Methodology for Amino Acids and Proteins

Estimation of total free amino acids


The amino acids are colorless ionic compounds that form the basic building blocks of protein. Apart from being bound as proteins, amino acids also exist in the free form in many tissues and are known as free amino acids. They are mostly water soluble in nature. Very often in plants during disease conditions, the free amino acid composition exhibits a change hence, the measurement of the total free amino acids gives the physiological and health status of the plants.



Principle
Ninhydrin, a powerful oxidizing agent, decarboxylates the alpha-amino acids and yields an intensely colored bluish purple product which is colorimetrically measured in 570nm.

Ninhydrin + alpha-amino acid
 

Hydrindantin + Decarboxylated Amino acid + Carbon dioxide + Ammonia


Hydrindantin + Ninhydrin + Ammonia


Purple colored product + Water


Materials
» Ninhydrin: dissolve 0.8g stannous chloride (SnCl2.2H2O) in 500mL of 0.2M citrate buffer (pH 5.0). add this solution to 20g of ninhydrin in 500mL of methyl cellosolve (2 methoxyethanol)
» 0.2M Citrate Buffer pH 5.0
» Diluent Solvent: Mix equal volumes of water and n-propanol, and use.


Procedure
Extraction of Amino Acids
Weigh 500mg of plant sample and grind it in a pestle and mortar with a small quantity of acid-washed sand. To this homogenate, add 5to 10mL of 80% ethanol. Filter or centrifuge. Save the filtrate or supernatant. Repeat the extraction twice with the residue and pool all the supernatants. Reduce the volume if needed by evaporation and use the extract for the quantitative estimation of total free amino acids. If the tissue is tough, use boiling 80% ethanol for extraction.




Estimation
1.
To 0.1mL of extract, add 1mL of ninhydrin solution.
2.
Make up the volume to 2mL with distilled water.
3.
Heat the tube in boiling water bath for 20min.
4.
Add 5mL of the diluents and mix the contents.
5.
After 15min read the intensity of the purple color against a reagent blank in a colorimeter at 570nm. The color is stable for 1h.
6.
Prepare the reagent blank as above by taking 0.1mL of 780% ethanol instead of the extract.


Standard

Dissolve 50mg leucine in 50mL of distilled water in a volumetric flask. Take 10mL of this stock standard and dilute to 100mL in another flask for working standard solution. A series of volume from 0.1 to 1mL of this standard solution gives a concentration range 10mg to 100mg. proceed as that of the sample and read the color.


Result

Draw a standard curve using absorbance versus concentration. Find out the concentration of the total free amino acids in the sample and express as percentage equivalent of leucine.


Notes

1. Ninhydrin is carcinogenic. Wear gloves while handling it.
2. Since this estimation includes only alpha-amino acids, non protein amino acids are not accounted.


References

1. Moore, S and Stein, W H (1984) In: Methods in Enzymol (Eds. Colowick, S P and Kaplan, N D) Academic Press New York 3 468.
2. Misra, P S, Mertz E T and Glower, D V (1975) Cereal Chem 52 844.
3. Theymoli Balasubramanian and Sadasivam, S (1987) Plant Food Hum Nutr  3741.