Aldolase catalyzes the following key reaction in glycolysis:

                           ALDOLASE
D-Fructose-1,6 diphosphate <------->  Dihydroxyacetone phosphate + D-glyceraldehyde-3-phosphate 

Aldolase from rabbit muscle has been extensively studied. It has a molecular weight of 161,000 and consists of four catalytically active sub-units.

Measurement of serum aldolase activity is of clinical significance. Elevated serum aldolase activity has been observed in certain carcinomas, muscular dystrophy, hepatitis and myocardial infarction (Kaldor, J., Clin. Chim. Acta, 28, 145, 1970). Aldolase is also used for determination of metabolites, in coupled enzyme reactions. The is 9.38 and isoelectric point is 6.1. It is inhibited by heavy metal ions, like copper, silver and zinc.

1. 0.1 M Triethanolamine HCl buffer, pH 7.6.
2. 0.008 M NADH, (5 mg/ml), NADH disodium salt in buffer.
3. 0.033 M Fructose-1,6-diphosphate, (11.22 mg/ml) in buffer.
4. Glycerol-3-phosphate dehydrogenase (G3PDH) (76 U/ml) in buffer. Prepare fresh.
5. Triose phosphate isomerase (TPI) (480 U/ml) in buffer. Prepare fresh.
6. Aldolase (enzyme) solution - dissolve in buffer to a final concentration of 0.1 U/ml. Prepare fresh immediately prior to assay.

1. Set the spectrophotometer (equipped with a strip chart recorder and temperature control) at 340 nm and 25°C.
2. Into a cuvette pipette the following:

   Triethanolamine buffer                  2.7 ml 
   NADH solution                           0.1 ml 
   Fructose 1,6-Diphosphate (substrate)    0.1 ml 
   

   Mix and incubate in the spectrophotometer at 25°C for 5 min. to achieve temperature equilibration. Record blank at 340 nm, if any.
3. Add the enzyme solutions to the cuvette as follows:

   Glycerol-3-phosphate dehydrogenase      0.01 ml
   Triose phosphate isomerase              0.01 ml
   Aldolase                                0.1  ml
   

4. Record the change in absorbance at 340 nm for 5-10 min.
5. Calculate E304nm/min