Profound Changes in Functional Structure and Dynamics of Serum Albumin in Children with Nephrotic Syndrome: An Exploratory Research Study.

Patients with nephrotic syndrome (NS) suffer from urinary loss of albumin. As a cause, previous studies focused on the glomerular filter rather than analyzing the molecular properties of albumin itself. Later one was initiated by clinical observations indicating unexplained molecular alterations of human serum albumin (HSA) in an NS pediatric patient. Therefore, we examined serum from eight pediatric patients with steroid-sensitive and -resistant NS and compared it with serum from healthy subjects as well as commercial HSA. We used dynamic and electrophoretic light scattering to characterize the protein size and effective surface charge and electron paramagnetic resonance spectroscopy to measure the local environment and binding dynamics of up to seven fatty acids associated with HSA. Our findings suggest that pronounced differences in binding behavior and surface charge of HSA could enhance their filtration through the GBM, leading to direct toxicity of HSA to podocytes.

Incorporation of ß-Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity.

Redox-active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α-amino acid Gly with the ß-amino acid ß-Ala at position 2 (Gly2→ß-Ala2) of the ATCUN motif reinstates ROS production (• OH and H2 O2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these ß-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for ß-Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where ß-Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP-MS measurements.

Access to New Cytotoxic Triterpene and Steroidal Acid-TEMPO Conjugates by Ugi Multicomponent-Reactions.

Multicomponent reactions, especially the Ugi-four component reaction (U-4CR), provide powerful protocols to efficiently access compounds having potent biological and pharmacological effects. Thus, a diverse library of betulinic acid (BA), fusidic acid (FA), cholic acid (CA) conjugates with TEMPO (nitroxide) have been prepared using this approach, which also makes them applicable in electron paramagnetic resonance (EPR) spectroscopy. Moreover, convertible amide modified spin-labelled fusidic acid derivatives were selected for post-Ugi modification utilizing a wide range of reaction conditions which kept the paramagnetic center intact. The nitroxide labelled betulinic acid analogue 6 possesses cytotoxic effects towards two investigated cell lines: prostate cancer PC3 (IC50 7.4 ± 0.7 µM) and colon cancer HT29 (IC50 9.0 ± 0.4 µM). Notably, spin-labelled fusidic acid derivative 8 acts strongly against these two cancer cell lines (PC3: IC50 6.0 ± 1.1 µM; HT29: IC50 7.4 ± 0.6 µM). Additionally, another fusidic acid analogue 9 was also found to be active towards HT29 with IC50 7.0 ± 0.3 µM (CV). Studies on the mode of action revealed that compound 8 increased the level of caspase-3 significantly which clearly indicates induction of apoptosis by activation of the caspase pathway. Furthermore, the exclusive mitochondria targeting of compound 18 was successfully achieved, since mitochondria are the major source of ROS generation.

Identification of Patients with Pancreatic Cancer by Electron Paramagnetic Resonance Spectroscopy of Fatty Acid Binding to Human Serum Albumin.

An effective biological marker for pancreatic adenocarcinoma (PAC) is not available so far. Here, we investigate how electron paramagnetic resonance (EPR) spectroscopy of spin-labeled fatty acid (FA) molecules binding to human serum albumin (HSA) in human serum is a suitable method for the identification of patients with PAC through detection of PAC-induced changes of FA binding to albumin. The functionality of HSA to bind FA is investigated in serum samples of 35 patients with PAC, 26 patients with benign pancreatic tumors (BPD), and 24 healthy individuals by continuous wave (CW) EPR spectroscopy by simply dissolving 16-DOXYL stearic acid as spin-labeled FA. It is found that FA binding to HSA in PAC is significantly modified when compared with healthy and BPD individuals. The PAC group could best be discriminated from the healthy group based on EPR characteristics at the loading ratio of 1:4 (HSA:FA), while patients with PAC and BPD are distinguishable at a loading ratio of 1:6. Using nanoscale distance measurements through double electron-electron resonance (DEER), it is found that the distribution of FAs in the HSA of one PAC patient is similar to that of FAs in healthy individuals. Combining all EPR spectroscopic data, this leads to a tentative molecular interpretation of only small changes in hydration at the protein's surface as origin of the detectable characteristics for PAC patients. Thus, EPR of FA/HSA binding is a simple and promising tool for clinical detection of patients with PAC and needs to be tested with larger ensembles of different patient groups.

