Protein Extraction and Quantification from Legumes

Protein extraction from the legumes was carried by a subsequent process. Primarily, the samples were cleaned followed by grinding and sieving. After, a known amount of the sieved sample was mixed with Milli-Q water (5 mL), and the pH of the sample solution was regulated to be pH 9 using sodium hydroxide (NaOH, 0.1 N) at 25 °C. Then, the solution was shaken for 50 min at room temperature followed by centrifugation (HERMLE, model Z32 HK, Wehingen, Germany) at 10,000 rpm for 10 min. The sample extraction and centrifugation methods were repeated twice for the residue to achieve better yields. In order to precipitate the protein, sample extracts were collected together and the pH value (4.5) was adjusted using hydrochloric acid HCl (1 N). Proteins was obtained by eradication of the sample supernatant by means of decantation. The achieved protein mass was cleaned two times with Milli-Q water and further centrifuged (10,000 rpm, 15 min). The obtained protein was then freeze-dried using a freeze-dryer model BenchTop Pro with Omnitronics (SP Scientific, New York, NY, USA), and used for further analysis [46 ,47 (link)].
Lowry’s reagent A: (2% Na₂CO₃ in 0.1 N NaOH) was prepared by the addition of NaOH (2 g) and Na₂CO₃ (10 g) in distilled water (5 mL) followed by dilution to 500 mL with distilled water. Lowry’s reagent B₁ was prepared with the addition of CuSO₄ (1 g) and distilled water (5 mL), diluted to 100 mL with distilled water. Lowry’s reagent B₂ was prepared with the addition of sodium potassium tartrate (2 mL) and distilled water (5 mL), diluted to 100 mL with distilled water. Lowry’s reagent C was freshly prepared with the addition of Lowry’s reagent B₁ (2 mL) and Lowry’s reagent B₂ (2 mL) while stirring solution was added to Lowry’s reagent A (200 mL). Then, 2 g of the extracted protein sample were added to reagent C followed by incubation for 45 min with continuous stirring in a dark room at room temperature. Then, reagent E (1 mL) was added to the sample, and incubated again for 45 min. Finally, the amounts of protein in the samples were determined using a spectrophotometer.
This method was performed by measuring the absorbance of all standards and samples using a spectrophotometer. The phenolic group of tyrosine and tryptophan residues (amino acid) in a protein produced a blue purple color complex, which had a maximum absorption in the region of the 660 nm wavelength with Folin–Ciocalteau reagent, which consists of sodium tungstate molybdate and phosphate. Thus, the intensity of the color depends on the amount of these aromatic amino acids present and will thus vary for different proteins. The reaction is dependent on pH and a working range of pH 9 to 10.5 is essential.