Protein glycation is a non-enzymatic covalent attachment of a reducing sugar to proteins. It is a post translational modification, commonly found in conditions of high blood glucose, such as diabetes mellitus. Glucose initially reacts with the side chain amino group of lysine to form glycosylamine, which dehydrates to a Schiff’s base that undergoes Amadori rearrangement to yield fructosamine. Human serum albumin (HSA) is an abundant serum protein that is susceptible to glycation. Its relatively longer half-life of 21 days makes it an effective marker for assessing glycemic status. Traditionally it is detected using expensive and time-consuming methods like NMR, HPLC and western blotting. To overcome these limitations, we used novel intrinsic chromophore termed as protein charge transfer spectra (ProCharTS). This chromophore exhibits a wide range of absorbance spectrum from 250–800 nm. It arises due to photoinduced electron transfer in charge-rich proteins. MD simulation and TD-DFT calculations reveal that this spectrum arises from multiple charge transfer states within the proteins. Here, we explore the possibility of using ProCharTS to detect different level of glycation in HSA. We obtained glycated HSA in two different ways: First, by incubating HSA with a fixed concentration of glucose for different days (day 1–7). Secondly by incubating HSA with different concentration of glucose for a fixed period (day 4). Glycation of HSA was confirmed using MALDI-ToF and NBT assay. We observed an increase in ProCharTS intensity across all wavelength following glycation of HSA, consistent with the trends observed in MALDI-ToF and NBT assay. The increase in charge transfer in glycated HSA reveals that addition of fructosamine enhances interaction of charges in the glycated protein. Further, we observed that the structural integrity was conserved after glycation, which was confirmed using CD spectroscopy and tryptophan fluorescence emission.
Saikia et al. (Sun,) studied this question.