How to calculate laccase activity spectrophotometrically?

Answer 1

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. PROCEDURE

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.

8. REFERENCES & ATTACHMENTS

Ride, J.P. (1980) Physiological Plant Pathology, 16, 187-196.

9. APPROVAL

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