Measuring local pH at interfaces from molecular tumbling: A concept for designing EPR-active pH-sensitive labels and probes.

Molecular probes and indicators are broadly employed for pH measurements in bulk media and at interfaces. The underlying physical principle of pH measurements of most of these probes is based on a change in the electronic structure that, for example, results in a shift of the emission peak of the fluorescence probes, changes in NMR chemical shifts due to the affected electronic shielding, or magnetic parameters of pH-sensitive nitroxides as measured by EPR. Here we explore another concept for measuring local protonation state of molecular tags based on changes in rotational dynamics of electron spin-bearing moieties that are readily detected by conventional continuous wave X-band EPR. Such changes are especially pronounced at biological interfaces, such as lipid bilayer membranes, due to the probe interactions with adjacent charges and polarizable dipoles. The concept was demonstrated by synthesizing a series of pH-sensitive nitroxides and spin-labelled phospholipids. EPR spectra of these newly synthesized nitroxides exhibit relatively small - about 0.5 G - changes in isotropic nitrogen hyperfine coupling constant upon reversible protonation. However, spin-labelled phospholipids incorporated into lipid bilayers demonstrated almost 6-fold change in rotational correlation time upon protonation, readily allowing for pKa determination from large changes in EPR spectra. The demonstrated concept of EPR-based pH measurements leads to a broader range of potential nitroxide structures that can serve as molecular pH sensors at the desired pH range and, thus, facilitates further development of spin-labelling EPR methods to study electrostatic phenomena at chemical and biological interfaces.

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and an inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.

Liquid Metal Nanoparticles as Initiators for Radical Polymerization of Vinyl Monomers.

Sonication of gallium or gallium-based liquid metals in an aqueous solution of vinyl monomers leads to rapid free radical polymerization (FRP), without the need for conventional molecular initiators. Under ambient conditions, a passivating native oxide separates these metals from solution and renders the metal effectively inert. However, sonication generates liquid metal nanoparticles (LMNPs) of ∼100 nm diameter and thereby increases the surface area of the metal. The exposed metal initiates polymerization, which proceeds via a FRP mechanism and yields high molecular weight polymers that can form physical gels. Spin trapping EPR reveals the generation of free radicals. Time-of-flight secondary ion mass spectrometry measurements confirm direct polymer bonding to gallium, verifying the formation of surface-anchored polymer grafts. The grafted polymers can modify the interfacial properties, that is, the preference of the metal particles to disperse in aqueous versus organic phases. The polymer can also be degrafted and isolated from the particles using strong acid or base. The concept of physically disrupting passivated metal surfaces offers new routes for surface-initiated polymerization and has implications for surface modification, reduction reactions, and fabrication of mechanically responsive materials.

Enhancing sensitivity of Double Electron-Electron Resonance (DEER) by using Relaxation-Optimized Acquisition Length Distribution (RELOAD) scheme.

Over the past decades pulsed electron-electron double resonance (PELDOR), often called double electron-electron resonance (DEER), became one of the major spectroscopic tools for measurements of nanometer-scale distances and distance distributions in non-crystalline biological and chemical systems. The method is based on detecting the amplitude of the primary (3-pulse DEER) or refocused (4-pulse DEER) spin echo for the so-called "observer" spins when the other spins coupled to the former by a dipolar interaction are flipped by a "pump" pulse at another EPR frequency. While the timing of the pump pulse is varied in steps, the positions of the observer pulses are typically fixed. For such a detection scheme the total length of the observer pulse train and the electron spin memory time determine the amplitude of the detected echo signal. Usually, the distance range considerations in DEER experiments dictate the total length of the observer pulse train to exceed the phase memory time by a factor of few and this leads to a dramatic loss of the signal-to-noise ratio (SNR). While the acquisition of the DEER signal seems to be irrational under such conditions, it is currently the preferred way to conduct DEER because of an effective filtering out of all other unwanted interactions. Here we propose a novel albeit simple approach to improve DEER sensitivity and decrease data acquisition time by introducing the signal acquisition scheme based on RELaxation Optimized Acquisition (Length) Distribution (DEER-RELOAD). In DEER-RELOAD the dipolar phase evolution signal is acquired in multiple segments in which the observer pulses are fixed at the positions to optimize SNR just for that specific segment. The length of the segment is chosen to maximize the signal acquisition efficiency according the phase relaxation properties of the spin system. The total DEER trace is then obtained by "stitching" the multiple segments into a one continuous trace. The utility of the DEER-RELOAD acquisition scheme has been demonstrated on an example of the standard 4-pulse DEER sequence applied to two membrane protein complexes labeled with nitroxides. While theoretical gains from the DEER-RELOAD scheme increase with the number of stitched segments, in practice, even dividing the acquisition of the DEER trace into two segments may improve SNR by a factor of >3, as it has been demonstrated for one of these two membrane proteins.

