Probing the effect of glycation on the pseudo-esterase activity of Human Serum Albumin

Human Serum Albumin (HSA), a major protein in blood plasma, exhibits pseudo-enzymatic activity such as pseudo-esterase activity. This study explores the impact of glycation on the pseudo-esterase activity of HSA. Glycation, a non-enzymatic reaction between reducing sugars and HSA was induced using glucose, fructose, and ribose. Glycated HSA samples (gHSA, fHSA, and rHSA) were analyzed for their pseudo-esterase activity using p-nitrophenyl acetate (PNPA) as a substrate. The kinetics of the enzyme-substrate reaction were studied, and structural changes monitored through UV-Vis, fluorescence, and circular dichroism (CD) spectroscopy. Glycation was confirmed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) mass spectrometry. Results indicated a significant reduction in the pseudo-esterase activity of glycated HSA compared to native HSA. Ribose-induced glycation exhibited the highest reduction in activity, attributed to the higher glycation efficiency of ribose. Fluorescence studies revealed changes in Trp fluorescence, suggesting conformational alterations in glycated as well as acetylated HSA. CD spectroscopy confirmed secondary structural changes upon glycation, followed by acetylation. A binding study was conducted to deduce the effect of glycation and acetylation of HSA on its binding activity. These findings highlight the significance of understanding how glycation modifies the biochemical properties of HSA, impacting its physiological roles and therapeutic applications in metabolic disorders. Our studies specifically reveal that glycation impairs the pseudo-esterase activity and alters the drug-binding properties of HSA.