7 Estimation of methionine


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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 grain legumes.

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


Materials and Reagents


2N Hydrochloric acid

10N NaOH (40%)

10% NaOH

10% Sodium nitroprusside

3% Glycine

Orthophosphoric acid (Sp.gr. 1.75)

Standard Methionine



Composition of reaction mixtures


A.  10N NaOH

Dissolve 40g NaOH in 100ml of distilled water


B.  Standard Methionine

Dissolve 100mg of DL-Methionine in 4ml of 20% HCI and dilute with water to 100ml.




1.  Weigh 0.5g of defatted grain sample into a 50ml conical flask. Add 6ml of 2N HCI and autoclave at 15 lb pressure for one hour.

2.  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.

3.  Neutralise the filtrate with 10N NaOH to pH 6.5. Make up the volume to 50ml with water after cooling to ambient temperature.

4.  Transfer 25ml of the made up solution into a 100ml conical flask.

5.  Add 3ml of 10% NaOH followed by 0.15ml sodium nitroprusside.

6.  After 10 min add 1ml of glycine solution.

7.  After another 10 min add 2ml orthophosphoric acid and shake vigorously.

8.  Read the intensity of red colour after 10 min at 520nm against a blank prepared in the same way but without nitroprusside.

9.  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 colour 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 from the graph x 6.4

Methionine content of the sample =   -------------------------------------------------- g / 16g N

                                   Percentage of N in the sample






Horn, M J, Jones, D B and Blum, A E (1946). Journal of Biological Chemistry 166: 313.