Estimation of Leghaemoglobin | Legumi­nous Plants | Biotechnology

In this article we will discuss about the principle, requirements and procedure for estimation of leghaemoglobin.

Kubo (1939) recognised the oxygen-binding haemoprotein in the root nodules of legumi­nous plants. Hence pan of the Leghaemoglobin is synthesised by the bacterium (Rhizobium) and the apoprotein (globin) is coded by plant DNA Oxygen is diffused by Lb across the nodule into the nitrogen fixing bacteroids to support oxidation and also ensures the oxygen sensitive nitrogenase activity without any damage.

Principle:

Haemoglobin reacts with pyridine in strong alkali and produces haemochrome which is- measured at 556 nm.

Requirements:

1. Alkaline pyridine reagent:

Dissolve 0.8g NaOH in 50 ml distilled water and cool. Dissolve 33.8 ml (33.2 g) of pyridine in distilled water and add to NaOH solu­tion and make up the volume to 100 ml. This forms 4.2 m pyridine in 0.2(M) NaOH.

2. Diluent buffer:

0.1 (M) Na/KP0₄ buffer (pH 7.4).

3. Sodium dithionate:

Grind the salt to a fine powder and store in small stoppered tubes in a desiccator.

4. Potassium hexacyanoferrate.

5. 30-60 days old root nodules of Phaseolus in liquid N₂ or ice cold 0.1 (M) P0₄ buffer (pH 7.4).

6. Mixer.

7. Centrifuge.

8. Cheese cloth.

Procedure:

1. Macerate fresh nodules in a mixer with 1-4 volumes of P0₄ buffer (pH 7.4) and filter through two layers of cheese cloth.

2. Discard the debris and clarify the red­dish brown filtrate by centrifugation at 1200 rpm for 10-20 minutes.

3. To 2-5 ml of the supernatant add an equal volume of alkaline pyridine rea­gent and mix.

4. Observe the greenish yellow colour formed due to the formation of ferric haemochrome.

5. Divide this haemochrome equally in two tubes.

6. Add a few crystals of sodium dithionate to one of the tubes which will reduce the haemochrome stir without aeration. After 2-5 minutes read at 556 nm against a reagent blank.

7. To the second tube add a few crystals of potassium hexacyanoferrate to oxidise the haemochrome. Read at 539 nm and calculate A₅₅₆ – A₅₃₉

Calculation:

Lb concentration (mM) = (A₅₅₆ – A ₅₃₉) 2D/ 234 where ‘D’ is the initial dilution.