Enzymatic Assay of Lysozyme (EC 3.2.1.17)

Description

This procedure may be used for Lysozyme products. The enzymatic rate determination (A450, Light path = 1 cm) is based on the following reaction:

Unit Definition – One unit of Lysozyme will produce a ΔA450 of 0.001 per minute at pH 6.24 at 25 °C using a suspension of Micrococcus lysodeikticus as substrate in a 2.6 ml reaction mixture.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Potassium phosphate, monobasic (Catalog No. P5379)
1 M potassium hydroxide (KOH) solution
Micrococcus lysodeikticus
, ATCC No. 4698, lyophilized cells (Catalog No. M3770)

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (66 mM Potassium Phosphate Buffer, pH 6.24, at 25 °C) – Prepare a 8.98 mg/ml solution in ultrapure water using Potassium phosphate, monobasic (Catalog No. P5379). Adjust the pH to 6.2 at 25 °C using 1 M potassium hydroxide (KOH) solution.

Substrate Suspension (Micrococcus lysodeikticus Cell Suspension) – Prepare a 0.01% (w/v) solution in Buffer using Micrococcus lysodeikticus, ATCC No. 4698, lyophilized cells (Catalog Number M3770). The A450 of this suspension must be between 0.6–0.7 versus a Buffer blank. If necessary, adjust the absorbance using appropriate amount of Buffer or Micrococcus lysodeikticus cells (Catalog No. M3770).

Enzyme Solution (Lysozyme) – Immediately before use, prepare a solution containing 200–400 units/ml of Lysozyme in cold (2–8 °C) Buffer.

Procedure

1. Pipette the following reagents into suitable cuvettes:

 

Reagent Blank (ml) Test (ml)
Substrate Suspension 2.50 2.50


2. Equilibrate cuvettes to 25 °C. Monitor the A450 until constant, using a suitably thermostatted spectrophotometer. Then add:

 

Reagent Blank (ml) Test (ml)
Buffer 0.10
Enzyme Solution 0.10


3. Immediately mix by inversion and record the decrease in A450 for ~5 minutes. Obtain the maximum linear rate (ΔA450/minute) for both the Test and Blank using at least a one minute interval and a minimum of 4 data points.

Results

Calculation

1.

Units/ml enzyme = (ΔA450/min Test – ΔA450/min Blank) (df)
(0.001) (0.1)


where:
df = dilution factor
0.001 = ΔA450 as per the Unit Definition
0.1 = Volume (in milliliters) of Enzyme Solution


2.

Units/mg solid = units/ml enzyme
mg solid/ml enzyme

Materials

     

 Reference

  • Shugar, D., Biochimica et Biophysica Acta, 8, 302-309 (1952).

 

03/14-1

Related Links