Methodology for Nitrogen Fixation

Allantoin and Allantoic acid

In nodulating legumes, ureides are synthesized from the products of nitrogen fixation via purine degradation. Roots are considered the main site of ureide synthesis from where they are translocated to different parts of the plant through xylem sap. Their synthesis in other plants also appears to proceed via purine degradation but is dependent on the inorganic nitrogen source fed to the plant; the greatest ureide production is associated with ammonia assimilation. Ureides serve as a source of nitrogen for amino acid and protein synthesis.


Allantoin in the presence of alkali forms allantoic acid. In turn allantoic acid in 'acidic medium forms glyoxylic acid and urea. Glyoxylic acid reacts with phenylhydrazine hydrochloride to form phenylhydrazone. This hydrazone on reaction with potassium ferricyanide produces a red color which is measured at 520nm.

» Sodium Hydroxide 0.5N
   Dissolve 20g NaOH in one liter distilled water.
» Hydrochloric Acid 0.65N
   Dilute 6.5mL of concentrated hydrochloric acid to 100mL with distilled water.
» Phenylhydrazine Hydrochloride, 0.33%
   Dissolve 0.05g in 15mL of distilled water. Only use fresh solution.
» Concentrated Hydrochloric Acid, 10N.
» Allantoin Stock Standard
   Dissolve 50mg allantoin in 100mL distilled water.
» Working Standard
   Dilute 1mL to 10mL (50mg/mL)
» Phosphate Buffer, 0.05M, pH 7.5

Excise the particular tissue of the plant in which allantoin has to be measured.
Grind with 10mL of 0.05M phosphate buffer (pH 7.5) and 0.05g polyclar AT per gram of tissue in a glass homogenizer immersed in boiling water.
Centrifuge the homogenate at 10,000g for 5 min and collect the supernatant.
Pipette out 0.5mL of the supernatant into a test tube (More than one volume may be taken in duplicate).
Dilute to 2.5mL with distilled water.
Add 0.5mL of 0.5N sodium hydroxide.
Place the tube in vigorously boiling water for 7 min.
Remove the tube and bring to room temperature by placing in a waterbath.
Add 0.5mL of 0.65N hydrochloric acid.
Then add 0.5mL of phenylhydrazine solution.
Shake well and place the tubes in a boiling water bath for exactly 2 min.
Immediately plunge the tube into an ice bath and chill it for 20 min.
Remove from the bath, add 2mL of already chilled ION hydrochloric acid and 0.5mL of potassium ferricyanide solution.
Mix the contents thoroughly.
After 30 min measure the absorbance in each tube at 520nm in a spectrophotometer.
Prepare a standard graph with 0 to 40mg concentration of allantoin.

Calculate the amount of allantoin in the unknown samples using the standard curve.

In this procedure allantoic acid present in the tissue is also estimated along with allantoin. Hence, the final value includes both allantoin and allantoic acid


Richard, J T and Larry, E S (1981) Phytochem 20 361. Young, E G and Conway, G F (1942) Jbiol Chem 142 839.