5-doxylstearic Acid Research Articles

Fatty acid binding to the human transport proteins FABP3, FABP4, and FABP5 from a Ligand’s perspective

Fatty acid binding proteins (FABPs) are a family of amphiphilic transport proteins with high diversity in terms of their amino acid sequences and binding preferences. Beyond their main biological role as cytosolic fatty acid transporters, many aspects regarding their binding mechanism and functional specializations in human cells remain unclear. In this work, the binding properties and thermodynamics of FABP3, FABP4 and FABP5 were analyzed under various physical conditions. For this purpose, the FABPs were loaded with fatty acids bearing fluorescence or spin probes as model ligands, comparing their binding affinities via microscale thermophoresis (MST) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy. The CW EPR spectra of non-covalently bound 5- and 16-DOXYL stearic acid (5/16-DSA) deliver in-depth information about the dynamics and chemical environments of ligands inside the binding pockets of the FABPs. EPR spectral simulations allow the construction of binding curves, revealing two different binding states (‘intermediately’ and ‘strongly’ bound). The proportion of bound 5/16-DSA depends strongly on the FABP concentration and the temperature, but with remarkable differences between the three isoforms. Additionally, the more dynamic state (‘intermediately bound’) seems to dominate at body temperature with thermodynamic preference. The ligand binding studies were supplemented by aggregation studies via dynamic light scattering (DLS) and bioinformatic analyses. Beyond the remarkably fine-tuned binding properties exhibited by each FABP, which were discernible with our EPR-centered approach, the results of this work attest the power of simple spectroscopic experiments to provide new insights into the ligand binding mechanisms of proteins in general on a molecular level.

Application of electron paramagnetic resonance spectroscopy for determining the relative nanoenvironment fluidity of polymeric micelles.

Polymeric micelles are nanocarriers for drug, protein and gene delivery due to their unique core/shell structure, which encapsulates and protects therapeutic cargos with diverse physicochemical properties. However, information regarding the micellar nanoenvironment's fluidity can provide unique insight into their makeup. In this study, we used electron paramagnetic resonance (EPR) spectroscopy to study free radical spin probe (5-doxylstearate methyl ester, 5-MDS, and 16-doxylstearic acid, 16-DS) behaviour in methoxy-poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (PEO-PBCL) and methoxy-poly(ethylene oxide)-poly(ε-caprolactone) (PEO-PCL) polymeric micelles. Spin probes provided information about the spectroscopic rotational correlation time (τ, s) and the spectroscopic partition parameter F. We hypothesized that spin probes would partition into the polymeric micelles, and these parameters would be calculated. The results showed that both 5-MDS and 16-DS spectra were modulated in the presence of polymeric micelles. Based on τ values, 5-MDS revealed that PEO-PCL (τ = 3.92 ± 0.26 × 10-8s) was more fluid than PEO-PBCL (τ = 7.15 ± 0.63 × 10-8s). The F parameter, however, could not be calculated due to the rotational hindrance of the probe within the micelles. With 16-DS, more probe rotation was observed, and although the F parameter could be calculated, it was not helpful to distinguish the micelles' fluidity. Also, doxorubicin-loading interfered with the spin probes, particularly for 16-DS. However, using simulations, we could distinguish the hydrophilic and hydrophobic components of the 16-DS probe. The findings suggest that EPR spectroscopy is a valuable method for determining core fluidity in polymeric micelles.

Quantification of Age-Related Changes in the Lateral Organization of the Lipid Portion of the Intact Membranes Isolated from the Left and Right Eye Lenses of the Same Human Donor.

