Binding of Doxyl Stearic Spin Labels to Human Serum Albumin: An EPR Study

Abstract

The binding of spin-labeled fatty acids (SLFAs) to the human serum albumin (HSA) examined by electron paramagnetic resonance (EPR) spectroscopy was studied to evaluate the potential of the HSA/SLFA/EPR technique as a biomarking tool for cancer. A comparative study was performed on two spin labels with nitroxide groups attached at opposite ends of the fatty acid (FA) chain, 5-doxyl stearic (5-DS) and 16-doxyl stearic (16-DS) acid. The effects of incubation time, different [SLFA]/[HSA] molar ratios, ethanol, and temperature showed that the position of the nitroxide group produces certain differences in binding between the two SLFAs. Spectra for different [SLFA]/[HSA] molar ratios were decomposed into two spectral components, which correspond to the weakly and strongly bound SLFAs. The reduction of SLFA with ascorbate showed the existence of a two component process, fast and slow, confirming the decomposition results. Warfarin has no effect on the binding of the two SLFAs, whereas ibuprofen significantly decreases the binding of 5-DS and has no effect on 16-DS. Together, the results of this study indicate that both SLFAs, 5-DS and 16-DS, should be used for the study of HSA conformational changes in blood induced by various medical conditions.

Exploring the pH-Induced Functional Phase Space of Human Serum Albumin by EPR Spectroscopy

“…Thus, all order parameters are found to continuously change with pH in a quite narrow range from 0.52 < S < 0.73 that would indicate a nematic behavior [122] of the HSA-bound spin probes in a figurative sense. Generally, S 21 values are a bit lower than S 11 , speaking in favor that the FA binding affinity (K A ) changes [39,133,134], or in EPR spectroscopic terms, the occupation of the weak immobilized spectral fraction (φ b2 ) increases with FA loading [43]. Whereas S 21 has an approximate two-state process-like curve shape, S 11 exhibits an additional, rather broad feature at about pH 2 that indicates a state of maximum spin probe disorder.…”

“…Here, the protein phase space [38] of HSA is explored in a very broad pH range from at least pH 1 to 12, mainly using spin-labeled fatty acids (FA) for monitoring the proteins solution properties. This spin probing approach allows to observe phenomena as ligand binding capabilities, rotational dynamics or changes in local polarity in continuous wave (CW) EPR [39][40][41][42][43]. Complementary nanoscale distance measurements with four-pulse double electron-electron resonance (DEER) reveal the solution structure [44][45][46][47] and may therefore also indirectly display functional changes in the solution shape of albumin from the viewpoint of spatial rearrangements of paramagnetic centers in EPR-active, albumin-bound FAs from the protein interior (5-DSA) or its surface [48][49][50].…”

Fatty Acid Triangulation in Albumins Using a Landmark Spin Label

“…The reaction was allowed to proceed under argon atmosphere at room temperature. Thorough observations of the reduction process were made in the time course of 1.0–1.5 hours (Figure S7) by using a quantitative kinetic approach including double integration routines (Supporting Information S1 and Table S1). The reduction efficiency of both spin probes was about 96–98 % after a maximum incubation time of about 40 min.…”

Investigation of the binding of cis/trans-[MCl4(1H-indazole)(NO)]− (M = Ru, Os) complexes to human serum albumin

“…This is an efficient and widely used method for studying the binding of FAs to albumin [18][19][20][21][22][23], as well as the competition between FAs and other compounds although its application for metal complexes is fairly rare in the literature [24]. Furthermore, displacement experiments with wellestablished markers for sites I and II, namely warfarin (WF) and dansylglycine (DG), respectively, were also performed and the data is discussed.…”

“…Since complexes 1-5 showed similar binding affinities for HSA (Table 1), the binding of complex 5 was further studied in the presence of a stearic FA, labeled with an aminoxyl radical at position C-16 (16-doxyl stearic acid, 16DS), a spin label that has been used in many previous studies [18][19][20]22,23]. It is important to note that the binding of FAs to HSA is not perturbed by spin labeling of FAs [20], hence the EPR spin labeling technique can provide reliable information about the amount of the FAs bound to HSA.…”