Methodology for Amino Acids and Proteins

Estimation of methionine in food grains

Methionine is one of the essential, sulphur-containing amino acids. Although it is present in many food proteins, methionine is the limiting amino acid in most of the grain legumes. Methionine estimation is a routine in nutritional screening programmes of the grain legumes.

The protein in the grain is first hydrolyzed under mild acidic condition. The liberated methionine gives an yellow color with nitroprisside solution under alkaline condition and turns red on acidification. Glycine is added to the reaction mixture in order to inhibit color formation with other amino acids.


» 2N Hydrochloric Acid
» 10N NaOH (40%)
» 10% NaOH
» 10% Sodium Nitroprusside
» 3% Glycine
» Orthophosphoric Acid (Sp. gr. 1.75)
» Standard Methionine: Dissolve 100mg of DL-Methionine in 4mL of 20% HCl and dilute with water to 100mL.

Weigh 0.5g of defatted sample into a 50mL conical flask. Add 6mL of 2N HCl and autoclave at 15lb pressure for one hour.
Add a pinch of activated charcoal to the hydrolysate (autoclaved sample) and heat to boil. Filter when hot and wash the charcoal with hot water.
Neutralize the filtrate with 10N NaOH to pH 6.5. Make up the volume to 50mL with water after cooling to ambient temperature.
Transfer 25mL of the made up solution into a 100mL conical flask.
Add 3mL of 10% NaOH followed by 0.15mL sodium nitroprusside.
After 10min add 1mL of glycine solution.
After another 10min add 2mL orthophosphoric acid and shake vigorously.
Read the intensity of red color after 10min at 520nm against a blank prepared in the same way but without nitroprusside.
Standard Curve: Pipette out 0, 1, 2, 3, 4 and 5mL of standard methionine solution and make up to 25mL with water. Follow steps 5 to 8 to develop the color in the standards. The 0 level serves as the blank.

Draw a standard curve and calculate the methionine content from the graph.
Methionine content in the sample = (Methionine content from the graph x 4) mg per g
Methionine is usually expressed as percentage of protein or g per 16g N.
Methionine content of the sample     =
Methionine content from the graph  x 6.4
Percent N in the sample
                                                            =          g per 16g N.

1. Horn, M J, Jones, D B and Blum, A E (1946) J Biol Chem 166 313.