RP-HPLC of fraction of protein content in biofertilizers

inoculated groundnut seeds were shown in the figure 34- 41.

Fig. 34. RP-HPLC fraction of protein content in control.

Control (Fig. 34) showed the minimum number of fraction of protein

content when compared to mono, dual and combined biofertilizers inoculations.

The chromatogram characterized by the three peaks. Number of peaks indicate the

number of protein present. The main peak with retention time at 2.242 minutes and

the subpeaks with retention time at 2.087 and 2.429 minutes. The amount of

protein in the highest peak was 41%. The amount of protein was also less in the

highest peak of control.

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Fig. 35. RP-HPLC fraction of protein content in Azotobacter.

In mono-inoculations Azotobacter showed in the figure 35. The

chromatogram characterized by the three peaks. The main peak with retention time

at 2.235 minutes and the subpeaks with retention time at 2.085 and 2.494 minutes

and also the amount of protein in the highest peak was 47%.

In mono inoculations Azotobacter showed minimum amount of protein

when compared to other two mono inoculations.

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Fig. 36. RP-HPLC fraction of protein content in Mycorrhizae.

The Mycorrhizae showed in the figure 36. The chromatogram characterized

by the three peaks. The main peak with retention time at 2.235 minutes and the

subpeaks with retention time at 2.084 and 2.434 minutes. The amount of protein in

the highest peak was 48%. The amount of protein present in Mycorrhizae was in

between Azotobacter and Rhizobium.

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Fig. 37. RP-HPLC fraction of protein content in Rhizobium

The Rhizobium showed in the figure 37. The chromatogram characterized

by the three peaks. The main peak with retention time at 2.245 minutes and the

subpeaks with retention time at 2.087 and 2.450 minutes. In mono inoculations

Rhizobium showed the maximum amount of protein fraction. The amount of

protein in the highest peak was 48%.

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Fig. 38. RP-HPLC fraction of protein content in Mycorrhizae + Azotobacter.

In dual inoculations Mycorrhizae + Azotobacter showed in the figure 38.

The chromatogram characterized by three peaks. The main peak with retention

time at 2.237 minutes and subpeaks with retention time at 2.100 and 2.443 minutes.

The amount of protein in the highest peak was 51%. In this dual inoculation the

amount of protein in highest peak was high, because the number of protein fraction

were low.
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Fig. 39. RP-HPLC fraction of protein content in Azotobacter + Rhizobium.

The Azotobacter + Rhizobium showed in the figure 39. The chromatogram

characterized by three peaks. The main peak with retention time at 2.242 minutes

and subpeaks with retention time at 2.085 and 2.485 minutes. The amount of

protein in the highest peak was 46%. In dual inoculations, Azotobacter +

Rhizobium showed the amount of protein was in between Azotobacter +

Mycorrhizae and Rhizobium + Mycorrhizae.

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Fig. 40. RP-HPLC fraction of protein content in Rhizobium + Mycorrhizae

The Rhizobium + Mycorrhizae showed in the figure 40. The chromatogram

characterized by four peaks. The main peak with retention time at 2.242 minutes

and subpeaks with retention time at 1.977, 2.082 and 2.492 minutes. The amount

of protein in highest peak was 43%. In dual inoculations (Rhizobium +

Mycorrhizae) showed the maximum number of protein fraction. But the amount of

protein present in highest peak was less than other two dual inoculations, because

the number of protein present in this inoculation is more.

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Fig. 41. RP-HPLC fraction of protein content in Azotobacter + Mycorrhizae +

Rhizobium

The combined inoculation (Azotobacter + Mycorrhizae + Rhizobium)

showed in the figure 41. The chromatogram showed four peaks. The main peak

with retention time at 2.250 minutes and subpeaks with retention time at 2.105,

2.505 and 2.780 minutes (but it was possible to get five peaks) because when the

number of peak increases the protein value decreases. In all the inoculations the

combined inoculation showed the maximum number of protein fraction. The

amount of protein in the highest peak was 40%.

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6.4 DISCUSSION

The Reverse HPLC pattern of groundnut samples showed the biofertilizers

inoculated seeds had higher protein content than control. In the present study the

maximum protein content was observed in plants treated with combined

inoculation of biofertilizers when compared to mono and dual inoculations.