The continuous wave EPR spin-labeling method was used to evaluate age-related changes in the amounts of phospholipids (PLs) and cholesterol (Chol) in domains present in intact, cortical, and nuclear fiber cell plasma membranes isolated separately from the left and right eye lenses of the same human donor. The relative amounts of boundary plus trapped PLs were evaluated with the PL analog 12-doxylstearic acid spin label (12-SASL) and the relative amounts of trapped Chol with the Chol analog androstane spin label (ASL). The donors ranged in age from 15 to 70 years. Both the left and right eye lenses from donors aged 60, 65, and 70 years had nuclear cataracts; additionally, the right eye lens only of the 60-year-old donor had a cortical cataract. In transparent lenses, the relative amounts of boundary plus trapped PLs increase monotonously with donor age, and, at all ages, this amount was greater in nuclear compared with cortical membranes. Moreover, in transparent lenses, the relative amount of trapped Chol increases with age in nuclear membranes. However, the EPR spectrum of ASL from cortical membranes of 15- to 60-year-old donors shows only the weakly immobilized component assigned to ASL in the bulk plus Chol bilayer domain. Only the cortical membranes of 61- to 70-year-old donors contain both weakly and strongly immobilized components. The strongly immobilized component is assigned to ASL in trapped lipids. We speculate that the age of 60 years may be considered as a "threshold" for appearance of trapped lipids in cortical membranes. The relative amounts of boundary plus trapped PLs in lenses with nuclear cataracts is lower than that predicted from the tendency of the age-dependent increase observed for transparent lenses. The differences in amounts of lipids in the indicated left and right eye domains of each donor are smaller than the differences in single donors of a similar age.

EPRI pristup u izučavanju prodiranja tretinoinskih lipozoma kroz duboke slojeve kože

Introduction: Tretinoin has a variety of skin-beneficial properties. However, when used at the dosages required to get the best outcomes, it also has significant adverse effects. Aim: Side-effects caused by tretinoin could be avoided or minimized by integratingit into lecithin liposomes, which have long-term stability and the ability to penetrate into the deep layers of skin. Material and Methods: Electron paramagnetic resonant spectroscopy imaging (EPRI) was used to study deep skin penetration of hydrogenated lecithin liposomes containing tretinoin, into the porcine skin. To make liposomes EPR-detectable, they were spin-labeled using 16-doxylstearic acid. This paper is a part of an academic (non commercial) study. Results and Conclusion: EPRI proved to be a powerful technique for tracking the spatial distribution of liposomes in the tissue (skin), with the potential to be applied in pharmaceutical trials. To the best of our knowledge, herein it was used for the first time to track the distribution of tretinoin liposomes through the skin.

Pentacyclic triterpenes modulate liposome membrane fluidity and permeability depending on membrane cholesterol content

Since the membrane-related processes represent an integral part of the biological activities of drugs, their effect on the membrane dynamics is actually considered. In this study, we investigated the effect of pentacyclic triterpenes (TTPs), oleanolic acid (OA) and erythrodiol (ER), on the fluidity and permeability of liposomes membranes differing by their cholesterol content. All liposomes were prepared by reverse phase evaporation technique (REV). Spin-labeled liposomes exposed or not to TTPs were used for fluidity studies by using 5- and 16-doxyl stearic acids (DSA). TTPs-loaded liposomes (phospholipid:cholesterol of 1:1), and preformed vesicles exposed to TTPs were used for permeability studies by monitoring the release of sulforhodamine B (SRB) at 37 °C. The apparent release constants of SRB were determined by Higuchi model based on a biphasic curve shape (0–10 h; 10–48 h). TTPs-loaded liposomes were characterized for their size and homogeneity. Results showed that ER increased the membrane fluidity at the upper region of the membrane while the both TTPs produced a condensing effect at the deeper region of the membrane. The membrane composition was a critical parameter modulating the effect of TTPs on the membrane permeability. Also, this study consolidated the fact that a fluidizing membrane agent is not necessarily a permeabilizing-membrane compound.

Electron Spin Resonance Evaluation of Buccal Membrane Fluidity Alterations by Sodium Caprylate and L-Menthol.

