Enzymatic Assay of Lysozyme (EC 3.2.1.17)

• Precautions
• Reagents and Equipment Required
• Preparation Instructions
• Procedure
• Results

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

Lysozyme

Micrococcus lysodeikticus Cells (Intact)  ––––––––>  Micrococcus lysodeikticus Cells (Lysed)

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

1.0 M Potassium phosphate monobasic solution (P8709)

1.0 M Potassium phosphate dibasic solution (P8584)

1 M Potassium hydroxide (KOH) solution

1 M HCl solution

Micrococcus lysodeikticus, ATCC No. 4698, lyophilized cells (M3770)

Preparation Instructions

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

Buffer (50 mM Potassium Phosphate Buffer, pH 6.24, at 25 °C) – To prepared 550 mL:

• Add 20.125 mL of 1.0 M Potassium phosphate monobasic solution (P8709).
• Add 7.375 mL of 1.0 M Potassium phosphate dibasic solution (P8584).
• Add ultrapure water to make up the final volume to 550 mL.
• Adjust the pH to 6.24 at 25 °C using 1 M KOH or 1 M HCl.

Substrate Suspension (0.015% [w/v] Micrococcus lysodeikticus Cell Suspension) – Prepare a 0.15 mg/mL suspension in Buffer using Micrococcus lysodeikticus, ATCC No. 4698, lyophilized cells (M3770).

Substrate Suitability – 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.

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 reagent into suitable cuvettes and equilibrate to 25 °C using a suitably thermostatted spectrophotometer:

Reagent	Blank (mL)	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
Substrate Suspension	2.50	2.50	2.50	2.50

2. Then add:

Reagent	Blank (mL)	Test 1 (mL)	Test 2 (mL)	Test 3 (mL)
Buffer	0.10	–	–	–
Enzyme Solution	–	0.10	0.10	0.10

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

Results

Calculation

1.

where:

df = dilution factor

0.001 = Change in absorbance (ΔA₄₅₀) as per the Unit Definition

0.1 = Volume (in milliliters) of Enzyme Solution

2.      

Reference

1. Shugar D. 1952. The measurement of lysozyme activity and the ultra-violet inactivation of lysozyme. Biochimica et Biophysica Acta. 8302-309. https://doi.org/10.1016/0006-3002(52)90045-0