Principle

i4753-invertase

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

Procedure

  1. Pipette 1.0 mL of substrate solution (A) into a test tube and equilibrate at 20 ℃ for about 5 minutes.
  1. Add 1.0 mL of the enzyme solution (pre-incubated at 20 ℃) and mix.
  2. 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).
  3. 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.
  4. 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.
  5. Keep for exactly 2 minutes in a boiling state and cool down to room temperature under running water.
  6. Add 2.0 mL 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

i4753-volume-activity

Weight activity (U/mg)=(U/mL)×1/C

This procedure is for informational purposes.

Materials
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