Enzymatic Assay of Esterase

1. Objective

To standardize a procedure for the enzymatic determination of esterase activity using ethyl butyrate as a substrate.

2. Scope

This procedure applies to all products that have a specification for esterase activity.

3. Definitions

3.1. Purified Water - Water from a deionizing system, resistivity ~18MΩּcm @25 °C

3.2. Unit Definition – One unit will hydrolyze 1.0 μmol of ethyl butyrate to butyric acid and ethanol per minute at pH 8.0 at 25 °C

4. Discussion

Ethyl Butyrate + H₂O ^Esterase >Butyric Acid + Ethanol

5. Responsibilities

Analytical services personnel should follow this protocol as written.

6. Safety

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

7. Procedure

7.1. CONDITIONS
T = 25 °C, pH 8.0

7.2. METHOD
Titrimetric Rate Determination

7.3. REAGENTS

7.3.1 10 mM Borate Buffer (Buffer)
Prepare a 0.62 mg/mL solution in purified water using boric acid, such as Product No. B0252. Adjust pH to 8.0 with 1 N NaOH

7.3.2 Ethyl Butyrate (EB Neat)
Use Butyric Acid Ethyl Ester (Product No. E15701).

7.3.3 Standardized Sodium Hydroxide
Prepare an appropriate dilution in purified water using 1 N sodium hydroxide (Product No. S2567).

7.3.4 0.01 N Sodium Hydroxide Titrant (NaOH)
Prepare a 0.01 N Sodium Hydroxide solution in purified water using the standardized Reagent 7.3.3. Prepare fresh.

7.3.5 Esterase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing approximately 50 units/mL in cold Reagent 7.3.1.

7.4. PROCEDURE

7.4.1. Using a suitable pH meter in conjunction with a magnetic stirrer and thermostated waterbath, pipette the following reagents (in milliliters) into a suitable titration vessel:

	Test
Reagent 7.3.1 (Buffer)	25.0

7.4.2. Equilibrate at 25 ºC with stirring. Then add (in milliliters):

Reagent 7.3.2 (EB Neat)

0.100

7.4.3. Equilibrate at 25 ºC with stirring. Adjust the pH within a range of 8.1-8.2 using Reagent 7.3.4 (NaOH). Then add (in milliliters):

Reagent 7.3.5 (Enzyme)

0.100

7.4.4. Monitor the pH of the reaction mix until the pH reaches 8.0. Using a timer in cumulative mode, simultaneously add 0.100 mL of Reagent 7.3.4 (NaOH) and start the timer. (Aliquots of NaOH titrant may be adjusted as necessary as stated in step 7.4.6)

7.4.5. When the pH of the reaction mix again reaches 8.0, add another 0.100 mL aliquot (or equivalent volume used in 7.4.4) of Reagent 7.3.4 (NaOH) and record the elapsed time.

7.4.6. Repeat steps 7.4.4 and 7.4.5 for approximately 5 minutes. There should be approximately 10 additions of titrant during this time period. A minimum of 5 additions of titrant during this time period is required. If the minimum addition requirements have not been met, adjust the volume of titrant used in steps 7.4.4 and 7.4.5.

7.4.7. Plot the volume (in milliliters) of titrant used versus time (in minutes) and determine the slope of the plot for each sample and control.

7.4.8. Perform a blank run by following steps 7.4.1 through 7.4.4, replacing the enzyme aliquot with purified water (enzyme diluent). If the pH of the reaction mix does not reach 8.0 in 60 minutes, record 60 minutes as the blank rate. This elapsed time (Tb) is used in the calculations. The blank run may be performed before the samples. If the blank rate appears to be negligible, the blank reaction mix can be set aside with a second timer. The pH should be checked periodically between sample runs.

7.5 CALCULATIONS

7.5.1. The slope is the rate of consumption of a 0.100 mL (or volume used in steps 7.4.4 and 7.4.5) aliquot of Reagent 7.3.4 (NaOH) divided by the time.

Blank rate correction: T =

(m) - (VT / Tb)

7.5.2.	Units/mL enzyme =	(T) x (NNaOH) x (1000)
		(0.001)

VT = Volume (in milliliters) of Reagent 7.3.4 (NaOH) used to maintain the pH of the reaction mix at 8.0
N_NaOH = Working concentration of Reagent 7.3.4 (NaOH) in normality
1000 = Conversion from millimoles to micromoles
m = Slope from 7.5.1
T_b = Elapsed time of blank from 7.4.8
0.1 = Volume (in milliliters) of enzyme used

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

7.5.4.	Units/mg protein =	Units/mL enzyme
		mg protein/mL enzyme

7.6 INITIAL ASSAY CONCENTRATION
In a 25.200 mL reaction mix, the initial concentrations are 10 mM borate, 0.4% (v/v)ethyl butyrate, and 5 units of esterase.