Enzymatic Assay of Laccase (EC 1.10.3.2)
1. OBJECTIVE
To standardize a procedure for the enzymatic assay of laccase at Sigma-Aldrich, St. Louis, Mo.
2. SCOPE
The scope of this procedure is all products that have a specification for laccase activity.
3. DEFINITIONS
One unit will produce a DA530nm of 0.001 per minute at pH 6.5 at 30°C in a 3 mL reaction volume using Syringaldazine as substrate.
4. DISCUSSION
Syringaldazine + O2 Laccase > Oxidized Syringaldazine + 2H2O.
5. RESPONSIBILITIES
It is the responsibility of Analytical Services personnel to follow this protocol as written.
6. SAFETY
Refer to Material Safety Data Sheets (MSDS) for hazards and appropriate handling precautions.
7.1 CONDITIONS:
T = 37°C, pH = 6.5, A530nm, Light path = 1 cm
7.2 METHOD:
Continuous Spectrophotometric Rate Determination
7.3 REAGENTS:
7.3.1 100 mM Potassium Phosphate buffer, pH 6.5 at 37°C (Buffer)
(Prepare 100 mL in deionized water using Potassium Phosphate, Monobasic, Anhydrous, Sigma-Aldrich Product Number P-5379. Adjust to pH 6.5 at 37°C with 1 M KOH.)
7.3.2 0.216 mM Syringaldazine Solution (SYR)
(Prepare 3 mL in absolute methanol using Syringaldazine, Sigma Product Number S7896.)
7.3.3 Laccase Enzyme Solution (Laccase)
(Immediately before use, prepare a solution containing 25-50 units/mL of Laccase in cold deionized water.)
7.4 TEST METHOD
Pipette the following reagents into suitable vials (in milliliters):
| Test | Blank | |
| Deionized water (Reagent 7.3.2) | ------- | 0.50 |
| Buffer (Reagent 7.3.2) | 2.20 | 2.20 |
| Laccase (Reagent 7.3.2) | 0.50 | ------- |
Equilibrate to 37°C .Monitor the A530nm until constant using a suitably thermostatted spectrophotometer.Then add:
| Test | Blank | |
| SYR | 0.30 | 0.30 |
Mix by inversion and record the increase in A530nm for approximately 10 minutes. Obtain the DA530nm/min. using the maximum linear rate for both the Test and Blank.
7.5 CALCULATIONS
| Units/ml enzyme = | ΔA530nm Sample = A530nm/min Test - A530nm/min Blank) (df) |
| (0.001)(0.5) |
df = dilution factor
0.001 = the change in A530nm/min. per unit of laccase at pH 6.5 at 30oC in a 3 mL reaction mix
0.5 = volume (in milliliters) of enzyme used
| 7.5.2.4 | Units/mg solid = | Units/mL enzyme |
| mg solid/mL enzyme |
7.6 FINAL ASSAY CONCENTRATION:
In a 3.00 mL reaction mix, the final concentrations are 73 mM potassium phosphate, 0.02 mM syringaldazine, 10% methanol, and 12.5 to 25.0 units laccase.
Ride, J.P. (1980) Physiological Plant Pathology, 16, 187-196.
| Print Name | Sign Name | Title | Date | |
| Prepared by: | Gene McNaughton | Gene McNaughton | Originator | 09/02/03 |
| Approved by: | David Lintz | David Lintz | Supervisor | 09/03/03 |
| Approved by: | Jeff D. Heiland | Jeff D. Heiland | Quality Assurance | 09/03/03 |
