Methodology for Nitrogen Fixation


The oxygen-binding hemoprotein was first recognized in root nodules by Kubo in 1939. Leghaemoglobin (Lb) occurs in the infected cells of legume root nodules. The concentration ranges l-3mg per g fresh weight depending on the plant species. Heme synthesis for Lb seems to be a bacterial property whereas, the apoprotein (globin) is coded for by the plant DNA and thus a well evolved coexistence mechanism between the host and the bacterium is seen in legume root nodules. Lb facilitates oxygen diffusion across the nodule into the nitrogen fixing bacteriods to support oxidation and at the same time ensures the oxygen sensitive nitrogenase activity without damage.


Haemoglobin reacts with pyridine in strong alkali to produce hemochrome. The hemochrome is measured at 556nm.

» Diluent Buffer: 0.1M sodium/potassium phosphate buffer (pH 7.4).
» Alkaline Pyridine Reagent: Dissolve 0.8g NaOH in 50mL water and cool. Add 33.8mL of pyridine (33.2g), dissolve and dilute to 100mL with water. This produces 4.2M pyridine in 0.2M NaOH.
» Sodium Dithionite: Grind finely and store in small stoppered tubes in a desiccator.
» Potassium Hexacyanoferrate

Extraction: Mix the fresh or thawed nodules with 1-3 volumes of phosphate buffer and macerate in a mixer. Filter through two layers of cheesecloth. Discard the nodule debris. Clarify the turbid reddish brown filtrate by centrifugation at 10,000g for 10-30 min. dilute suitably.
To a suitable volume (2 to 5mL) of the extract add an equal volume of alkaline pyridine reagent and mix. The solution becomes greenish-yellow due to the formation of ferric hemochrome.
Divide the hemochrome equally between two tubes.
To one portion add a few crystals of sodium dithionite to reduce the hemochrome. Stir without aeration.
Read at 556nm after 2-5 min against a reagent blank.
To the other portion add a few crystals of potassium hexacyanoferrate to oxidize the hemochrome and read at 539nm.
Calculate A556 minus A539.

Lb concentration (mM) =

A556 - A539 x 2 D
where D is the initial dilution.
(The calculation is based upon the equation E = 23.4 X 103 mol-1 cm-1)


Best yields of Lb are obtained when the nitrogen fixing activity is near the maximum. For most legume crops, 30-35 day old nodules are suitable.
To test field-grown crops it is essential that the soil is low in nitrogen and heavily inoculated with Rhizobium.
Nodules can be picked either into liquid nitrogen or ice-cold 0.1M phosphate buffer pH 7.4. Nodules picked in this way can be stored frozen for short periods.
In purified preparations, Lb can be estimated by measuring the reduced hemochrome (step 4). The formula
Lb concentration (mM) =

A556 x 2 D

is used for calculation since E = 33.9 x 103 mol-1 cm-1 for reduced hemochrome.


Appleby, C A and Bergersen, F J (1980) In: Methods for Evaluating Biological Nitrogen Fixation (Ed. Bergersen F J) John Wiley and Sons New York p 315.