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
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.
2N Hydrochloric acid
10N NaOH (40%)
10% Sodium nitroprusside
Orthophosphoric acid (Sp.gr.
Composition of reaction mixtures
NaOH in 100ml of distilled water
Dissolve 100mg of DL-Methionine in 4ml of 20% HCI and
dilute with water to 100ml.
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.
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.
Neutralise 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 10 min add 1ml of glycine
After another 10 min add 2ml
orthophosphoric acid and shake vigorously.
Read the intensity of red colour
after 10 min 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 colour in
the standards. The 0 level serves as the blank.
Draw a standard curve and calculate the methionine content from
content in the sample = (Methionine content from the graph x 4) mg
is usually expressed as percentage of protein or g per 16g N.
Methionine content from the graph x 6.4
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.