Tyrosinase Essay

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  • Topic: Enzyme assay, Sodium, Enzyme
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  • Published : April 5, 2012
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Enzymatic Assay of TYROSINASE (EC 1.14.18.1) PRINCIPLE: L-Tyrosine + O2 L-DOPA Tyrosinase Tyrosinase

> L-DOPA

> L-DOPA-quinone + H20

Abbreviation used: L-DOPA = L-3,4-Dihydroxyphenylalanine CONDITIONS: METHOD: REAGENTS: A. 50 mM Potassium Phosphate Buffer, pH 6.5 at 25° C (Prepare 50 ml in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Sigma Prod. No. P5379. Adjust to pH 6.5 at 25° C with 1 M KOH.) 1 mM L-Tyrosine Solution (Prepare 100 ml in deionized water using L-Tyrosine, Free Base, Sigma Prod. No. T-3754.) Tyrosinase Enzyme Solution (Immediately before use, prepare a solution containing 500 - 1,000 units/ml of Tyrosinase in cold Reagent A.) T = 25° C, pH = 6.5, A280nm, Light path = 1 cm

Continuous Spectrophotometric Rate Determination

B.

C.

PROCEDURE: Prepare a reaction cocktail by pipetting (in milliliters) the following reagents into a suitable container: Deionized Water Reagent A (Buffer) Reagent B (Tyrosine) 9.00 10.00 10.00

SPTYRO01.001 Revised: 02/22/94

Page 1 of 3

Enzymatic Assay of TYROSINASE (EC 1.14.18.1) PROCEDURE: (continued) Mix and adjust to pH 6.5 at 25° C with 1 M HCl or 1 M NaOH, if necessary. Immediately before use, oxygenate by bubbling 99.9% pure O2 through the reaction cocktail for 3 to 5 minutes. Pipette (in milliliters) into suitable quartz cuvettes:1 Test Blank Reaction Cocktail 2.90 2.90

Equilibrate to 25° C. Monitor the A280nm until constant, using a suitably thermostatted spectrophotometer. Then add: Reagent A (Buffer) Reagent C (Enzyme Solution) -----0.10 0.10 ------

Immediately mix by inversion and record the increase in A280nm for approximately 10 minutes. Obtain the r A280nm/minute using the maximum linear rate for both the Test and Blank. CALCULATIONS: Units/ml enzyme = (0.001) (0.1) df = Dilution factor 0.001 = The change in A280nm/minute per unit of Tyrosinase at pH 6.5 at 25° C in a 3 ml reaction mix 0.1 = Volume (in milliliters) of enzyme used units/ml enzyme Units/mg...
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