Tyrosinase Essay
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
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