Lysozyme dimer formation on lysozyme oxidation with Br2 radical as studied by fluorescence evolution

Area of Science:

• Biochemistry
• Protein chemistry
• Free radical chemistry

Background:

• Lysozyme is a key enzyme in the innate immune system.
• Oxidation can alter protein structure and function.
• Bi-tyrosine fluorescence is a marker for protein cross-linking.

Purpose of the Study:

• To investigate the formation and structure of lysozyme dimers produced by Br2 radical oxidation.
• To elucidate the kinetic mechanisms governing dimer formation across a range of pH values.

Main Methods:

• Oxidation of lysozyme using Br2 radical in aqueous solutions.
• Spectroscopic analysis of fluorescence emission spectra (lambda max = 400 nm).
• Kinetic studies of dimer formation using time-dependent fluorescence measurements after pulsed-electron irradiation over a wide pH range.

Main Results:

• Lysozyme dimers exhibited fluorescence spectra similar to bi-tyrosine, indicating a tyrosine-tyrosine bond.
• At pH 10.7-12.5, dimer fluorescence showed second-order growth, consistent with radical combination being rate-determining.
• At pH 6.8-10.2, dimer fluorescence exhibited first-order growth, attributed to the enolization of the dimer's keto-form.

Conclusions:

• The formation of lysozyme dimers involves the combination of tyrosine phenoxy radicals.
• The kinetics of dimer formation are pH-dependent, with distinct mechanisms dominating at high and low pH ranges.
• A reaction scheme for lysozyme dimer formation has been proposed based on kinetic data.