Refocused Out-Of-Phase (ROOPh) DEER: A pulse scheme for suppressing an unmodulated background in double electron-electron resonance experiments.

EPR pulsed dipolar spectroscopy (PDS) is indispensable for measurements of nm-scale distances between electronic spins in biological and other systems. While several useful modifications and pulse sequences for PDS have been developed in recent years, DEER experiments utilizing pump and observer pulses at two different frequencies remain the most popular for practical applications. One of the major drawbacks of all the available DEER approaches is the presence of a significant unmodulated fraction in the detected signal that arises from an incomplete inversion of the coupled spins by the pump pulse. The latter fraction is perceived as one of the major sources of error for the reconstructed distance distributions. We describe an alternative detection scheme - a Refocused Out-Of-Phase DEER (ROOPh-DEER) - to acquire only the modulated fraction of the dipolar DEER signal. When Zeeman splitting is small compared to the temperature, the out-of-phase magnetization components cancel each other and are not observed in 4-pulse DEER experiment. In ROOPh-DEER these components are refocused by an additional pump pulse while the in-phase component containing an unmodulated background is filtered out by a pulse at the observed frequency applied right at the position of the refocused echo. Experimental implementation of the ROOPh-DEER detection scheme requires at least three additional pulses as was demonstrated on an example of a 7-pulse sequence. The application of 7-pulse ROOPh-DEER sequence to a model biradical yielded the interspin distance of 1.94 ±â€¯0.07 nm identical to the one obtained with the conventional 4-pulse DEER, however, without the unmodulated background present as a dominant fraction in the latter signal.

Glycol Chitosan Engineered Autoregenerative Antioxidant Significantly Attenuates Pathological Damages in Models of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the foremost cause of irreversible blindness in people over the age of 65 especially in developing countries. Therefore, an exploration of effective and alternative therapeutic interventions is an unmet medical need. It has been established that oxidative stress plays a key role in the pathogenesis of AMD, and hence, neutralizing oxidative stress is an effective therapeutic strategy for treatment of this serious disorder. Owing to autoregenerative properties, nanoceria has been widely used as a nonenzymatic antioxidant in the treatment of oxidative stress related disorders. Yet, its potential clinical implementation has been greatly hampered by its poor water solubility and lack of reliable tracking methodologies/processes and hence poor absorption, distribution, and targeted delivery. The water solubility and surface engineering of a drug with biocompatible motifs are fundamental to pharmaceutical products and precision medicine. Here, we report an engineered water-soluble, biocompatible, trackable nanoceria with enriched antioxidant activity to scavenge intracellular reactive oxygen species (ROS). Experimental studies with in vitro and in vivo models demonstrated that this antioxidant is autoregenerative and more active in inhibiting laser-induced choroidal neovascularization by decreasing ROS-induced pro-angiogenic vascular endothelial growth factor (VEGF) expression, cumulative oxidative damage, and recruitment of endothelial precursor cells without exhibiting any toxicity. This advanced formulation may offer a superior therapeutic effect to deal with oxidative stress induced pathogeneses, such as AMD.

