BBI Solutions, Blaenavon AP 24 Analytical procedures Title: Glucose Oxidase Issue date: 1 st May 2013

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1 Issue date: 1 st May PRODUCT DETAILS 1.1 Enzyme Name: GLUCOSE OXIDASE 1.2 Systematic Name: -Glucose : oxygen 1-oxidoreductase 1.3 E.C. Number: Source: Aspergillus Niger 1.5 Suitable for BBI Solutions codes: all Glucose Oxidase codes. 2.0 ASSAY PRINCIPLE The procedure for the analysis of glucose oxidase is based on the method of Bergmeyer. 1 Glucose Oxidase (GO) catalyses the oxidation of glucose to produce D-glucono-1,5-lactone and hydrogen peroxide. This procedure utilises the hydrogen peroxide produced to convert a reduced dye (o-dianisidine) to an oxidised dye in the presence of peroxidase, the formation of which can be followed spectrophotometrically. -glucose + O 2 + H 2 O H 2 O 2 + reduced dye peroxidase glucose oxidase D-glucono-1,5-lactone + H 2 O 2 2 H 2 O + oxidised dye 3.0 UNIT DEFINITION That amount of enzyme causing the oxidation of one micromole of glucose per minute at 25 o C and ph EQUIPMENT REQUIRED Double beam UV/Vis recording spectrophotometer, with accuracy of 0.005A and a sensitivity of 0.001A and a cell compartment thermostatically controlled at 25 o C ( 0.1 o C). Water bath set at 25 o C ( 0.1 o C) Heated magnetic stirrer and magnetic followers. ph meter, readable to 0.01 ph unit Analytical balance, readable to 0.1mg Disposable plastic cuvettes and test tubes. Glass pipettes and test tubes. Automatic pipettes and tips. Glass beakers, bottles and volumetric flasks. 5.0 REAGENTS REQUIRED 5.1 Reagent details When using the following reagents please refer to the manufacturer s instructions for safe handling and disposal. Version 03 Page 1 of 5

2 o-dianisidine.dihydrochloride (3 3 -Dimethylbenzidine; Fast Blue B). Supplier : Sigma. Product No. : D F.W. = Glucose, AnalaR. Supplier : VWR International. Product No. : F.W. = Oxygen. Supplier : British Oxygen Company. Peroxidase. Supplier : BBI Solutions, Blaenavon. Product No. : code HRP2 or HRP3C Dipotassium hydrogen orthophosphate, trihydrate, AnalaR. Supplier : VWR International Product No. : F.W. = Potassium dihydrogen orthophosphate, AnalaR. Supplier : VWR International Product No. : F.W. = Preparation of reagents M potassium phosphate, ph M dipotassium hydrogen orthophosphate trihydrate (K 2 HPO 4.3H 2 O). Dissolve 11.4g of dipotassium hydrogen phosphate in 450ml of USP purified water and make up to 500ml. 0.1M potassium dihydrogen phosphate (KH 2 PO 4 ). Dissolve 6.8g of potassium dihydrogen phosphate in 450ml of USP purified water and make up to 500ml. Using the ph meter titrate the 0.1M K 2 HPO 4 with the 0.1M KH 2 PO 4 at 25 o C until ph 7.0 is obtained. Stable for 2 weeks at 2 o C to 8 o C M o-dianisidine.dihydrochloride. Caution: This material is a possible carcinogen, may cause inheritable genetic damage, and can cause irritation to the eyes, skin and respiratory system. Do not breathe dust. Handle the powder in the fume hood. Seek medical advice if you feel unwell after usage. Accurately weigh approximately 66mg of o-dianisidine into a clean glass vial and dissolve up to 6.6mg/ml in USP purified water. Stable for 5 days at 2 o C to 8 o C. Version 03 Page 2 of 5

