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  Section: Plant Lab Protocols
 
 
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Methodology for Enzymes

 
     
 
Nitrite Reductase
(NAD(P)H: Nitrite Oxidoreductase EC 1.6.6.4)
 
Nitrite is directly reduced to ammonia without the liberation of free intermediates by nitrite reductase
NO-2
Nitrite reductase
NH+4

+6e
 
The enzyme present in both eukaryotic and prokaryotic cells accepts electrons from photosynthetically reduced ferredoxin but not from reduced pyridine nucleotides.
 
 



Principle
The disappearance of nitrite is measured in the reaction. Reduced methyl viologen is used as electron donor.
 
 



Materials
Tris-HCl Buffer 0.5M (pH 7.5)
Sodium Nitrite Solution:
    Dissolve 43.2mg NaNO2 in 20mL distilled water.
Methyl Viologen Solution:
    Dissolve 60.1mg methyl viologen in 20mL water.
Sodium Dithionite-Bicarbonate Solution:
    Dissolve 250mg each of Na2S2O4 and NaHCO3 in 10mL water.
Enzyme Extract
    Homogenize the leaf tissue (10g/100mL) with Tris-HCl buffer (pH 7.5) in a waring blender at high speed for 3min and force the homogenate to flow through eight layers of cheese cloth at 4°C. Use the filtrate as enzyme source.
 
 
Procedure
1.
Prepare a reaction mixture by mixing 6.25mL of Tris-HCl buffer, 2mL of sodium nitrite solution, 2mL methyl viologen solution and 14.75mL water.
2.
Pipette out 1.5mL reaction mixture and 0.3mL of enzyme preparation into a test tube.
3.
Run a blank without the enzyme.
4.
Start   the   reaction   by   adding 0.2mL of recently prepared dithionite-sodium bicarbonate solution.
5.
Incubate for 15 min at 30°C.
6.
Stop the reaction by vigorous shaking (vortex mixer) until blue color disappears.
7.
Use a 2mL aliquot for nitrite determination.
8.
Follow steps 5-8 as under nitrate reductase.
9.
Estimate the amount of nitrite disappeared using blank as reference.
 
 
Calculation
The enzyme activity is expressed as the amount of nitrite (mM reduced per min per mg protein.
 
 


References
1.  Guerrero, MG (1982) In: Techniques in Bioproductivity and Photosynthesis {Ed Coombs, J and Hall, D O) Pergamon Press I edition p 127.
2.  Vega, J M, Jacobo, C and Manuel, L (1980) In: Methods in Enzymology (Ed Anthony San Pietro) Academic Press New York 69 p 255.
 
     
 
 
     




     
 
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