Spin Probe Multi-Frequency EPR Study of Unprocessed Cotton Fibers.

Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity-Tempo and its more hydrophilic derivative Tempol-using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the strategy of spectral parameter sharing could be potentially applicable to other heterogeneous biological systems in addition to the cotton fibers studies here.

IKMTSL-PTE, a Phospholipid-Based EPR Probe for Surface Electrostatic Potential of Biological Interfaces at Neutral pH: Effects of Temperature and Effective Dielectric Constant of the Solvent.

The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0 of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0 of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17 °C, pKa0 = 6.16 ± 0.03 vs 20 °C, pKa0 = 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48 °C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKa and ε has been established and found to be essentially the same at 17 and 48 °C. The polarity term contributing to the pKa of IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKa values than those previously reported for IMTSL-PTE (e.g., at 17 °C, pKai = 7.80 ± 0.03 vs pKa0 = 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKa values close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes.

Effect of Solution Ionic Strength on the pKa of the Nitroxide pH EPR Probe 2,2,3,4,5,5-Hexamethylimidazolidin-1-oxyl.

Spin probe and spin labeling Electron Paramagnetic Resonance methods are indispensable research tools for solving a wide range of bioanalytical problems-from measuring microviscosity and polarity of phase-separated liquids to oxygen concentrations in tissues. One of the emerging uses of spin probes are the studies of proton transfer-related and surface electrostatic phenomena. The latter Electron Paramagnetic Resonance methods rely on molecular probes containing an additional functionality capable of reversible ionization (protonation, in particular) in the immediate proximity to an Electron Paramagnetic Resonance-active reporter group, such as (N-O•) for nitroxides. The consequent formation of protonated and nonprotonated nitroxide species with different magnetic parameters (A iso, g iso) could be readily distinguished by Electron Paramagnetic Resonance. Bioanalytical Electron Paramagnetic Resonance studies employing pH-sensitive paramagnetic probes typically involve determination of the equilibrium constant (pK a) between the protonated and nonprotonated forms of the nitroxide. However, any chemical equilibrium involving charged species, such as ionization of acids and bases, and so the reversible protonation of the nitroxide, is known to be affected by an ionic strength of the solution. Currently, only scarce data for the effect of the solution ionic strength on the experimental pK a's of the ionizable nitroxides can be found in the literature. Here we have carried out a series of Electron Paramagnetic Resonance titration experiments for aqueous solutions of 2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl (HMI) nitroxide known for one of the largest differences in the isotropic nitrogen hyperfine coupling constant A iso between the protonated and nonprotonated forms. Electrolyte concentration was varied over an exceptionally large range (i.e., from 0.05 to 5.0 M) to elucidate the effect of ionic strength on the ionization constant of this pH-sensitive Electron Paramagnetic Resonance probe and the data were compared to the Debye-Hückel limiting law. Effects of the ionic strength on the magnetic parameters of the ionizable nitroxides are also discussed.

The Hydroxyl Radical is a Critical Intermediate in the Voltammetric Detection of Hydrogen Peroxide.

Cyclic voltammetry is a widely used and powerful tool for sensitively and selectively measuring hydrogen peroxide (H2O2). Herein, voltammetry was combined with electron paramagnetic resonance spectroscopy to identify and define the role of an oxygen-centered radical liberated during the oxidation of H2O2. The spin-trap reagents, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO), were employed. Spectra exhibit distinct hyperfine patterns that clearly identify the DMPO(•)-OH and EMPO(•)-OH adducts. Multiple linear regression analysis of voltammograms demonstrated that the hydroxyl radical is a principal contributor to the voltammetry of H2O2, as signal is attenuated when this species is trapped. These data incorporate a missing, fundamental element to our knowledge of the mechanisms that underlie H2O2 electrochemistry.

