Methodology for Amino Acids and Proteins

Estimation of lysine

Lysine is the limited amino acid in the cereal grains. Assessment of lysine in cereal grains for nutritional quality is hence one of the procedures adopted for screening varieties. Though an ideal simple method for direct estimation of lysine is yet to be found, the given procedure is sufficient for routine screening.

The protein in the grain sample is hydrolyzed with a proteolytic enzyme, papain. The alpha-amino group of the derived amino acids are made to form a complex with copper. The e -amino group of lysine which does not couple with copper is made to form e -dinitropyridyl derivative of lysine with 2-chloro-3, 5-dinitropyridine. The excess pyridine is removed with ethyl acetate and the color of e-dinitropyridyl derivative is read at 390nm.

» Solution A: 2.89 CuCl2.2H2O in 100mL water
» Solution B: 13.6g Na3PO4.12H2O in 200mL water
» Odium Borate Buffer 0.05M pH9.0
» Copper Phosphate Reagent: Pour solution A (100mL) to B (200mL) with swirling, centrifuge and discard the supernatant. Resuspend the pellet three timesin 15mL borate buffer and centrifuge after each suspension. After third washing resuspend the pellet in 80mL of borate buffer. The reagent must be prepared fresh for every seven days.
» 3% solution of 2-Chloro-3, 5-Dinitropyridine in methanol. Prepare fresh just prior to use.
» 0.05M Sodium Carbonate Buffer pH 9.0
» Amino Acid Mixture: grind in a motor 30mg alanine, 50mg glutamic acid, 60mg aspartic acid, 20mg cystein, 300mg glutamic acid, 40mg glycine, 30mg histidine, 30mg isoleucine, 80mg leucine, 30mg methionine, 40mg phenylalanine, 80mg proline, 50mg serine, 30mg threonine, 30mg tyrosine and 40mg valine. Dissolve 100mg of this mixture in 10mL of sodium carbonate buffer (0.05M, pH 9.0)
» Dissolve 400mg technical grade papain in 100mL 0.1M sodium acetate buffer (pH 7.0)
» 1.2N HCl
» Ethyl acetate

To 100mg of defatted grain sample add 5mL of papain solution and incubate overnight at 65°C. cool to room temperature, centrifuge and decant the clear digest.
To one mL digest taken in a centrifuge tube add 0.5mL carbonate buffer and 0.5mL copper phosphate suspension.
Shake the mixture for 5min in a vortex mix and centrifuge.
To one mL supernatant add 0.1mL pyridine reagent, mix well and shake for 2h.
Add 5mL of 1.2N HCl and mix.
Extract three times with 5mL ethyl acetate and discard the ethyl acetate (top) layer.
Read the absorbance of aqueous layer at 390nm.
Prepare a blank with 5mL papain alone repeating steps 1 to 7.
Dissolve 62.5mg lysine monohydrochloride in 50mL carbonate buffer (1mg lysine/mL). Pipette out 0.2, 0.4, 0.6, 0.8 and 1mL and make up to 1mL with carbonate buffer. Add 4mL papain to each tube and mix, pipette out one mL from each and add 0.5mL of amino acid mixture and 0.5mL of copper phosphate suspension. Carry out steps 3 to 7. The standard curve represents absorbance values for 40, 80, 120, 160 and 200mg lysine.

Prepare a standard curve from the readings of the standard lysine. Subtract the absorbance of the blank from that of the sample and calculate the lysine content in the aliquot from the graph.

Lysine content of the sample =
Lysine value from graph in mg x 0.16
Percent N in the sample
                                                 =    g per 16g N.

1. Mertz, E T, Jambunathan, R and Misra, P S (1975) In: Protein quality, Agric Experiment Stn Bull No 70 Purdue Univ USA p 11.
2. Theymoli, Balasubramanian and Sadasivam, S (1987) Plant Foods Hum Nutr 37 41.