Enzymatic Assay of Invertase

1. Objective

To standardize a procedure for the enzymatic assay of Invertase.

2. Scope

This procedure applies to all products that have specification for the enzymatic activity of Invertase.

3. Definitions

3.1. Purified Water — water from a deionizing system, resistivity ~ 18MΩ•cm @ 25 ºC

3.2. Unit Definition — One unit will hydrolyze 1.0 μmole of sucrose to invert sugar per minute at pH 4.5 at 55 ºC

4. Discussion

Sucrose + H₂O ^Invertase > Glucose + Fructose
Both glucose and fructose are detected as reducing sugars.

5. Responsibilities

It is the responsibility of all trained analytical services personnel to follow this procedure as written.

6. Safety

Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1. CONDITIONS: T = 55 ºC, Abs₄₁₀, pH = 4.5, Light Path= 1cm

7.2 METHOD:
Spectrophotometric Stop Reaction

7.3 REAGENTS

7.3.1. 100 mM Sodium Acetate, pH 4.5 at 55 ºC (BUFFER)
Prepare a 13.6 mg/mL solution of Sodium Acetate Trihydrate, Sigma-Aldrich Product Number such as S8625, in purified water. Adjust pH to 55 ºC with 1M HCl.

7.3.2. 1.0% w/v Sucrose Substrate (SUCROSE)
Prepare a 10 mg/mL solution of Sucrose, Sigma-Aldrich Product Number such as S7903, in Reagent 7.3.1. Prepare Fresh.

7.3.3. Invertase (ENZYME)
Immediately before use, prepare a solution containing 0.3 – 0.4 units/mL of Invertase in cold purified water. Suggestion: For increased sample solubility weigh product into a wig-l-bug and dilute accordingly.

7.3.4. 0.5 M NaOH (NaOH)
Prepare a 1:2 dilution of 1N NaOH, Sigma-Aldrich Product Number such as S2567, in purified water.

7.3.5. 0.5% w/v PAHBAH (PAHBAH)
Prepare a 5 mg/mL solution of p-Hydroxybenzhydrazide, Sigma-Aldrich Product Number such as H9882, in reagent 7.3.4. Prepare Fresh

7.3.6. 5.56 mM Glucose Standard Solution (STD SOLN)
Prepare a 1.0 mg/mL solution of Glucose Standard, Sigma-Aldrich Product Number such as G8270, in purified water.

7.4 Test Method

7.4.1. Pipette the following, in milliliters, into suitable containers:

	Test Blank	Test	Std Blank	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6
Purified Water	0.10	---	1.00	0.95	0.90	0.80	0.70	0.60	0.50
Reagent 7.3.2 (SUCROSE)	0.90	0.90	---	---	---	---	---	---	---
Reagent 7.3.6 (STD SOLN)	---	---	---	0.05	0.10	0.20	0.30	0.40	0.50

7.4.2. Mix by swirling and equilibrate to 55 ºC. Then add:

	Test Blank	Test	Std Blank	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6
Reagent 7.3.3 (ENZYME)	---	0.10	---	---	---	---	---	---	---

7.4.3. Mix by swirling and incubate at 55 ºC for exactly 20 minutes. Swirl and invert tubes after the 20-minute incubation.

7.4.4. Remove 0.10mL from each reaction mixture and place in separate tubes containing:

	Test Blank	Test	Std Blank	Std 1	Std 2	Std 3	Std 4	Std 5	Std 6
Reagent 7.3.5 (PAHBAH)	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90	2.90

7.4.5. Immediately mix by swirling. Place a marble or vented cap over each tube and place in a boiling water bath.

7.4.6. Boil tubes for 5 minutes. Tubes can be boiled together within 15 minutes of stopping reaction.

7.4.7. Remove from the boiling water bath and cool to room temperature.

7.4.8. Mix by swirling and transfer the solution to suitable cuvettes. Read the Abs410 for all reaction mixtures using a suitable spectrophotometer.

7.5 CALCULATIONS

7.5.1. Standard Curve:

Corrected Absorbance: ΔA_410nm Std = A_410nm Std - A_410nm Std Blank

Prepare a standard curve by plotting the ΔA_410nm Std versus the μmoles of glucose.

7.5.2. Corrected ΔA_410nm Test = A_410nm Test - A₄₁₀nm Test Blank

7.5.3.	µmoles glucose liberated =	(ΔA410nm/ Test - (y - intercept)
		(slope)

7.5.4.	Units/mL enzyme=	(µmoles glucose)(df)
		(20)(0.1)(2)

where:
df = dilution factor
20 = Time (in minutes) of assay
0.1 = Volume (in milliliters) of enzyme used
2 = Conversion Factor for 1 μmole of sucrose being hydrolyzed to glucose and fructose

7.5.5.	Units/mg solid =	Units/mL enzyme
		mg solid/mL enzyme

7.5.6.	Units/mg protein =	Units/mg Solid
		mg protein/mg Solid

7.5.7. Final Assay Concentrations:
In a 1.00 mL reaction mix, the final concentrations are 90 mM sodium acetate, 0.90% (w/v) sucrose and 0.03 - 0.04 unit invertase.