The ability of sodium caprylate and l-menthol to fluidize phospholipid bilayers composed of lipids simulating the buccal epithelium was investigated using electron spin resonance (ESR) to evaluate the action of these agents as permeation enhancers. 5-Doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA) were used as spin labels to identify alterations in membrane fluidity near the polar head groups or inner acyl regions of the lipid bilayer, respectively. The molecular motion of both 5-DSA and 16-DSA showed increased disorder near the polar and inner hydrophobic regions of the bilayer in the presence of sodium caprylate suggesting fluidization in both the regions, which contributes to its permeation enhancing effects. L-menthol decreased the order parameter for 16-DSA, showing membrane fluidization only in the inner acyl regions of the bilayer, which also corresponded to its weaker permeation enhancing effects. The rapid evaluation of changes in fluidity of the bilayer in the presence of potential permeation enhancers using ESR enables improved selection of effective permeation enhancers and enhancer combinations based on their effect on membrane fluidization.

Radical reactions induced by ketoprofen in phospholipid membranes under ultraviolet light irradiation

2-(3-Benzoylphenyl)propanoic acid (ketoprofen), one of the nonsteroidal anti-inflammatory drugs, causes photocontact dermatitis by ultraviolet (UV) light as a side effect. In this study, we examined radical reactions induced by ketoprofen in the lipid membranes under UV irradiation using egg yolk phosphatidylcholine (egg-PC) liposomal membranes containing 5- or 16-doxyl stearic acid (5- or 16-DSA), which carry nitroxyl radical at the 5- or 16-position of the fatty acid chain, respectively. When the suspension of liposomal membrane was mixed with ketoprofen and irradiated with UV, electron spin resonance signal of 5- and 16-DSA in the membrane decreased. The decay consisted of fast decay and subsequent slow decay. The overall decay for 5-DSA was faster than that for 16-DSA. The rate of slower decay of 16-DSA increased with ketoprofen concentration. The bulk lipid in the membrane affected the rate of slower decay of 5-DSA; the rate increased with the amount of egg-PC and decreased in the rigid membrane composed of dipalmitoylphosphatidylcholine. When spin trapping studies with α-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN) and 5,5-dimetyl-1-pyrroline-N-oxide (DMPO) were performed in ketoprofen solution, C-centered radical adducts of POBN and superoxide anion radical adducts of DMPO were detected after UV irradiation. POBN suppressed the signal decay of 5-DSA in the liposomal membrane, whereas superoxide dismutase accelerated it. These results support that ketoprofen penetrates the lipid membrane and induces a radical reaction near the polar region in the membrane, and that ketoprofen-related C-centered radical is involved in the radical reaction.

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.

Systematic Quantification of Electron Transfer in a Bare Phospholipid Membrane Using Nitroxide-Labeled Stearic Acids: Distance Dependence, Kinetics, and Activation Parameters.

In this report, we present a method to characterize the kinetics of electron transfer across the bilayer of a unilamellar liposome composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The method utilizes synthetic phospholipids containing noninvasive nitroxide spin labels having the >N–O• moiety at well-defined distances from the outer surface of the liposome to serve as reporters for their local environment and, at the same time, permit measurement of the kinetics of electron transfer. We used 5-doxyl and 16-doxyl stearic acids. The paramagnetic >N–O• moiety is photo-oxidized to the corresponding diamagnetic oxoammonium cation by a ruthenium electron acceptor formed in the solution. Electron transfer is monitored by three independent spectroscopic methods: by both steady-state and time-resolved electron paramagnetic resonance and by optical spectroscopy. These techniques allowed us to differentiate between the electron transfer rates of nitroxides located in the outer leaflet of the phospholipid bilayer and of those located in the inner leaflet. Measurement of electron transfer rates as a function of temperature revealed a low-activation barrier (ΔG‡ ∼ 40 kJ/mol) that supports a tunneling mechanism.

Nanoscopic Characterization of Stearic Acid Release from Bovine Serum Albumin Hydrogels.

