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In this article we will discuss about the principle, requirements and procedure for estimation of total free amino acids that exist in plant and animal tissues.
Amino acids exist free as well as bound form as proteins in plant and animal tissues. They are colourless ionic compounds that form the building blocks of proteins. Most of them are water soluble.
Principle:
A powerful oxidising agent, ninhydrin, decarboxylates the alfa-amino acids giving rise to an intensely coloured bluish purple product which can be estimated colorimetrically at 570 nm. Alfa-aminoacid + Ninhydrin —> Decarboxylated amino acid + Hydrindantin + CO2 + NH3 ;Hydrindantin + Ninhydrin + NH3 —> Purple product + Water.
Requirements:
1. Plant material-callus.
2. 80% ethanol.
3. Water bath.
4. Ninhydrin-0.8 g stannous chloride in 500 ml 0.2(M) Citrate buffer (pH 5.0). Add this solution to 20.0 g ninhydrin in 500 ml methyl cellosolve (2 methoxy ethanol).
5. 0.2(M) citrate buffer pH 5.0.
6. Diluent solvent: Mix equal volumes of water and n-propanol.
7. Sand.
8. Mortar and pestle.
9. Water bath.
10. Test tubes and stands.
11. Graduated pipettes.
12. Spectronic 20.
13. Glass marking pencil.
14. Filter paper.
15. Standards: Dissolve 50 mg leucine in 50 ml distilled water. Dilute 10 ml of this stock to 100 ml-working standard.
Procedure:
1. Homogenate callus (500 g) by grinding it with acid washed sand in a mortar and pestle. Add 5-10 ml of 80% ethanol and filter. Collect the filtrate. Grind the material twice, filter and pool the filtrates. Evaporate on a water bath to reduce the volume.
2. Take a series of 0.1 ml to 1.0 ml of the working standard in tubes giving a concentration range of 10 µg to 100 µg and label.
3. Take 0.1 ml of extract and 0.1 ml of ethanol (80%) (blank) in different tubes.
4. Add 1.0 ml of ninhydrin to 0.1 ml of extract as well as the blank and standards and make up the volume to 2.0 ml with distilled water.
5. Heat the tubes in boiling water bath for 20 minutes.
6. Add 5 ml of diluent solvent and mix well.
7. Keep for 15-20 minutes.
8. The purple colour developed is read at 570 nm against the blank. Colour is stable for one hour.
Plot a graph of standard concentration against absorbance and find the concentration of total free amino acids in the sample and express as percentage equivalent of leucine.