Aldolase Assay

Method: Based on Boyer's modification of the hydrazine assay (Jagannathan et al. 1956) in which 3-phosphoglyceraldehyde reacts with hydrazine to form a hydrazone which absorbs at 240 nm. The advantages of the system are its simplicity and specificity. One unit is described as a change in absorbance of 1.00 per minute at 25°C and pH 7.5 under the specified conditions. Alternate methods for the assay of aldolase have been described by Rutter (1961). The coupled a-glycerophosphate dehydrogenase assay (Richards et al. 1961) has been used.

Reagents 

• 0.0001 M EDTA, pH 7.5 containing 0.0035 M hydrazine sulfate
• Substrate: 0.012 M Fructose-1,6-bisphosphate pH 7.5

Enzyme

Immediately before use, dilute in water to a concentration of 0.5-2.0 units per ml.

Procedure

Set spectrophotometer at 240 nm and 25°C.

Pipette into cuvettes as follows:

	Blank	Test
Fructose-1,6-bisphosphate	1.0 ml	1.0 ml
Hydrazine sulfate	2.0 ml	2.0 ml
Distilled water	0.1 ml	----
Record ΔA240 for 10 minutes
Add enzyme	----	0.1 ml
Record ΔA240 for 10 minutes

Subtract ΔA₂₄₀/min of the blank from ΔA₂₄₀/min of the test.

Calculation