The release behavior of 16-doxyl stearic acid (16-DSA) from hydrogels made from bovine serum albumin (BSA) is characterized. 16-DSA serves as a model tracer molecule for amphiphilic drugs. Various hydrogel preparation procedures are tested and the fatty acid release from the different gels is compared in detail. These comparisons reach from the macroscopic level, the viscoelastic behavior via rheological characterization to changes on the nanoscopic level concerning the secondary structure of the protein during gelation through infrared (ATR-IR) spectroscopy. 16-DSA-BSA interaction via continuous wave electron paramagnetic resonance (CW EPR) spectroscopy in addition gives a nanoscopic view of small molecule-hydrogel interaction. The combined effects of fatty acid concentration, hydrogel incubation time, and gelation procedures on release behavior are studied via CW EPR spectroscopy and dynamic light scattering (DLS) measurements, which provide deep insight on the interaction of 16-DSA with BSA hydrogels and the nature and size of the released components, respectively. It is found that the release rate of the fatty acid from BSA hydrogels depends on and can thus be tuned through its loading percentage, duration of hydrogel formation and the type of gelation methods. All of the results confirm the potential of these gels as delivery hosts in pharmaceutical applications allowing the sustained release of drug.

Interaction of Catechins with Human Erythrocytes.

The aim of this study was to characterize the interaction of chosen catechins ((+)-catechin, (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCG)) with human erythrocytes and their protective effects against oxidative damage of erythrocytes. Uptake of the catechins by erythrocytes was studied by fluorimetry, their interaction with erythrocyte membrane was probed by changes in erythrocyte osmotic fragility and in membrane fluidity evaluated with spin labels, while protection against oxidative damage was assessed by protection against hemolysis induced by permanganate and protection of erythrocyte membranes against lipid peroxidation and protein thiol group oxidation. Catechin uptake was similar for all the compounds studied. Accumulation of catechins in the erythrocyte membrane was demonstrated by the catechin-induced increase in osmotic resistance and rigidification of the erythrocyte membrane detected by spin labels 5-doxyl stearic acid and 16-doxyl stearic acid. (−)-Epigallocatechin and EGCG inhibited erythrocyte acetylcholinesterase (mixed-type inhibition). Catechins protected erythrocytes against permanganate-induced hemolysis, oxidation of erythrocyte protein thiol groups, as well as membrane lipid peroxidation. These results contribute to the knowledge of the beneficial effects of catechins present in plant-derived food and beverages.

ESR Spectroscopy Insight on the Anticancer Drug Mitoxantrone Location in Membrane Mimetic Systems

The location of mitoxantrone molecule in micelles formed by bile salts (sodium taurocholate (NaTC) and sodium taurodeoxycholate (NaTDC)) and sodium dodecyl sulfate (SDS) have been investigated by electron spin resonance (ESR) spectroscopy, using three doxylstearic acid probes (5-, 12- and 16-doxylstearic acid abbreviated as 5-DSA, 12-DSA and 16-DSA). The analysis of ESR parameters of these spin probes evidenced slower dynamics induced by mitoxantrone that vary in the following order: 12-DSA > 5-DSA > 16-DSA for both bile salts micelles and 5-DSA > 12-DSA > 16-DSA for SDS micelles. The ESR parameters are slightly sensitive to variation of pH. These results indicate that the spin probes target different regions in these aggregates. Keywords: mitoxantrone, bile salts micelles, SDS micelles, ESR spectroscopy

Structural Analysis of the Stratum Corneum using EPR and EPR Imaging with Stable Spin Probes.

X-band (9.4 GHz) electron paramagnetic resonance (EPR) and electron paramagnetic resonance imaging (EPRI) were used for elucidating structural aspects of the stratum corneum (SC). We found that psoriasis vulgaris (PV)-SC has a less-ordered structure than that of the control SC, indicating the abnormal architecture of PV-SC. Different spectra were observed for the PV-SC. The three-line spectral pattern suggests that the 5-doxylstearic acid (5-DSA) is mobile or less rigid in the SC. The simulated order parameter (S0) value obtained for 5-DSA in the SC was approximately 0.20. The statistical analysis suggests that the value of the PV-SC is significantly smaller than that of the control (p < 0.01). Thus, we suggest that this EPR assay is of great use for evaluating SC function. In addition, the EPRI of various SC samples provides a useful image concerning SC status. A strong red image was observed for the PV skin. No red lesion region was observed in the control. The EPR images differentiated various sizes and number distribution concerning the disordered states in the SC.