Ionizable Nitroxides for Studying Local Electrostatic Properties of Lipid Bilayers and Protein Systems by EPR.

Electrostatic interactions are known to play a major role in the myriad of biochemical and biophysical processes. Here, we describe biophysical methods to probe local electrostatic potentials of proteins and lipid bilayer systems that are based on an observation of reversible protonation of nitroxides by electron paramagnetic resonance (EPR). Two types of probes are described: (1) methanethiosulfonate derivatives of protonatable nitroxides for highly specific covalent modification of the cysteine's sulfhydryl groups and (2) spin-labeled phospholipids with a protonatable nitroxide tethered to the polar head group. The probes of both types report on their ionization state through changes in magnetic parameters and degree of rotational averaging, thus, allowing the electrostatic contribution to the interfacial pKa of the nitroxide, and, therefore, the local electrostatic potential to be determined. Due to their small molecular volume, these probes cause a minimal perturbation to the protein or lipid system. Covalent attachment secures the position of the reporter nitroxides. Experimental procedures to characterize and calibrate these probes by EPR, and also the methods to analyze the EPR spectra by simulations are outlined. The ionizable nitroxide labels and the nitroxide-labeled phospholipids described so far cover an exceptionally wide range of ca. 2.5-7.0 pH units, making them suitable to study a broad range of biophysical phenomena, especially at the negatively charged lipid bilayer surfaces. The rationale for selecting proper electrostatically neutral interface for probe calibration, and examples of lipid bilayer surface potential studies, are also described.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Surface electrostatics of lipid bilayers by EPR of a pH-sensitive spin-labeled lipid.

Many biophysical processes such as insertion of proteins into membranes and membrane fusion are governed by bilayer electrostatic potential. At the time of this writing, the arsenal of biophysical methods for such measurements is limited to a few techniques. Here we describe a, to our knowledge, new spin-probe electron paramagnetic resonance (EPR) approach for assessing the electrostatic surface potential of lipid bilayers that is based on a recently synthesized EPR probe (IMTSL-PTE) containing a reversibly ionizable nitroxide tag attached to the lipids' polar headgroup. EPR spectra of the probe directly report on its ionization state and, therefore, on electrostatic potential through changes in nitroxide magnetic parameters and the degree of rotational averaging. Further, the lipid nature of the probe provides its full integration into lipid bilayers. Tethering the nitroxide moiety directly to the lipid polar headgroup defines the location of the measured potential with respect to the lipid bilayer interface. Electrostatic surface potentials measured by EPR of IMTSL-PTE show a remarkable (within ±2%) agreement with the Gouy-Chapman theory for anionic DMPG bilayers in fluid (48°C) phase at low electrolyte concentration (50 mM) and in gel (17°C) phase at 150-mM electrolyte concentration. This agreement begins to diminish for DMPG vesicles in gel phase (17°C) upon varying electrolyte concentration and fluid phase bilayers formed from DMPG/DMPC and POPG/POPC mixtures. Possible reasons for such deviations, as well as the proper choice of an electrostatically neutral reference interface, have been discussed. Described EPR method is expected to be fully applicable to more-complex models of cellular membranes.

Unique oxidation of imidazolidine nitroxides by potassium ferricyanide: strategy for designing paramagnetic probes with enhanced sensitivity to oxidative stress.

Potassium ferricyanide (PF), routinely employed for the oxidation of sterically-hindered hydroxylamines to nitroxides, is considered to be chemically inert towards the latter. In the present study, we report on an unexpected oxidative fragmentation of the imidazolidine nitroxides containing hydrogen atom in the 4-position of the heterocycle (HIMD) by PF resulting in the loss of the EPR signal. The mechanistic EPR, spectrophotometric, electrochemical and HPLC-MS studies support the assumption that the HIMD fragmentation is facilitated by the proton abstraction from the 4-position of the oxoammonium cation formed as a result of the initial one-electron HIMD oxidation. Increase in steric hindrance around the radical fragment by introducing ethyl substituents decreased the rate of ascorbate-induced HIMD reduction by more than 20 times, but did not affect the rate of ferricyanide-induced HIMD oxidation. This preferential sensitivity of HIMDs to oxidative processes has been used to detect peroxyl radicals in the presence of high concentration of the reducing agent, ascorbate. HIMD-based EPR probes capable to discriminate oxidative and reductive processes might find application in biomedicine and related fields for monitoring the oxidative stress and reactive radical species in biological systems.

Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity.

Emerging applications of nanosized iron oxides in nanotechnology introduce vast quantities of nanomaterials into the human environment, thus raising some concerns. Here we report that the surface of γ-Fe(2)O(3) nanoparticles 20-40 nm in diameter mediates production of highly reactive hydroxyl radicals (OH(•)) under conditions of the biologically relevant superoxide-driven Fenton reaction. By conducting comparative spin-trapping EPR experiments, we show that the free radical production is attributed primarily to the catalytic reactions at the nanoparticles' surface rather than being caused by the dissolved metal ions released by the nanoparticles as previously thought. Moreover, the catalytic centers on the nanoparticle surface were found to be at least 50-fold more effective in OH(•) radical production than the dissolved Fe(3+) ions. Conventional surface modification methods such as passivating the nanoparticles' surface with up to 935 molecules of oleate or up to 18 molecules of bovine serum albumin per iron oxide core were found to be rather ineffective in suppressing production of the hydroxyl radicals. The experimental protocols developed in this study could be used as one of the approaches for developing analytical assays for assessing the free radical generating activity of a variety of nanomaterials that is potentially related to their biotoxicity.

EPR detection of cellular and mitochondrial superoxide using cyclic hydroxylamines.

Superoxide (O2ⁱ⁻) has been implicated in the pathogenesis of many human diseases, but detection of the O(2)(•-) radicals in biological systems is limited due to inefficiency of O2ⁱ⁻ spin trapping and lack of site-specific information. This work studied production of extracellular, intracellular and mitochondrial O2ⁱ⁻ in neutrophils, cultured endothelial cells and isolated mitochondria using a new set of cationic, anionic and neutral hydroxylamine spin probes with various lipophilicity and cell permeability. Cyclic hydroxylamines rapidly react with O2ⁱ⁻, producing stable nitroxides and allowing site-specific cO2ⁱ⁻ detection in intracellular, extracellular and mitochondrial compartments. Negatively charged 1-hydroxy-4-phosphono-oxy-2,2,6,6-tetramethylpiperidine (PP-H) and positively charged 1-hydroxy-2,2,6,6-tetramethylpiperidin-4-yl-trimethylammonium (CAT1-H) detected only extramitochondrial O2ⁱ⁻. Inhibition of EPR signal by SOD2 over-expression showed that mitochondria targeted mitoTEMPO-H detected intramitochondrial O2ⁱ⁻ both in isolated mitochondria and intact cells. Both 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CM-H) detected an increase in cytoplasm O2ⁱ⁻ stimulated by PMA, but only CM-H and mitoTEMPO-H showed an increase in rotenone-induced mitochondrial O2ⁱ⁻. These data show that a new set of hydroxylamine spin probes provide unique information about site-specific production of the O2ⁱ⁻ radical in extracellular or intracellular compartments, cytoplasm or mitochondria.

Design of liposome-based pH sensitive nanoSPIN probes: nano-sized particles with incorporated nitroxides.

Liposome-based nanoSized Particles with Incorporated Nitroxides, or nanoSPINs, were designed for EPR applications as pH probes in biological systems. Phospholipid membrane of the liposomes with incorporated gramicidin A showed selective permeability to a small analyte, H(+), while protecting entrapped sensing nitroxide from biological reductants. An application of the pH-sensitive nanoSPIN in an ischemia model in rat heart homogenate allows for monitoring ischemia-induced acidosis while protecting encapsulated nitroxide against bioreduction.

