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Enzymatic Assay of Choloylglycine Hydrolase

1. Objective

To standardize a procedure for the determination of the enzymatic assay of choloylglycine hydrolase.

2. Scope

This scope of this procedure applies to products that have a specification for the enzymatic activity of choloylglycine hydrolase.

3. Definitions

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

3.2 EDTA = Ethylenediaminetetraacetic Acid

3.3 CH = Choloylglycine Hydrolase

4. Discussion

Glycoholic Acid + H2O    CH   >Glycine + Cholic Acid

5. Responsibilities

It is the responsibility of all trained Analytical Services laboratory personnel to 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 = 37ºC, pH = 5.6, A600nm, Light path = 1 cm

7.2. METHOD: Colorimetric Stop Reaction

7.3. REAGENTS

7.3.1.   25 mM Sodium Acetate Buffer, pH 5.6 at 37 ºC (Buffer) 
Prepare a 3.4 mg /ml solution of Sodium Acetate, Sigma-Aldrich Product Number such as S8625, in purified water. Adjust pH to 5.6 at 37ºC with 1N HCl.

7.3.2.   144 mM Sodium Glycocholate (Glycoch) 
Prepare a 70 mg/ml solution of Sodium Glycocholate, Sigma-Aldrich Product Number such as G7132, in Reagent 7.3.1 (Buffer). PREPARE FRESH.

7.3.3.   107 mM β-Mercaptoethanol (β-ME) 
Prepare a 1:133 dilution of β-Mercaptoethanol, Sigma-Aldrich Product Number such as M3148, in purified water

7.3.4.   55 mM Ethylenediaminetetraacetic Acid (EDTA) 
Prepare a 22.9 mg/ml solution of EDTA, Sigma-Aldrich Product Number such as ED2SS, in purified water.

7.3.5.   4% (w/v) Ninhydrin (Ninhydrin) 
Prepare a 40 mg/ml solution of Ninhydrin, Sigma-Aldrich Product Number such as N4876, in 2-Methoxyethanol, Sigma-Aldrich Product Number such as 270482. (Store under Nitrogen/Argon) KEEP IN DARK.

7.3.6.   200 mM Sodium Citrate Buffer, pH 5.0 at 37 ºC (Citrate Buffer) 
Prepare a 38.4 mg/ml solution of Sodium Citrate, Sigma-Aldrich Product Number such as C0759, in purified water. Adjust pH to 5.0 at 37ºC with 5N NaOH.

7.3.7.   0.16% (w/v) Stannous Chloride (Stannous) 
Prepare a 1.6 mg/ml solution of Stannous Chloride, Sigma-Aldrich Product Number such as 208256, in Reagent 7.3.6 (Citrate Buffer).

7.3.8.   Color Reagent Solution (CRS) 
Prepare solution by combining equal volumes of Reagent 7.3.5 (Ninhydrin) and Reagent 7.3.7 (Stannous). Prepare immediately before use and KEEP IN DARK.

7.3.9.   50% (v/v) n-Propanol (Prop) 
Prepare a 1:2 dilution of n-Propanol, Sigma-Aldrich Product Number such as 190764, in purified water.

7.3.10.   2.66 mM Glycine Standard (Standard) 
Prepare a 0.2 mg/ml solution of Glycine, Sigma-Aldrich Product Number such as G7126, in purified water.

7.3.11.   20% (v/v) Trichloroacetic Acid (TCA)
 Prepare a 1:5 dilution of TCA, Sigma-Aldrich Product Number such as T0699, in purified water.

7.3.12.   5 mM Sodium Phosphate Buffer, pH 7.0 at 37 ºC (Enz Dil) 
Prepare a 0.6 mg/ml solution of Sodium Phosphate, Sigma-Aldrich Product Number such as S0751, in purified water. Adjust pH to 7.0 at 37ºC with 1N NaOH.

7.3.13.   Choloylglycine Hydrolase Enzyme Solution (Enzyme) 
Immediately before use, prepare a 15 – 30 unit/ml solution of Choloylglycine Hydrolase in cold Reagent 7.3.12 (Enz Dil).

