Enzymatic Assay of Esterase
3.1. Purified Water - Water from a deionizing system, resistivity ~18MΩּcm @25°C
3.2. Unit Definition – One unit will hydrolyze 1.0 μmole of ethyl butyrate to butyric acid and ethanol per minute at pH 8.0 at 25ºC
T = 25ºC, pH 8.0
Titrimetric Rate Determination
7.3.1 10mM Borate Buffer (Buffer)
Prepare a 0.62 mg/mL solution in purified water using Boric Acid such as Sigma-Aldrich Product Number B0252. Adjust pH to 8.0 with 1 N NaOH
7.3.2 Ethyl Butyrate (EB Neat)
Use Butyric Acid Ethyl Ester such as Sigma-Aldrich Product Number E15701.
7.3.3 Standardized Sodium Hydroxide
Prepare an appropriate dilution in purified water using 1N Sodium Hydroxide, such as Sigma-Aldrich Product Number S2567. Standardize according Sigma-Aldrich Operating Procedure, PROC-DEK-OP-002861.
7.3.4 0.01N Sodium Hydroxide Titrant (NaOH)
Prepare a 0.01N 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.
7.4.1. 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:
|Reagent 7.3.1 (Buffer)||25.0|
7.4.2. Equilibrate at 25ºC with stirring. Then add (in milliliters):
|Reagent 7.3.2 (EB Neat)||0.100|
7.4.3. Equilibrate at 25ºC 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. At this point, using a timer in cumulative mode, simultaneously add 0.100ml 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 0.100ml 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.
7.5.1. The slope is the rate of consumption of a 0.100ml (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)|
VT = Volume (in milliliters) of Reagent 7.3.4 (NaOH) used to maintain the pH of the reaction mix at 8.0
NNaOH = Working concentration of Reagent 7.3.4 (NaOH) in normality
1000 = Conversion from millimoles to micromoles
m = Slope from 7.5.1
Tb = 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 25.200ml reaction mix, the initial concentrations are 10mM Borate, 0.4% (v/v)ethyl butyrate, and 5 units of esterase.
8.1. Replaces PEETHY02.
8.2. Adler, A.J. and Kistiakowsky, G.B. (1962) Journal of the American Chemicals Society 84, 695-703.
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