Spin-labeled pH-sensitive phospholipids for interfacial pKa determination: synthesis and characterization in aqueous and micellar solutions.

The synthesis and characterization of spin-labeled phospholipids (SLP)--derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE)--with pH-reporting nitroxides that are covalently attached to the lipid's polar headgroup are being reported. Two lipids were synthesized by reactions of PTE with thiol-specific, pH-sensitive methanethiosulfonate spin labels methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL) and S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl)benzyl methanethiosulfonate (IKMTSL). The pKa values of the IMTSL-PTE lipid measured by EPR titration in aqueous buffer/isopropyl alcohol solutions of various compositions were found to be essentially the same (pKa approximately 2.35), indicating that in mixed aqueous/organic solvents, the amphiphilic lipid molecules could be shielded from changing bulk conditions by a local shell of solvent molecules. To overcome this problem, the spin-labeled lipids were modeled by synthesizing IMTSL- and IKMTSL-2-mercaptoethanol adducts. These model compounds yielded the intrinsic pKa0's for IMTSL-PTE and IKMTSL-PTE in aqueous buffers as 3.33 +/- 0.03 and 5.98 +/- 0.03, respectively. A series of EPR titrations of IMTSL-PTE in mixed water/isopropyl alcohol solution allowed for calibrating the polarity-induced pKa shifts, deltapKapol, vs bulk solvent dielectric permittivity. These calibration data allowed for estimating the local dielectric constant, epsilon(eff), experienced by the reporter nitroxide of the IMTSL-PTE lipid incorporated into the nonionic Triton X-100 micelles as 60 +/- 5 and 57 +/- 5 at 23 and 48 degrees C, respectively. For micelles formed from an anionic surfactant sodium dodecyl sulfate (SDS) the electrostatic-induced pKa shift, deltapKael = 2.06 +/- 0.04 units of pH, was obtained by subtracting the polarity-induced contribution. This shift yields psi = -121 mV electric potential of the SDS micelle surface.

Mapping local protein electrostatics by EPR of pH-sensitive thiol-specific nitroxide.

A first thiol-specific pH-sensitive nitroxide spin-label of the imidazolidine series, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL), has been synthesized and characterized. X-Band (9 GHz) and W-band (94 GHz) EPR spectral parameters of the new spin-label in its free form and covalently attached to an amino acid cysteine and a tripeptide glutathione were studied as a function of pH and solvent polarity. The pKa value of the protonatable tertiary amino group of the spin-label was found to be unaffected by other ionizable groups present in side chains of unstructured small peptides. The W-band EPR spectra were shown to allow for pKa determination from precise g-factor measurements. Is has been demonstrated that the high accuracy of pKa determination for pH-sensitive nitroxides could be achieved regardless of the frequency of measurements or the regime of spin exchange: fast at X-band and slow at W-band. IMTSL was found to react specifically with a model protein, iso-1-cytochrome c from the yeast Saccharomyces cerevisiae, giving EPR spectra very similar to those of the most commonly employed cysteine-specific label MTSL. CD data indicated no perturbations to the overall protein structure upon IMTSL labeling. It was found that for IMTSL, g iso correlates linearly with A iso, but the slopes are different for the neutral and charged forms of the nitroxide. This finding was attributed to the solvent effects on the spin density at the oxygen atom of the NO group and on the excitation energy of the oxygen lone-pair orbital.

Interfacial surface properties of thiol-protected gold nanoparticles: a molecular probe EPR approach.

We present a molecular probe technique for accessing interfacial surface electrostatics of ligand-protected gold nanoparticles. A series of ligands with variable length of the hydrocarbon bridge between the anchoring sulfur and the reporting pH-sensitive nitroxide is described. The protonation state of this probe is directly observed by EPR spectroscopy. For tiopronin-protected Au nanoparticles, we observed an increase in pKa of up to ca. 1.1 pH units that was affected by the position of the reporter moiety with respect to the monolayer interface.