Probing the Y2 Receptor on Transmembrane, Intra- and Extra-Cellular Sites for EPR Measurements.

The function of G protein-coupled receptors is intrinsically linked to their conformational dynamics. In conjugation with site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy provides powerful tools to study the highly dynamic conformational states of these proteins. Here, we explored positions for nitroxide spin labeling coupled to single cysteines, introduced at transmembrane, intra- and extra-cellular sites of the human neuropeptide Y2 receptor. Receptor mutants were functionally analyzed in cell culture system, expressed in Escherichia coli fermentation with yields of up to 10 mg of purified protein per liter expression medium and functionally reconstituted into a lipid bicelle environment. Successful spin labeling was confirmed by a fluorescence assay and continuous wave EPR measurements. EPR spectra revealed mobile and immobile populations, indicating multiple dynamic conformational states of the receptor. We found that the singly mutated positions by MTSL ((1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methanesulfonothioate) have a water exposed immobilized conformation as their main conformation, while in case of the IDSL (bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide) labeled positions, the main conformation are mainly of hydrophobic nature. Further, double cysteine mutants were generated and examined for potential applications of distance measurements by double electron-electron resonance (DEER) pulsed EPR technique on the receptor.

Concentration Effects in the Interaction of Monoclonal Antibodies (mAbs) with their Immediate Environment Characterized by EPR Spectroscopy.

Monoclonal antibodies (mAbs) are often needed and applied in high concentration solutions, >100 mg/mL. Due to close intermolecular distances between mAbs at high concentrations (~10-20 nm at 200 mg/mL), intermolecular interactions between mAbs and mAbs and solvent/co-solute molecules become non-negligible. Here, EPR spectroscopy is used to study the high-concentration solutions of mAbs and their effect on co-solvated small molecules, using EPR "spin probing" assay in aqueous and buffered solutions. Such, information regarding the surrounding environments of mAbs at high concentrations were obtained and comparisons between EPR-obtained micro-viscosities (rotational correlation times) and macroscopic viscosities measured by rheology were possible. In comparison with highly viscous systems like glycerol-water mixtures, it was found that up to concentrations of 50 mg/mL, the mAb-spin probe systems have similar trends in their macro- (rheology) and micro-viscosities (EPR), whereas at very high concentrations they deviate strongly. The charged spin probes sense an almost unchanged aqueous solution even at very high concentrations, which in turn indicates the existence of large solvent regions that despite their proximity to large mAbs essentially offer pure water reservoirs for co-solvated charged molecules. In contrast, in buffered solutions, amphiphilic spin probes like TEMPO interact with the mAb network, due to slight charge screening. The application of EPR spectroscopy in the present work has enabled us to observe and discriminate between electrostatic and hydrophobic kinds of interactions and depict the potential underlying mechanisms of network formation at high concentrations of mAbs. These findings could be of importance as well for the development of liquid-liquid phase separations often observed in highly concentrated protein solutions.

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

One of the functions of Human Serum Albumin (HSA) is binding and transport of fatty acids. This ability could be altered by the presence of several blood components such as toxins or peptides - which in turn alters the functionality of the protein. We aim at characterizing HSA and its fatty acid binding in native serum environment. Native ligand binding and deviations from normal function can be monitored by electron paramagnetic resonance (EPR) spectroscopy using spin labeled fatty acids (FAs). Blood serum from healthy individuals is used to examine healthy HSA in its natural physiological conditions at different loading ratios of protein to FAs. Among the EPR spectroscopic parameters (like hyperfine coupling, line shape, rotational correlation time and population of different binding sites) the rotational correlation time is found to differ significantly between binding sites of the protein, especially at loading ratios of four FAs per HSA. Although differences are observed between individual samples, a general trend regarding the dynamics of healthy HSA at different loading ratios could be obtained and compared to a reference of purified commercially available HSA in buffer.

