key: cord-0260642-0a9w0rk2 authors: Gligorijević, Nikola; Šukalović, Vladimir; Miljuš, Goran; Nedić, Olgica; Penezić, Ana title: Dihydro-alpha-lipoic acid binds to human serum albumin at Sudlow I binding site date: 2020-10-16 journal: bioRxiv DOI: 10.1101/2020.10.16.342121 sha: 5a4cb69a429356c9d797623753b74e57d5e35fce doc_id: 260642 cord_uid: 0a9w0rk2 Binding of dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised in detail in this study. Binding process was monitored by spectroscopic methods and molecular docking approach. HSA binds DHLA with moderate affinity, 0.80 ± 0.007 × 104 M−1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Hydrogen bonds and electrostatic interactions were identified as the key binding interactions. DHLA binding thermally stabilized HSA, yet it had no effect on HSA structure and its susceptibility to trypsin digestion. Molecular docking confirmed that Sudlow I site accommodated DHLA in a certain conformation in order for binding to occur. Molecular dynamic simulation showed that formed complex is stable. Reported results offer future perspectives for investigations regarding the use of DHLA as a dietary intervention but also raise concerns about the effectiveness of alpha-lipoic acid and DHLA in treatment of patients with COVID-19. spinach. Humans can also synthetize LA in small amounts. LA is readily absorbed from foods 48 and its oral administration as a drug is a viable therapeutic option, including the treatment of Although the ability of albumin to bind DHLA is well known (Kawabata & Packer, 1994) , no 62 detailed analysis of this interaction has been reported so far. In the case of bovine albumin 63 (BSA), DHLA was shown to bind at IIIA site (Suji et al., 2008) , however no binding 64 experiments in the presence of the specific ligand for this site were performed. Taking into 65 account structural similarity of DHLA and octanoic fatty acid, it was proposed that DHLA binds 66 3 to IIA site (Atukeren, Aydin, Uslu, Gumustas, & Cakatay, 2010), however, IIIA site was also 67 considered (Suji et al., 2008) . 68 Having in mind that DHLA is a very potent antioxidant and its use can alleviate a number of 69 conditions related to oxidative stress, it seemed relevant to elucidate its mode of interaction with 70 HSA, a universal transporter in the circulation. The properties of this interaction, are still 71 unknown and undefined, so the present study aimed to investigate characteristics of the DHLA-72 HSA binding in detail, by using spectroscopic and molecular docking approach. where T is temperature in Kelvins (K) and R is a universal gas constant (8.314 Jmol -1 K -1 ). ΔH The presence of DHLA quenches intrinsic fluorescence of HSA, as can be seen from Figure 1A . increases, which may affect its susceptibility to proteolytic cleavage. In order to be proteolyzed, 278 peptide bonds in the protein need to be flexible and exposed enough to enable its accurate 279 accommodation in the active site of a protease. Some ligands, such as bilirubin, reduce the 280 susceptibility of HSA to cleavage by trypsin (Sjödin, Hansson, & Sjöholm, 1977) . According to 281 the results of this study, it seems that DHLA, although it thermally stabilizes HSA, has no 282 significant effect on HSA proteolysis by trypsin ( Figure 4D ). Thus, it may be expected that the The obtained results describe in detail the binding of DHLA to HSA for the first time. 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