Characterization of methylglyoxal-modified human IgG by physicochemical methods

Abstract

Human IgG is a defence protein and quite reactive to dicarbonyls. In this study, methylglyoxal-induced modification of IgG was examined by various biochemical and biophysical methods. The methylglyoxal-induced changes in IgG were monitored by UV-visible and fluorescence spectroscopy, Fourier transform infrared spectroscopy, 1-anilinonaphthalene-8-sulfonic acid (ANS), and thermal denaturation studies. Aggregate formation was studied by Thioflavin T (ThT), Congo red (CR) and scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Spectroscopic studies suggested gross changes in MGO-modified IgG. Fluorogenic AGEs appeared during modification and the MGO-modified IgG gained thermostability. The reaction produced oxidative stress in the medium because carbonyl content increased manifold and sulfhydryl groups decreased. Enhanced binding of the MGO-modified IgG by Congo red and Thioflavin T suggests crosslinking and aggregation. This was supported by SEM and TEM results.

Keywords:

• Human IgG
• methylglyoxal
• hyperglycemia
• protein aggregate
• crosslink

Abbreviations:

• IgG: Immunoglobulin G
• MGO: Methylglyoxal
• AGEs: Advanced glycation end products
• SEM: Scanning electron microscope
• TEM: Transmission electron microscope
• ANS: 8-anilinonaphthalene-1-sulfonic acid
• DNPH: Dinitrophenyl hydrazine
• FT-IR: Fourier transform infrared spectroscopy
• DTNB: 5,5’-dithiobis-(2-nitrobenzoic acid)
• NBT: Nitroblue tetrazolium

Acknowledgements

MAK is thankful to the CSIR, New Delhi for NET-Junior/Senior Research Fellowship awarded vide letter no. 09/112 (0506)/2013-EMR-I. Authors are thankful to the USIF of the AMU for SEM and TEM, as well as the instrument facility of the “DST-FIST” program of the department.