Cysteine racemization on IgG heavy and light chains.

Area of Science:

• Biochemistry
• Immunology
• Protein Chemistry

Background:

• The hinge region of immunoglobulin G1 (IgG1) contains a critical disulfide bond (H220-L214).
• Basic pH conditions are known to affect protein structure and stability.
• Previous studies suggest potential modifications to cysteine residues under alkaline conditions.

Purpose of the Study:

• To investigate the impact of basic pH on IgG1 hinge region disulfide bonds and cysteine residues.
• To compare the susceptibility of IgG1λ and IgG1κ antibodies to these modifications.
• To elucidate the mechanism of base-catalyzed racemization and thioether formation in antibody hinge regions.

Main Methods:

• Incubation of monoclonal IgG1 antibodies (IgG1λ and IgG1κ) under basic pH conditions.
• Analysis of cysteine racemization and thioether formation at specific hinge region residues (H220, L214).
• Examination of endogenous human IgG1λ and IgG1κ antibodies from serum.

Main Results:

• Basic pH induces thioether formation from the H220-L214 disulfide bond in IgG1.
• Racemization of H220 cysteine occurs under basic conditions, with additional L214 racemization observed in IgG1λ but not IgG1κ.
• Similar patterns of racemization were found in endogenous human IgG1λ and IgG1κ antibodies.
• Low levels of D-cysteines were detected in IgG2 hinge regions under basic conditions and in serum-isolated antibodies.

Conclusions:

• Base-catalyzed reversible β-elimination on cysteine explains both racemization and thioether formation at the hinge disulfide.
• The light chain type (λ vs. κ) influences the extent of cysteine racemization in the IgG1 hinge region under basic conditions.
• These findings have implications for antibody stability, characterization, and potential degradation pathways.