Methodology for Enzymes

Malate Dehydrogenase
(Lwalate: NAD+ oxidoreductase EC 1.1.1.37)

Malate dehydrogenase (MDH) is one of the enzymes involved in TCA cycle. It catalyses the reversible conversion of oxaloacetic acid to malic acid.

Oxaloacetic acid + NADH
Mg2+
Malic acid + NAD+



This enzyme is also involved in carbon dioxide assimilation in C4 plants. It is coupled with PEPcase in CO2 assimilation in the chloroplasts of mesophyll cells. Here it uses NADPH as the coenzyme.


Principle
Since it is an oxidoreductase involving nicotinamide adenine dinucleotide the decrease in absorbance due to the oxidation of NADH is followed.





Materials
» Oxaloacetic Acid (5mmole ) 66mg/50mL dis. water
» Magnesium Chloride (10mmole ) 203.5mg/50mL dis. water
» Tris-HCl Buffer (0.1M) pH 7.8
» NADH (0.4/*mole) 5.32mg/10mL


Procedure
1 Pipette out all reagents as shown in table.
S.No.
Reagents
Test
Blank


(mL)
(mL)
1.
Oxaloacetic acid
0.5
0.5
2.
Magnesium chloride
0.5
0.5
3.
Tris-HCl buffer, pH 7.8
1.3
1.8
4.
Enzyme extract
0.2
0.2
5.
NADH (Refer step 3)
0.5
Omit
2.
Set the spectrophotometer to zero absorbance at 340nm without adding NADH in the test against the blank in the reference cuvette.
3.
Add NADH as quickly as possible into the test, mix well and record the initial absorbance.
4.
Record the absorbance every 30sec for at least 3 min.





Notes
Crude Enzyme Extract
Grind the tissue thoroughly with acid-washed sand in a pre-chilled pestle and mortar in grinding medium (1mL/1g tissue) containing 50mM Tris-HCl (pH 8.0) 50mM MgCl2, 5mM 2-mercap-toethanol and 1mM EDTA. Pass the homogenate through four layers of cheese cloth, and centrifuge the filtrate at 3000g for 20 min at 5°C. Use the supernatant as enzyme source.


Calculation
Calculate the enzyme activity as follows with decrease in absorbance for one min.
mmoles NADH oxidized per min per 0.2ml enzyme extract
= Absorbance decrease/min X 0.1613 x 3 (Volume of the reaction mixture in mL)
Determine the protein content by the method of Lowry et at in the enzyme extract. Compute the value for mg protein to calculate the specific activity.


References
1.  Gnanam, A and Francis, K (1976) Plant Biochem J 3 11.
2.  Sadasivam, S and Gowri, G (1981) Photosynthetica 15 453.