Abstract
The aim of this study was attempted to investigate both the glycation kinetics and protein secondary conformational changes of human serum albumin (HSA) after the reaction with ribose. The browning and fluorescence determinations as well as Fourier transform infrared (FTIR) microspectroscopy with a curve-fitting technique were applied. Various concentrations of ribose were incubated over a 12-week period at 37 ± 0.5 <sup>o</sup>C under dark conditions. The results clearly shows that the glycation occurred in HSA-ribose reaction mixtures was markedly increased with the amount of ribose used and incubation time, leading to marked alterations of protein conformation of HSA after FTIR determination.<br /> In addition, the browning intensity of reaction solutions were colored from light to deep brown, as determined by optical observation. The increase in fluorescence intensity from HSA–ribose mixtures seemed to occur more quickly than browning, suggesting that the fluorescence products were produced earlier on in the process than compounds causing browning. Moreover, the predominant α-helical composition of HSA decreased with an increase in ribose concentration and incubation time, whereas total β-structure and random coil composition increased, as determined by curve-fitted FTIR microspectroscopy analysis. We also found that the peak intensity ratios at 1044 cm<sup>−1</sup>/1542 cm<sup>−1 </sup>markedly decreased prior to 4 weeks of incubation, then almost plateaued, implying that the consumption of ribose in the glycation reaction might have been accelerated over the first 4 weeks of incubation, and gradually decreased. This study first evidences that two unique IR peaks at 1710 cm<sup>−1</sup> [carbonyl groups of irreversible products produced by the reaction and deposition of advanced glycation end products (AGEs)] and 1621 cm<sup>−1</sup> (aggregated HSA molecules) were clearly observed from the curve-fitted FTIR spectra of HSA-ribose mixtures over the course of incubation time. This study clearly suggests that FTIR spectroscopic curve-fitting technique may be easily used to allow determining the marked changes in the secondary conformational structure and protein aggregation of HSA during ribosylation as well as the production of AGEs.
Highlights
Advanced glycation end products (AGEs) have received considerable attention since glycation can occur both outside and inside the body [1]
It is well known that the Maillard reaction may result in the formation of final browning compounds due to an advanced glycation reaction, so the UV absorbance and browning of Maillard reaction products (MRPs) were measured by UV spectroscopy using several reported methods [36,37]
The absorbance values (A420) at 420 nm were used as an indicator of browning development in the final stage of the browning reaction
Summary
Advanced glycation end products (AGEs) have received considerable attention since glycation can occur both outside (exogenously) and inside (endogenously) the body [1]. The glycating ability of reducing sugars was found to increase in the following order: D-glucose < D-mannose < D-galactose < D-xylose < D-fructose < D-arabinose < D-ribose [14,15] Among these reducing sugars, D-ribose is the most reactive in the glycation of proteins and results in a more rapid production of AGEs than other sugars in vitro and in vivo [16,17,18]. Several controversial results have led to a debate on whether glycation or ribosylation alters the protein conformational structure [21,22,23,24]. The effects of ethanol and/or captopril on the secondary structure of HSA before and after protein binding were investigated by Fourier transform infrared (FTIR) microspectroscopy [32,33]. UV absorbance, browning, and fluorescence development of Maillard reaction products (MRPs) in the HSA–ribose system were determined
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