The appearance of phenolphthalein is measured spectrophotometrically at 540nm (2).

Transcription

1 Document Title β-glucuronidase Page 1 of 5 Originating Department QC Approval Departments QA, QC Approval Date 23 rd May 2017 Effective Date 8 th June PRODUCT DETAILS 1.1 Enzyme Name: β-glucuronidase 1.2 Systematic Name: β-d-glucuronidase glucuronosohydrolase 1.3 E.C. Number: Source: Helix pomatia 2.0 ASSAY PRINCIPLE β-glucuronidase Phenolphthaleine glucuronide + H 2 O D-Glucuronate + Phenolphthalein The appearance of phenolphthalein is measured spectrophotometrically at 540nm (2). 3.0 UNIT DEFINITION That amount of enzyme which will liberate one microgram of Phenolphthalein from Phenolphthalein glucuronide per hour at 37 C and ph EQUIPMENT REQUIRED Double beam UV/VIS spectrophotometer with chart recorder. Water bath set to achieve a reaction temperature of 37 C ( 0.1 C). Thermometer Silica cuvettes Test tubes Manual pipettes and tips

2 Document Title β-glucuronidase Page 2 of REAGENTS REQUIRED When using the following reagents, refer to the manufacturer s instructions for safe handling and disposal. Reagent details Chemical / Reagent Supplier Product No. F.W. Glycine free base Sigma G Hydrochloric acid VWR Ethanol B (Denatured) Albion Chemicals DEN9602NF N/A Sodium hydroxide VWR Sodium acetate, anhydrous VWR Phenolphthalein β-d-glucuronide Sigma P Sodium chloride VWR Phenolphthalein Sigma P PREPARATION OF REAGENTS 6.1 6M Hydrochloric Acid Use as required and refer to the manufacturer s expiry date M Sodium Acetate Dissolve 820.3mg of anhydrous sodium acetate in 90ml of water. Adjust to ph 5.0 at 37 C with 6M HCl, and make up to 100ml with water. Stable at 2 C to 8 C for two weeks mM Phenolphthalein glucuronide Dissolve Phenolphthalein glucuronide acid up to a concentration of mg/ml water. Stable at 2 C to 8 C for 3 days M NaOH. Use as required and refer to the manufacturer s expiry date M Glycine ph 10.4 Dissolve 7.51g of Glycine in approximately 450ml of water. Adjust to ph 10.4 with 2M NaOH at 37 C and make up to 500ml with the same. Stable at 2 C to 8 C for two weeks.

3 Document Title β-glucuronidase Page 3 of % Sodium chloride (enzyme diluent) Dissolve 1g of Sodium chloride in approximately 450ml of water and make up to 500ml with the same. Stable at 2 C to 8 o C for 2 weeks % Phenolphthalein standard solution Dissolve 25mg of Phenolphthalein in 40ml of Ethanol. Make up to 50ml volumetrically. Prepare fresh daily. 6.8 Enzyme solutions Freeze-dried powders. Into new glass vials accurately weigh at least 10mg of freeze dried powder, each test sample to be weighed in triplicate. Dissolve each to a concentration of 5mg/ml in 0.2% NaCl. Store on ice and use within 1 hour of dissolution. Immediately prior to assay, dilute to approximately 375U/ml in 0.2% Ammonium sulphate suspensions. Immediately prior to assay, dilute to approximately 375U/ml in 0.2% NaCl. 7.0 TEST PROCEDURE Temperature = 37 C Wavelength = 540nm light path = 1 cm 7.1 Standard curve Prepare a standard curve by pipetting the following reagents into disposable test tubes. Reagent (ml) Std 1 Std 2 Std 3 Std 4 15µg PheP 25µg PheP 35µg PheP 35µg PheP Blank 0.1M Na acetate ph PheP-Glu Standard solution: 0.05% PheP Ethanol M glycine ph Mix, then transfer the standards into plastic cuvettes and record the absorbance against water at 540nm. Prepare a standard curve by plotting the absorbance for each standard vs micrograms of Phenolphthalein. (Where: Absorbance =A 540 standard A 540 blank).

4 Document Title β-glucuronidase Page 4 of Assay Into disposable test tubes pipette the following: Test Reference 0.1M Sodium acetate 0.70ml 0.70ml 1.2mM Phep-Gluc 0.70ml 0.70ml Stopper the tubes and mix, then allow to equilibrate for 5 minutes at 37 C, then at zero time add: Enzyme solution (diluted to approx. 375U/ml) 0.10ml 0.00ml Stopper the tubes and mix, then incubate for exactly 30 min at 37 C, then add: 0.2M Glycine buffer 5.00ml 5.00ml Enzyme solution (diluted to approx. 375U/ml) 0.00ml 0.10ml Mix, then transfer the solutions into plastic cuvettes and immediately record the absorbance at 540nm both the test and blank solution versus water. (Sample absorbance = A 540 sample A 540 blank). Determine the total micrograms of Phenolphthalein liberated using the standard curve. 8.0 CALCULATION 8.1 Volume activity (U/ml) = µg phenolphthalein liberated x T x dilution factor V s Where : V s = sample volume in cuvette = (0.10ml) T = Time correction of assay (unit definition = 1 hour) = 2 Volume activity (U/ml) = µg phenolphthalein liberated x 20 x dilution factor Weight activity (U/mg material) = Specific activity (U/mg protein) = U/ml mg material/ml U/mg material mg material/ml 9.0 PROTEIN DETERMINATION Protein is determined by the method of Lowry et al in accordance with Analytical Procedure No, AP62 2.

5 Document Title β-glucuronidase Page 5 of A 1% 280 DETERMINATION This is determined in accordance with Analytical Procedure AP REFERENCES 1. O.H. Lowry et al. J. Biol. Chem. (1951), 193, Fishman, W.H. and Bernfield, P. (1955) Methods of Enzymology, 1, Combie, J., Blake, J.W., Nugent, T.E., and Tobin, T. (1982) Clin. Chem., 28, Fishman, W.H. (1974) Method of Enzymatic Analysis (Bergmeyer, H.U.) 2 nd ed., II, REVISION HISTORY Document version number Section number Global Approval Panel Summary of Changes Reformat throughout. Header and footer updated to current format. New section added for Approval Panel. 1.5 Section removed as it is not necessary to record BBI Solutions codes. 2.0 Section 2.0 added for Assay Principle. 4.0 Equipment required amended to reflect current requirements 5.0 Reagent details amended to reflect current suppliers Preparation of Reagents named as Section M Hydrochloric acid amended to 6M Hydrochloric acid. Paragraph amended to read Use as required and refer to the Manufacturer s expiry date M Hydrochloric acid amended to 6M Hydrochloric acid. 6.2 to 6.6 Removed reference to Analytical grade water and replaced with water & 7.2 Amended approximately to at least. 20mg of freeze-dried powder amended to10mg of freeze-dried powder. Plastic test tubes amended to disposable test tubes. Standard sol n : 0.05% PheP amended to read Standard solution: 0.05% PheP in section Volume activity referenced as section Section amended from E 1% Determination to A 280 1% Determination New section added for Revision History.