Glycation Alters the Fatty Acid Binding Capacity of Human Serum Albumin.

Glycation significantly alters the physicochemical and biofunctional properties of proteins in foods and in vivo. In the present study, human serum albumin (HSA) as the major transporter of fatty acids was modified with glyoxal under physiological conditions. Reversibly albumin-bound glyoxal was removed, and advanced glycation end products were quantitated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total modification of protein-bound lysine and arginine residues reached up to 4.2 and 9.6%, respectively. The impact of these modifications on the transport capacity of long-chain fatty acids was characterized by spin-labeled fatty acid probes via electron paramagnetic resonance spectroscopy. With increasing degree of glycation, the equivalence of the seven binding sites of native HSA with a dissociation constant of 0.74 ± 0.09 µM was set off with only the three high-affinity sites 2, 4, and 5 remaining (0.46 ± 0.07 µM). The other four sites were shifted to low affinities with significantly higher dissociation constants (1.32 ± 0.35 µM). Tryptic peptide mapping enabled us to relate these findings to molecular changes at specific binding sites. Modification hotspots identified were lysine 351, 286, 159 and arginine 144, 485, 117. Further investigation of plasma protein samples of uremic patients vs healthy controls gave first insights into the in vivo situation.

Hydrogels from serum albumin in a molten globule-like state.

We demonstrate that a molten globule-like (MG) state of a protein, usually described as a compact yet non-folded conformation that is only present in a narrow and delicate parameter range, is preserved in the high concentration environment of the protein hydrogel. We reveal mainly by means of electron paramagnetic resonance (EPR) spectroscopy that bovine serum albumin (BSA) retains the known basic MG state after a hydrogel has been formed from 20 wt% precursor solutions. At pH values of ~11.4, BSA hydrogels made from MG-BSA remain stable for weeks, while gels formed at slightly different (~0.2) pH units above and below the MG-state value dissolve into viscous solutions. On the contrary, when hydrophobic screening agents are added such as amphiphilic, EPR-active stearic acid derivatives (16-DOXYL-stearic acid, 16-DSA), the MG-state based hydrogel is the least long-lived, as the hydrophobic interaction of delicately exposed hydrophobic patches of BSA molecules is screened by the amphiphilic molecules. These bio- and polymer-physically unexpected findings may lead to new bio-compatible MG-based hydrogels that display novel properties in comparison to conventional gels.

The Effect of Ethanol on Gelation, Nanoscopic, and Macroscopic Properties of Serum Albumin Hydrogels.

Serum albumin has shown great potential in the development of new biomaterials for drug delivery systems. Different methods have been proposed to synthesis hydrogels out of serum albumin. It has been observed that ethanol can also act as a trigger for serum albumin denaturation and subsequent gelation. In this study, we focus on basic mechanisms of the albumin gelation process at 37 °C when using the chemical denaturant ethanol. The temperature of 37 °C was chosen to resemble human body temperature, and as under physiological conditions, albumin is in a non-denatured N conformation. As established in our previous publication for the triggers of pH and temperature (and time), we here explore the conformational and physical properties space of albumin hydrogels when they are ethanol-induced and show that the use of ethanol can be advisable for certain gel properties on the nanoscopic and macroscopic scale. To this end, we combine spectroscopic and mechanically (rheology) based data for characterizing the gels. We also study the gels' binding capacities for fatty acids with electron paramagnetic resonance (EPR) spectroscopy, which implies observing the effects of bound stearic acids on gelation. Ethanol reduces the fraction of the strongly bound FAs in bovine serum albumin (BSA) hydrogels up to 52% and induces BSA hydrogels with a maximum storage modulus of 5000 Pa. The loosely bound FAs in ethanol-based hydrogels, besides their relatively weak mechanical properties, introduce interesting new materials for fast drug delivery systems and beyond.