Monitoring the effect of glyoxal and methylglyoxal glycation on the secondary structure of human serum albumin: an analysis based on CD, ESI‐MALDI‐TOF/MS, thermal melting profiles and UV fluorescence spectroscopy

Abstract

Glyoxal and methylglyoxal (MG) are reactive dicarbonyl compounds, which modify amino groups of proteins to form advanced glycation endproducts (AGEs), that are involved in diabetes complications. The objective of this investigation was to determine the effects of nonenzymatic glycation of glyoxal and MG on the secondary structure of human serum albumin (HSA) and to evaluate the factors that influence the modification of HSA with glyoxal and MG. The formation of AGEs was monitored by HPLC, MALDI‐TOF/MS, UV, and fluorescence spectroscopy. The change in the secondary structure of HSA was determined by circular dichroism (CD) and thermal melting (Tm) profiles of the native and glycated proteins. UV and fluorescence demonstrate that the extent of AGE formation was dependent on both glyoxal and MG concentration and incubation time. The CD profiles demonstrated that HSA was a‐helical in structure, and low concentrations (2 mM) of glyoxal or methylglyoxal destabilized HSA while high concentrations (40 mM) preserved and stabilized the a‐helical structure of the protein. This was confirmed by thermal melting profiles. Finally, MALDI‐TOF/MS was successfully applied for site‐ and product‐specific relative quantification of AGEs. Supported in part by NCRR/NIH Grant # P20 RR16457.