3 5.2.3 Dye-Buffer solution. To approximately 95ml of 0.1M potassium phosphate, ph 7.0 in a 100ml volumetric flask, add 1ml of M o-dianisidine. Mix, make up to 100ml with the 0.1M potassium phosphate, ph 7.0 and store in a dark bottle. Stable for 1 week at 2 o C to 8 o C % -glucose solution. Pour ~90ml of USP purified water into a suitable glass beaker and stir on a magnetic stirrer. -glucose and add to the water whilst still stirring. Continue to stir until completely dissolved. Make up to 100ml. Allow to stand at room temperature for at least one hour to mutarotate before using. Stable for 1 week at 2 o C to 8 o C Peroxidase solution. (approximately 60 pyrogallol U/ml) Code HRP2: dissolve up to a concentration of 1mg/ml in 0.1M potassium phosphate, ph 7.0 Code HRP3C: dissolve enzyme up to a concentration of 5mg/ml in 0.1M potassium phosphate, ph 7.0, and dilute further to 60 pyrogallol U/ml in 0.1M potassium phosphate, ph 7.0. Store on ice and prepare fresh daily Enzyme solution. Freeze-dried powders: Into clean glass vials accurately weigh 10-15mg of freeze-dried powder, each test sample to be weighed in triplicate. Dissolve each up to a concentration of 5mg/ml in 0.1M potassium phosphate, ph 7.0. Store on ice. Immediately prior to assay, dilute to approximately 0.2 U/ml in 0.1M potassium phosphate, ph 7.0. Liquid preparations: Immediately prior to assay, dilute to approximately 0.2 U/ml in 0.1M potassium phosphate, ph TEST PROCEDURE Temperature = 25 o C. Wavelength ( Light path = 10mm Sparge the Dye-Buffer solution with oxygen for approximately 10 minutes before use. Hold at 2 o C to 8 o C and resparge every 30 minutes. Into disposable test tubes pipette the following: Test Reference Oxygenated Dye-Buffer: 2.40ml 2.40ml 0.1M potassium phosphate, ph 7.0: 0.00ml 0.10ml -glucose: 0.50ml 0.50ml Peroxidase solution: 0.10ml 0.10ml Allow the solutions to equilibrate to 25 o C for approximately 5 minutes, then add: Enzyme solution, diluted to ~0.2U/ml: 0.10ml 0.00ml Total volume (V t ): 3.10ml 3.10ml Version 03 Page 3 of 5

4 Without vortexing, transfer to a disposable cuvette. Mix by gently pouring the reaction mixture into another disposable cuvette, which is then placed in the spectrophotometer. Record the increase in absorbance at 436nm, reading the test solution against the reference solution for approximately 5 minutes. Measure the change in absorbance per minute ( 436/min) over the linear portion of the curve and use this value in the calculation. 7.0 CALCULATION 7.1 Volume activity (U/ml) = 436/min x V t x dilution factor V s Where: V t = final volume of the reaction mix (3.10ml) V s = sample volume (0.10ml) -dianisidine (8.3cm 2 436/min x 3.72 x dilution factor For freeze-dried samples: Weight activity (U/mg material) = U/ml mg/ml Specific activity (U/mg protein) = U/mg material mg protein/mg material 7.3 For liquid samples: Specific activity (U/mg protein) = U/ml mg protein/ml 8.0 PROTEIN DETERMINATION Protein is determined by the method of Lowry et al 2, (see Procedure No. AP 62, Analytical Procedures Manual). 9.0 E 1% DETERMINATION The E is the absorbance of a sample measured at nm, and is an estimate of the protein concentration. It is determined according to AP 63, Analytical Procedures Manual. The ratio U/E is an indication of the purity of the sample. The E of a 1% (ie 10mg material/ml of a freeze-dried sample, or a 10mg protein/ml liquid sample) solution is known as the E 1%. The E 1% is determined in 0.1M potassium phosphate, ph 7.0. The spectrophotometer is zeroed at nm with 0.1M potassium phosphate, ph 7.0. The enzyme is diluted in this buffer to give a reading between 0.1A and 2.0A. E = Absorbance at nm x dilution Version 03 Page 4 of 5

5 For freeze-dried samples (at 5mg/ml): E 1% = E of a 10mg material/ml solution = E x 2 For liquid samples: E 1% = E of a 10mg protein/ml solution = E x protein concentration (mg protein/ml) REFERENCES 1. Bergmeyer, H.-U., Gawehn, K., & Grassl, M., (1974) Methods of Enzymatic Analysis. 2 nd edn (Bergmeyer,H.-U., ed) Vol.1, p.457, Academic Press, New York 2. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951) J. Biol. Chem. 193, 265 Version 03 Page 5 of 5