A novel dual mode X-band EPR resonator for rapid in situ microwave heating.

A unique dual mode X-band Continuous Wave (CW) EPR resonator designed for simultaneous EPR measurement and rapid microwave (MW) induced sample heating is described. Chemical reactions subjected to a flow of energy and matter can be perturbed away from the thermodynamic equilibrium by imposing a rapid shock or physical change to the system. Depending on the magnitude of the perturbation, these changes can dictate the subsequent evolution of the entire system, allowing for instance to populate non-equilibrium reactive intermediate states. Temperature jump (T-jump) experiments are a common method to achieve such perturbations. Most T-jump experiments are based on Joule Heating methods or IR lasers. Here we demonstrate the principle of rapid sample heating based on microwaves. The benefits of MW heating include (i) rapid and efficient heating (i.e. using a tuned resonant cavity, >99% efficient power transfer to the sample can be achieved), and (ii) volumetric heating (i.e. the entire sample volume rises in temperature at once, since heat is generated in the sample instead of being transferred to it). Accordingly, the key concept of the design is the use of a cavity resonator allowing EPR detection (at 9.5GHz) and simultaneous sample heating (at 6.1GHz). Temperature increments of 50°C within a few seconds are possible. This is evidenced and illustrated here by probing the temperature-induced variation of the rotational dynamics of 16-doxyl stearic acid methyl ester (16-DSE) spin probe grafted on the surface of sodium dodecyl sulphate (SDS) micelles in water, as well as copper (II) acetylacetonate in chloroform. Rapid changes in the rotational dynamics of the paramagnetic centres provide direct evidence for the in situ and simultaneous EPR measurement-heating capabilities of the resonator. Improvements afforded by the use of pulsed MW sources will enable faster heating time scales to be achieved. In the longer term, this current study demonstrates the simple and direct possibilities for using MW heating as a means of performing T-jump experiments.

The Physicochemical Characteristics of Serum Albumin and Erythrocyte Cell Membranes under Normal and Heart Failure Symptom Conditions

Abstract—Spin labeling EPR spectroscopy was used to study the structural and conformational characteristics of human serum albumin and human erythrocyte membranes under normal conditions and with symptoms of heart failure. 5-Doxyl stearic acid and 16-doxyl stearic acid were used as spin labels, whose paramagnetic NO fragments are bound to different sites of the hydrocarbon chain. The EPR spectra of 16-doxyl stearic acid indicate that in the physiological temperature range serum albumin molecules are characterized by several types of fatty acid binding sites, which differ in parameters of spin-label rotational diffusion. This distribution of fatty-acid binding sites was typical for the blood serum of all patients who participated in our study, regardless of deviations from the normal blood parameters. The microviscosity of erythrocyte membranes from patient blood was measured using both 5-doxyl stearic and 16-doxyl stearic spin labels, whose paramagnetic fragments are located at different depths inside the lipid bilayer. It was found that in patients with an increased erythrocyte distribution width, the membrane lipid microviscosity is statistically significantly higher than under normal conditions.

Evaluation of heating effects on the morphology and membrane structure of Escherichia coli using electron paramagnetic resonance spectroscopy

Bacterial cell characteristics, such as size, morphology, and membrane integrity, are affected by environmental conditions. Thermal treatment results in related structural changes, extent of which is determined by the microorganism's survival skills and inactivation kinetics. The objective of this study was to characterize changes in cell structure of Escherichia coli during heating using the combined analysis of dynamic light scattering (DLS), electron paramagnetic resonance (EPR) spectroscopy, and transmission electron microscopy (TEM) techniques. The size of E. coli cells increased from 2.3 μm to 3.0 μm with heating up to 50 °C followed by a shrinkage with further heating up to 70 °C. The morphological changes were verified using transmission electron microscopy. Related changes in membrane integrity was quantified via the mobility of 16-doxylstearic acid (16-DSA) spin probe using EPR spectroscopy. Two order parameters S1 and S2 defined on x- and y-axes, respectively, decreased with increasing temperature indicating loss of membrane integrity. The combined techniques as in this study can be used to further understand factors that play role in survival behavior of microorganisms.