7.4. TEST METHOD

7.4.1   Prepare the following cocktail as follows (in milliliters):

Reagent 7.3.1. (Buffer) 4.0
Reagent 7.3.2. (Glycoch) 4.0
Reagent 7.3.3. (β-ME) 4.0
Reagent 7.3.4. (EDTA) 4.0


Mix by stirring and adjust pH to 5.6 at 37ºC with 1N NaOH.

 

7.4.2.   Pipette (in milliliters) the following reagents into suitable containers

  Test Blank
Cocktail (step 7.4.1) 0.90 0.90

 

Mix by swirling and equilibrate to 37ºC. Then add:

Reagent 7.3.13. (Enzyme) 0.10 ----

 

Mix by swirling and incubate at 37ºC for exactly 5 minutes. Then add:

Reagent 7.3.11. (TCA) 1.00 1.00
Reagent 7.3.13. (Enzyme) ---- 0.10

 

Mix by swirling and filter test and blank reactions with 0.45 µm PVDF filters. The filtrate will be used for the color development.

7.5. COLOR DEVELOPMENT

7.5.1   Prepare a Standard Curve by pipetting (in milliliters) the following reagents into suitable vials:

  Std 1 Std 2 Std 3 Std 4 Std 5 Std 6 Std Blank
Reagent 7.3.10. (Standard) 0.01 0.03 0.05 0.10 0.15 0.20 ----
Purified Water 0.19 0.17 0.15 0.10 0.05 ---- 0.20
Reagent 7.3.8 (CRS) 2.00 2.00 2.00 2.00 2.00 2.00 2.00

 

7.5.2.   Mix by swirling and heat in a boiling water bath for 20 minutes. Cool to room temperature and add 10 ml of Reagent 7.3.9 (Prop). Mix by swirling.

7.5.3.   For sample color development, pipette (in milliliters) the following reagents into suitable vials:

  Test Blank
Test Supernatant 0.20 ----
Blank Supernatant ---- 0.20
Reagent 7.3.8 (CRS) 2.00 2.00

 

7.5.4.   Mix by swirling and heat in a boiling water bath for 20 minutes. Cool to room temperature and add 10 ml of Reagent 7.3.9 (Prop). Mix by swirling.

7.5.5.   Transfer standard curve, test and blank samples to suitable cuvettes and determine the A600nm for each.

7.6. CALCULATIONS

7.6.1.   Corrected ΔA600nm Standard = ΔA600nm Standard - ΔA600nm Standard Blank

7.6.2.   Plot the Corrected ΔA600nm Standard vs. µmoles of Glycine

7.6.3.   Corrected ΔA600nm Test = ΔA600nm Test - ΔA600nm Test Blank

7.6.4 Units/ml enzyme = (Corrected ΔA600nmTest - y - intercept)(2)(df)
(slope)(0.2)(0.1)

 

where,
   df = Dilution Factor
   2 = volume (in milliliters) of stopped reaction
   0.2 = Volume (in milliliters) of the Test used in color development
   0.1 = Volume (in milliliters) of the enzyme used
   slope = slope of standard curve
   y-intercept = y-intercept of the standard curve

7.6.5 Units / mg solid = Units / ml enzyme
mg solid/ ml enzyme

 

7.6.6 Units / mg protein = Units / mg solid
mg protein/ mg solid

 

UNIT DEFINITION
One unit will hydrolyze 1.0 µmole of glycocholic acid to glycine and cholic acid in 5 minutes at pH 5.6 at 37 °C.

7.8 FINAL ASSAY CONTENTRATION
In a 1.00 ml reaction mix, the final concentrations are 10 mM sodium acetate, 29 mM sodium glycocholate, 21 mM β-mercaptoethanol, 11 mM ethylenediaminetetraacetic acid, 0.5 mM sodium phosphate, and 1.5 – 3.0 units choloylglycine hydrolase.

8. References & Attachments

8.1. Replaces SSGLYC04.

8.2. Nair, P.P, Gordon, M., and Reback, J. (1967) Journal of Biological Chemistry 242, 7-11.

9. Approval

Review, approvals and signatures for this document will be generated electronically using EDMS. Print a “For Use” copy if hardcopy with signature verification is required.

Materials

     
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