Assay Procedure for Invertase

Principle

The appearance of reducing sugars is measured by the modified Fehling-Lehmann-Schoorl method.7)

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

 

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

Procedure

  1. Pipette 1.0ml 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
  1. Add 1.0ml of the enzyme solution (pre-incubated at 20℃) and mix.
  2. After exactly 3 minutes at 20℃, add 2.0ml of alkaline solution (B) to stop the reaction. At the same time, prepare the blank by first mixing the substrate solution with 2.0ml 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).
  3. Transfer the stopped reaction mixture from the test tube to a 100ml volume of Erlenmeyer flask. Rinse the inside of the test tube with about 3ml of distilled water and transfer the rinsings to the flask. Repeat this procedure three times.
  4. Add 2.0ml of CuSO4 Solution (C) and place the flask directly on a electrothermic heater (1.2 KWH) in the presence of a glass bead (5mmφ) to prevent bumping.
  5. Keep for exactly 2 minutes in a boiling state and cool down to room temperature under running water.
  6. Add 2.0ml each of KI solution (D) and H2SO4 solution (E) in this order.
  7. 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.
  8. 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 (1.0ml)
0.600 :Titration difference (ml) of 50mM Na2S2O3 solution (F=1.00) for 1.0mg of reducing sugar (glucose)
F :Concentration factor of 50mM 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. For a current copy of Sigma’s quality control procedure contact our Technical Service Department.

Materials

     
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