Investigation of Human Albumin-Induced Circular Dichroism in Dansylglycine

Abstract

Induced circular dichroism (ICD), or induced chirality, is a phenomenon caused by the fixation of an achiral substance inside a chiral microenvironment, such as the hydrophobic cavities in proteins. Dansylglycine belongs to a class of dansylated amino acids, which are largely used as fluorescent probes for the characterization of the binding sites in albumin. Here, we investigated the ICD in dansylglycine provoked by its binding to human serum albumin (HSA). We found that the complexation of HSA with dansylglycine resulted in the appearance of an ICD band centred at 346 nm. Using this ICD signal and site-specific ligands of HSA, we confirmed that dansylglycine is a site II ligand. The intensity of the ICD signal was dependent on the temperature and revealed that the complexation between the protein and the ligand was reversible. The induced chirality of dansylglycine was susceptive to the alteration caused by the oxidation of the protein. A comparison was made between hypochlorous acid (HOCl) and hypobromous acid (HOBr), and revealed that site II in the protein is more susceptible to alteration provoked by the latter oxidant. These findings suggest the relevance of the aromatic amino acids in the site II, since HOBr is a more efficient oxidant of these residues in proteins than HOCl. The three-dimensional structure of HSA is pH-dependent, and different conformations have been characterised. We found that HSA in its basic form at pH 9.0, which causes the protein to be less rigid, lost the capacity to bind dansylglycine. At pH 3.5, HSA retained almost all of its capacity for binding to dansylglycine. Since the structure of HSA at pH 3.5 is expanded, separating the domain IIIA from the rest of the molecule, we concluded that this separation did not alter its binding capacity to dansylglycine.

Albumin-induced circular dichroism in Congo red: Applications for studies of amyloid-like fibril aggregates and binding sites

“…Here, a confirmation of the complexation between Congo red and BSA was obtained by the displacement experiment of the fluorescent site markers dansylamide (DNSA, for site I in BSA) and dansylglycine (DG, for site II in BSA). These site markers have their fluorescence quantum yield increased and the maximum fluorescence band shifted to a lower wavelength when bound to the hydrophobic microenvironment in the protein [24,25]. In this experimental model, the displacement of these fluorescent probes by the addition of competitor drugs provokes a decrease in the fluorescence quantum yield of the probes.…”

“…Determination of the binding sites of Congo red in BSA was performed using the fluorescent probes dansylamide for site I [24] and dansylglycine for site II [25]. The spectrofluorimeter was adjusted to the following parameters: excitation at 340 nm and emission in the range 400-650 nm, slit widths of 2.5 nm for excitation and 10 nm for emission wavelengths, total volume 3 mL in a quartz cuvette with a 10 mm path length and magnetic stirring.…”

“…After the addition of Congo red, the mixtures were incubated for 5 min at 25°C before the measurements were taken. The fluorescence spectra were obtained using a Perkin Elmer LS 55 spectrofluorimeter (Shelton, CT, USA) [25].…”

“…After the addition of ligands, the mixtures were incubated for 5 min at 25°C before the ICD measurements. The ligands used were phenylbutazone [26] and warfarin [27] for site I, and ibuprofen [28], naproxen [29] and dansylglycine [25] for site II.…”

Study of relative stability of tautomeric forms of phenylbutazone and calculation of UV-Vis and ECD spectra

“…ECD tem sido utilizado com frequência na caracterização de estruturas com atividade óticas interagindo com sítios enzimáticos. 2,10,11 Deste modo, pode-se dizer que, graças aos cálculos mecânico-quânticos de dicroísmo circular eletrônico tem sido possível a determinação de configurações absolutas de compostos quirais. 12 As transições eletrônicas na região do ultravioleta e do visível, UV-Vis, e de dicroísmo circular eletrônico estão relacionados à absorção de fótons.…”