Assay Procedure for Invertase

Principle

The appearance of reducing sugars is measured by the modified Fehling-Lehmann-Schoorl method.

Unit Definition

One unit causes the formation of one milligram of reducing sugars equivalent to the glucose at 3 minutes under the conditions described below (This activity is equivalent to the international unit that hydrolyzes one micromole of saccharose per minute at the same temperature).

Method

Reagents

Reagents
A. Saccharose solution	：5.0%［5.0 g saccharose/100 mL of 80 mM acetate buffer, pH 4.5 (add 2－3 drops of toluene for preservation)］
B. Alkaline solution	：(103 g NaOH, 346 g Rochelle salt・4H2O/1,000 mL of H2O)
C. CuSO4 solution	：6.93% (69.3 g CuSO4・5H2O/1,000 mL of H2O)
D. KI solution	：30% (300 g KI/1,000 mL of H2O)(Store in an amber bottle)
E. H2SO4 solution	：25%
F. Na2S2O3 solution	：50 mM (49.638 g Na2S2O3・5H2O, 4.0g Na2CO3 (stabilizer)/4,000 mL of H2O) (Store in an amber bottle and keep for 3－4 days before use)
G. Soluble starch solution	：2.0% (Dissolve by boiling) (prepare fresh)
F. Enzyme diluent	：50 mM acetate buffer, pH 4.5

Procedure

1. Pipette 1.0 mL of substrate solution (A) into a test tube and equilibrate at 20 ℃ for about 5 minutes.

Concentration in Assay Mixture
Acetate buffer	65 mM
Saccharose	73 mM

2. Add 1.0 mL of the enzyme solution (pre-incubated at 20 ℃) and mix.
   
3. After exactly 3 minutes at 20 ℃, add 2.0 mL of alkaline solution (B) to stop the reaction. At the same time, prepare the blank by first mixing the substrate solution with 2.0 mL of alkaline solution after a 3 min-incubation at 20 ℃, followed by the addition of the enzyme solution and carry out the same procedure as the test (Procedure 4－8).
   
4. Transfer the stopped reaction mixture from the test tube to a 100 mL volume of Erlenmeyer flask. Rinse the inside of the test tube with about 3 mL of distilled water and transfer the rinsings to the flask. Repeat this procedure three times.
   
5. Add 2.0 mL of CuSO₄ Solution (C) and place the flask directly on a electrothermic heater (1.2 KWH) in the presence of a glass bead (5 mmφ) to prevent bumping.
   
6. Keep for exactly 2 minutes in a boiling state and cool down to room temperature under running water.
   
7. Add 2.0 mL each of KI solution (D) and H₂SO₄ solution (E) in this order.
   
8. Shake the flask and determine the amount of residual Cu^＋＋ by titration with Na₂S₂O₃ solution (F) in the presence of a few drops of soluble starch (G) as an indicator.
   
9. Record the titers (mL) of the test (t) and the blank (b), and calculate the titration difference (Δtiter, mL).
   

* Dissolve the enzyme preparation in ice-cold distilled water and dilute to 2.0－9.0 U/mL with the enzyme dilute (H), immediately before assay.

Calculation

Activity can be calculated by using the following formula

Weight activity (U/mg)＝(U/mL)×1/C

Vs	：Sample volume (1.0 mL)
0.600	：Titration difference (mL) of 50 mM Na2S2O3 solution (F＝1.00) for 1.0 mg of reducing sugar (glucose)
F	：Concentration factor of 50 mM Na2S2O3 (F should be determined by the iodotimetry method in each time of the preparation).
C	：Enzyme concentration in dissolution (c mg/mL)

This procedure is for informational purposes.