To standardize a procedure for the determination of the enzymatic assay of choloylglycine hydrolase.
This scope of this procedure applies to products that have a specification for the enzymatic activity of choloylglycine hydrolase.
3.1 Purified water = water from a deionizing system, resistivity ~18MΩ•cm @ 25 ºC
3.2 EDTA = Ethylenediaminetetraacetic Acid
3.3 CH = Choloylglycine Hydrolase
Glycoholic Acid + H2O CH >Glycine + Cholic Acid
It is the responsibility of all trained Analytical Services laboratory personnel to follow this protocol as written.
Refer to the Safety Data Sheet (SDS) for hazards and appropriate handling precautions.
7.1. CONDITIONS: T = 37 ºC, pH = 5.6, A600nm, Light path = 1 cm
7.2. METHOD: Colorimetric Stop Reaction
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, Product Number such as S8625, in purified water. Adjust pH to 5.6 at 37 ºC with 1N HCl.
7.3.2. 144 mM Sodium Glycocholate (Glycoch)
Prepare a 70 mg/mL solution of Sodium Glycocholate, 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, 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, Product Number such as ED2SS, in purified water.
7.3.5. 4% (w/v) Ninhydrin (Ninhydrin)
Prepare a 40 mg/mL solution of Ninhydrin, Product Number such as N4876, in 2-Methoxyethanol, 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, Product Number such as C0759, in purified water. Adjust pH to 5.0 at 37 ºC with 5N NaOH.
7.3.7. 0.16% (w/v) Stannous Chloride (Stannous)
Prepare a 1.6 mg/mL solution of Stannous Chloride, 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, 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, Product Number such as G7126, in purified water.
7.3.11. 20% (v/v) Trichloroacetic Acid (TCA)
Prepare a 1:5 dilution of TCA, 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, Product Number such as S0751, in purified water. Adjust pH to 7.0 at 37 ºC 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):
Mix by stirring and adjust pH to 5.6 at 37 ºC with 1N NaOH.
7.4.2. Pipette (in milliliters) the following reagents into suitable containers
Mix by swirling and equilibrate to 37 ºC. Then add:
Mix by swirling and incubate at 37 ºC for exactly 5 minutes. Then add:
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:
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:
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.
7.6.1. 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)
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|
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.
8.1. Replaces SSGLYC04.
8.2. Nair, P.P, Gordon, M., and Reback, J. (1967) Journal of Biological Chemistry 242, 7-11.
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