Redox properties and human serum albumin binding of nitro-oleic acid

Nitro-fatty acids modulate inflammatory and metabolic stress responses, thus displaying potential as new drug candidates. Herein, we evaluate the redox behavior of nitro-oleic acid (NO2-OA) and its ability to bind to the fatty acid transporter human serum albumin (HSA). The nitro group of NO2-OA underwent electrochemical reduction at −0.75 V at pH 7.4 in an aqueous milieu. Based on observations of the R–NO2 reduction process, the stability and reactivity of NO2-OA was measured in comparison to oleic acid (OA) as the negative control. These electrochemically-based results were reinforced by computational quantum mechanical modeling. DFT calculations indicated that both the C9-NO2 and C10-NO2 positional isomers of NO2-OA occurred in two conformers with different internal angles (69° and 110°) between the methyl- and carboxylate termini. Both NO2-OA positional isomers have LUMO energies of around −0.7 eV, affirming the electrophilic properties of fatty acid nitroalkenes. In addition, the binding of NO2-OA and OA with HSA revealed a molar ratio of ~7:1 [NO2-OA]:[HSA]. These binding experiments were performed using both an electrocatalytic approach and electron paramagnetic resonance (EPR) spectroscopy using 16-doxyl stearic acid. Using a Fe(DTCS)2 spin-trap, EPR studies also showed that the release of the nitro moiety of NO2-OA resulted in the formation of nitric oxide radical. Finally, the interaction of NO2-OA with HSA was monitored via Tyr and Trp residue electro-oxidation. The results indicate that not only non-covalent binding but also NO2-OA-HSA adduction mechanisms should be taken into consideration. This study of the redox properties of NO2-OA is applicable to the characterization of other electrophilic mediators of biological and pharmacological relevance.

Correction of Cold Damage to Mammalian Erythrocytes by Chlorpromazine to Influence the Dynamic Structure of a Membrane

Abstract—The resistance of mammalian erythrocytes (human, dog, horse, bull, and rabbit) to cold shock in the presence of chlorpromazine was studied; its effect on the state of erythrocyte membranes was investigated. The bond strength between membrane phospholipids of mammalian erythrocytes and the characteristics of the cold shock of these cells was determined with the correlation analysis. It has been established that erythrocytes of mammals whose membranes contain a high content of sphingomyelin and phosphatidylethanolamine and a low content of phosphatidylcholines are more resistant to cold shock. Chlorpromazine showed high efficiency in protecting mammalian erythrocytes against cold shock. The dynamic structure of membranes of human erythrocytes treated with the chlorpromazine was studied by EPR of spin-probes. The use of a set of spin-labeled probes: palmitic acid amide, 5-DOXYL stearic acid, and 16-DOXYL stearic acid, which allow studying the micro-viscosity parameters of the lipid bilayer of erythrocyte membranes in the transmembrane direction, made it possible to establish the locations of chlorpromazine action, namely, in the region of polar heads and hydrophobic tails of membrane phospholipids.

The Influence of Dinitrosyl Iron Complexes on the Physicochemical Characteristics of Rat Blood Components

Abstract—The effect of injected intravenously dinitrosyl iron complexes with glutathione ligand on the structural and conformational characteristics of albumin, which is a primary ligand of these complexes in plasma, and on molecules of the lipid phase in the erythrocyte membrane, which constitute the microenvironment of dinitrosyl iron complexes in erythrocytes, was analyzed. The analysis of EPR spectra of 5- and 16-DOXYL stearic acid spin probes in plasma and erythrocyte suspensions before and after injection of dinitrosyl iron complexes with glutathione was performed in the experiments. It has been found that injection of these complexes into an organism does not affect the molecular motion in albumin hydrophobic regions in plasma and erythrocyte membranes, while at the same time they stimulate incorporation of charged spin labels into the erythrocyte membrane. Apparently, this was due to a change in the charge on the outer surface of the erythrocytes.

Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.