The combined effect of biofertilizers (Rhizobium + Farmyard manure +

Phosphate solublising bacteria) proved the best giving 39.86% grain protein in

soybean (Sharma and Namadeo, 1999a). Increase in protein content with

Rhizobium inoculation, nitrogen and phosphorus application might be due to

Rhizobium fixed atmosphere nitrogen, increased nitrogen content in seed as well as

in other plant parts. The application of nitrogen resulted in higher content of

leghaemoglobin, was a heme containing protein which produced the symbiotic

association of both the partners. Phosphorus increases fixed nitrogen in plant

(Singh et al., 2007).

Maximum protein content was obtained by conjoint use of 100 KgN/ha plus

Azotobacter inoculation in barley (Tiwari et al., 2008). Rhizobial inoculation also

increased a grain protein content in cowpea (Naik et al., 2007).

The maximum value of crude protein was obtained by application of

compost and biofertilizers in sweet marjoram (Gharib et al., 2008). The significant

increases in seed protein content due to bacterial inoculation supported the

hypothesis that biological nitrogen fixation by the Rhizobium and PGPR – root
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associations could be responsible for the observed higher Nitrogen uptake of

inoculated plants (Kantar et al., 2003).

The highest seed protein in groundnut obtained by using 25% chemical

fertilizer and 75% of bioorganic fertilizers (El-Karamany et al., 2007). .

Protein content was increased at all the biofertilizers inoculation. The

highest protein content was showed in combined effect of Rhizobium and

phosphobacteria (Selvakumar et al., 2009). It was found that uptake of nitrogen

and phosphorous is increased with application of phosphate solublizing bacteria

(Mahendran and Chandramani, 1998). The trend of variation in protein content due

to absorption of nitrogen and phosphorous content by plants (Ulyanoz, 2007).

Protein content in seed was progressively increased with increasing level of

nitrogen (Bachhar and Sabale, 2006 and Selvakumar et al., 2009).

Seed protein content was increased in response to biofertilizers application

to soybean CV. MALS 13 (Sharma and Namedo, 1999b). Bacterial culture alone

or in combination with nitrogen with nitrogen fertilizer increased crude protein

(Chela et al., 1993). The increase in the crude protein yield is an expected result to

the successive increase in nitrogen level in response to biofertilizers treatment

(Patel et al., 1992). The total soluble protein content was significantly increased in

sunflower, the increase was more pronounced with fertilization with biogien

(Shehata and El-Khawas, 2003).

Application of biofertilizers significantly increased the seed protein content

and protein yield per hectare over the untreated control (Akbari et al., 2011).

Sugiyama et al. (1984) stated that the soluble proteins are increased with better
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nitrogen supply and favourable growth condition. Greef (1994) reported that high

values of the reduced nitrogen fraction (Protein fraction) were found in

photosynthetic active leaf tissue. This is true especially under conditions of

favourable nitrate supply. These results suggest that the high nitrogen rate

increases amino acid synthesis in leaves and this stimulates the accumulation of

protein in the seed (Akbari et al., 2011).

The rhizobacteria enhanced protein concentrations in plants (Sanazzaro et

al., 2006). Bacillus pumilus RS3 (BPRS3) inoculation increased with 60% of total

amount of seed soluble protein, probably due to stimulation of protein synthesis

processes in soybean plants (Stefan et al., 2010). The highest amount of total crude

protein contents were obtained in plants treated with biogien (Shetewi and Tawfik,

2007). The highest protein percentage increased due to application of organic

manure alone and when it combined with biofertilizers (Mekki and Ahmed, 2005).

Biofertilization combined with half the recommended dose of the chemical

fertilizer was usually more effective and also increased protein contents of

produced pea seeds (Osman et al., 2010).

Trichoderma sp. induced genes were associated with protein metabolism

(Vinale et al., 2008). The positive correlation between PSB and protein content

was reported by Jutur and Reddy (2001). Chikpea inoculated with biofertilizers

have significantly higher grain protein content (Mohamed, 2010). The present

study revealed that combined effect of biofertilizers definitely improve the protein

content in Arachis hypogaea L. var TMV-7.