Electronic and Geometric Structures of Paramagnetic Diazadiene Complexes of Lithium and Sodium.

The electronic and molecular structures of the lithium and sodium complexes of 1,4-bis(2,6-diisopropylphenyl)-2,3-dimethyl-1,4-diazabutadiene (Me2DADDipp) were fully characterized by using a multi-frequency electron paramagnetic resonance (EPR) spectroscopy approach and crystallography, together with density functional theory (DFT) calculations. EPR measurements, using T1 relaxation-time-filtered pulse EPR spectroscopy, revealed the diagonal elements of the A and g tensors for the metal and ligand sites. It was found that the central metals in the lithium complexes had sizable contributions to the SOMO, whereas this contribution was less strongly observed for the sodium complex. Such strong contributions were attributed to structural specifications (e.g. geometrical data and atomic size) rather than electronic effects.

Spin-labelled diketopiperazines and peptide-peptoid chimera by Ugi-multi-component-reactions.

For the first time, spin-labelled coumpounds have been obtained by isonitrile-based multi component reactions (IMCRs). The typical IMCR Ugi-protocols offer a simple experimental setup allowing structural variety by which labelled diketopiperazines (DKPs) and peptide-peptoid chimera have been synthesized. The reaction keeps the paramagnetic spin label intact and offers a simple and versatile route to a large variety of new and chemically diverse spin labels.

New insights into the chemistry of Coenzyme Q-0: A voltammetric and spectroscopic study.

Coenzyme Q-0 (CoQ-0) is the only Coenzyme Q lacking an isoprenoid group on the quinoid ring, a feature important for its physico-chemical properties. Here, the redox behavior of CoQ-0 in buffered and non-buffered aqueous media was examined. In buffered aqueous media CoQ-0 redox chemistry can be described by a 2-electron-2-proton redox scheme, characteristic for all benzoquinones. In non-buffered media the number of electrons involved in the electrode reaction of CoQ-0 is still 2; however, the number of protons involved varies between 0 and 2. This results in two additional voltammetric signals, attributed to 2-electrons-1H(+) and 2-electrons-0H(+) redox processes, in which mono- and di-anionic compounds of CoQ-0 are formed. In addition, CoQ-0 exhibits a complex chemistry in strong alkaline environment. The reaction of CoQ-0 and OH(-) anions generates several hydroxyl derivatives as products. Their structures were identified with HPLC/MS. The prevailing radical reaction mechanism was analyzed by electron paramagnetic resonance spectroscopy. The hydroxyl derivatives of CoQ-0 have a strong antioxidative potential and form stable complexes with Ca(2+) ions. In summary, our results allow mechanistic insights into the redox properties of CoQ-0 and its hydroxylated derivatives and provide hints on possible applications.

Hydroxylated derivatives of dimethoxy-1,4-benzoquinone as redox switchable earth-alkaline metal ligands and radical scavengers.

Benzoquinones (BQ) have important functions in many biological processes. In alkaline environments, BQs can be hydroxylated at quinoid ring proton positions. Very little is known about the chemical reaction leading to these structural transformations as well as about the properties of the obtained hydroxyl benzoquinones. We analyzed the behavior of the naturally occurring 2,6-dimethoxy-1,4-benzoquinone under alkaline conditions and show that upon substitution of methoxy-groups, poly-hydroxyl-derivatives (OHBQ) are formed. The emerging compounds with one or several hydroxyl-substituents on single or fused quinone-rings exist in oxidized or reduced states and are very stable under physiological conditions. In comparison with the parent BQs, OHBQs are stronger radical scavengers and redox switchable earth-alkaline metal ligands. Considering that hydroxylated quinones appear as biosynthetic intermediates or as products of enzymatic reactions, and that BQs present in food or administered as drugs can be hydroxylated by enzymatic pathways, highlights their potential importance in biological systems.