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

Abstract

A systematic study on the self-assembled solution system of human serum albumin (HSA) and paramagnetic doxyl stearic acid (5-DSA and 16-DSA) ligands is reported covering the broad pH range 0.7–12.9, mainly using electron paramagnetic resonance (EPR) methods. It is tested to which extent the pH-induced conformational isomers of HSA reveal themselves in continuous wave (CW) EPR spectra from this spin probing approach in comparison to an established spin-labeling strategy utilizing 3-maleimido proxyl (5-MSL). Most analyses are conducted on empirical levels with robust strategies that allow for the detection of dynamic changes of ligand, as well as protein. Special emphasis has been placed on the EPR spectroscopic detection of a molten globule (MG) state of HSA that is typically found by the fluorescent probe 8-Anilino- naphthalene-1-sulfonic acid (ANS). Moreover, four-pulse double electron-electron resonance (DEER) experiments are conducted and substantiated with dynamic light scattering (DLS) data to determine changes in the solution shape of HSA with pH. All results are ultimately combined in a detailed scheme that describes the pH-induced functional phase space of HSA.

Fatty Acid Binding to Human Serum Albumin in Blood Serum Characterized by EPR Spectroscopy

“…Moreover, ethanol above a certain concentration in the sample can cause HSA denaturation . Examining the pH of the samples showed that they all range between pH 7.4 to 8.0, in which HSA is in its native form, so the natural conformation and environment of the protein is completely preserved …”

“…Shorter NÀ O bond length and the presence of negative charges close to NO * moiety and the deviation of NÀ O from planarity (the so called out of plane À OOP-angle) can be named as other factors which increase the hyperfine coupling values. [25][26][27][28][29][30][31] At physiological pH, HSA is negatively charged (~19e) [14,32,33] and it is a well-established fact that hydrophilic negative headgroups and hydrophobic moieties are required for binding to the fatty acid binding sites of HSA. The high affinity binding site of FAs to the protein are the ones who have hydrogen bond and /or electrostatic interactions with the ligand carboxylate head group.…”

“…[46] Examining the pH of the samples showed that they all range between pH 7.4 to 8.0, in which HSA is in its native form, so the natural conformation and environment of the protein is completely preserved. [14] To know if the obtained results are reproducible on the one hand and to ascertain stability of the blood samples on the other hand, we chose three samples at random at a loading ratio of 1 : 4 and repeated the measurements after three months. During this time interval samples were kept at À 80°C and were thawed before reexamining them.…”

“…[11] Electron paramagnetic Resonance (EPR) is a powerful tool which can monitor such structural and therefore functional modifications. [12][13][14][15][16] EPR spectroscopy as a magnetic resonance method detects spins of unpaired electron, hence (unlike in nuclear magnetic resonance (NMR) spectroscopy) one needs to introduce paramagnetic species into the sample. Here, the labeled stearic acid (16-doxyl stearic acid, 16-DSA) is our main probe molecule of choice and is used as a spin probe throughout this study.…”

Fatty Acid Triangulation in Albumins Using a Landmark Spin Label

“…Generally, this study was inspired by the earliest spin‐labeling experiments on albumin with the 3‐amino‐proxyl, or 3‐maleimido‐proxyl spin labels . However, these reagents typically generate a mixture of lysine‐ and cysteine‐labeled albumins resulting in the emergence of rather unspecific labeling sites . Selective labeling of the highly conserved Cys34 residue in HSA with the cysteine‐specific methanethiosulfonate spin label (MTSSL) was reported earlier, serving as a tool for investigations on e. g. albumin fragment dynamics .…”