Dysprosium and Holmium Vanadate Nanoprobes as High-Performance Contrast Agents for High-Field Magnetic Resonance and Computed Tomography Imaging.

We describe a wet chemical method for the synthesis of uniform and well-dispersed dysprosium vanadate (DyVO4) and holmium vanadate (HoVO4) nanoparticles with an almost spherical shape and a mean size of ∼60 nm and their functionalization with poly(acrylic acid). The transverse magnetic relaxivity of both systems at 9.4 T is analyzed on the basis of magnetic susceptibility and magnetization measurements in order to evaluate their potential for application as high-field MRI contrast agents. In addition, the X-ray attenuation properties of these systems are also studied to determine their capabilities as computed tomography contrast agent. Finally, the colloidal stability under physiological pH conditions and the cytotoxicity of the functionalized NPs are also addressed to assess their suitability for bioimaging applications.

Novel Dy2O3/ZnO-Au ternary nanocomposites: Green synthesis using pomegranate fruit extract, characterization and their photocatalytic and antibacterial properties.

In this study for the first time, high efficient, eco-friendly and novel Dy2O3/ZnO-Au ternary nanocomposites (Dy/ZnO-AuNCs) were prepared in presence of pomegranate fruit (PF) extract as capping and reducing agents (Dy/ZnO-AuNCs@PF). The influence of various parameters such as basic agents, reducing agents, sonication power, and sonication time were performed to reach the optimum condition. The formation of the products was characterized by FT-IR, HRTEM, XRD, FE-SEM, TEM, EDX and DRS techniques. The XRD and TEM analysis showed that the morphology and crystallite size of nanocomposites were spherical morphology and 85-90 nm, respectively. The obtained Dy/ZnO-AuNCs@PF were investigated as a nanocatalyst for degradation of erythrosine (ES) as anionic dye and basic violet 10 (BV10) as cationic dye under UV and visible light irradiations. The Dy/ZnO-AuNCs@PF exhibited higher photodegradation against ES (89.6%) and BV10 (91.3%) than pure Dy2O3 (63.1% for ES, 66.5% for BV10) and Dy2O3/ZnO (64.5% for ES, 70.8% for BV10) under UV irradiation. It was found that gold nanoparticles have significant effect on Dy/ZnO-AuNCs@PF catalytic performance for decomposition of organic pollutants. In addition, Dy/ZnO-AuNCs@PF showed excellent in-vitro antibacterial activity against A. baumannii, S. aureus and P. mirabilis with MIC and MBC values of (5, 80 mg/ml), (5, 40 mg/ml) and (2.5, 20 mg/ml), respectively. Generally, according to its excellent antibacterial and catalytic activity, Dy/ZnO-AuNCs@PF can be used in biomedical and environmental applications.

Biodegradable Zn-5Dy Alloy with Enhanced Osteo/Angio-Genic Activity and Osteointegration Effect via Regulation of SIRT4-Dependent Mitochondrial Function.

Zinc (Zn)-dysprosium (Dy) binary alloys are promising biodegradable bone fracture fixation implants owing to their attractive biodegradability and mechanical properties. However, their clinical application is a challenge for bone fracture healing, due to the lack of Zn-Dy alloys with tailored proper bio-mechanical and osteointegration properties for bone regeneration. A Zn-5Dy alloy with high strength and ductility and a degradation rate aligned with the bone remodeling cycle is developed. Here, mechanical stability is further confirmed, proving that Zn-5Dy alloy can resist aging in the degradation process, thus meeting the mechanical requirements of fracture fixation. In vitro cellular experiments reveal that the Zn-5Dy alloy enhances osteogenesis and angiogenesis by elevating SIRT4-mediated mitochondrial function. In vivo Micro-CT, SEM-EDS, and immunohistochemistry analyses further indicate good biosafety, suitable biodegradation rate, and great osteointegration of Zn-5Dy alloy during bone healing, which also depends on the upregulation of SIRT4-mediated mitochondrial events. Overall, the study is the first to report a Zn-5Dy alloy that exerts remarkable osteointegration properties and has a strong potential to promote bone healing. Furthermore, the results highlight the importance of mitochondrial modulation and shall guide the future development of mitochondria-targeting materials in enhancing bone fracture healing.

Outstanding MRI contrast with dysprosium phosphate nanoparticles of tuneable size.

The use of high-field magnets for magnetic resonance imaging (MRI) is expected to experience the fastest growth rate during the present decade. Although several CAs for MRI scanners using high magnetic fields have been reported, they are mostly based on fluoride matrices, which are known for their low chemical stability in aqueous suspensions. Chemically stable MRI CAs for high-field magnets are therefore needed to enable the advances in MRI technique. Herein, we synthesized uniform DyPO4 nanoparticles (NPs) with tuneable sizes between 23 and 57 nm using homogeneous precipitation in butanol. The NPs were successfully functionalized with polyacrylic acid (PAA) and showed good colloidal stability in aqueous suspensions. Chemical stability was also assessed in PBS, showing negligible solubility. The effect of particle size on the transversal relaxivity value (r2) was further explored at 9.4 T, finding a clear increase in r2 with particle size. The r2 value found for the largest NPs was 516 mM-1 s-1, which is, to the best of our knowledge, the highest r2 value ever reported at 9.4 T for any Dy-based nanometric particles in the literature. Finally, the latter NPs were submitted to biosafety studies after polyethylene glycol (PEG) functionalization. Cell morphology, induction of necrotic/late apoptotic cells, and mitochondrial activity were thoroughly analyzed. The results clearly indicated negligible toxicity effects under the assayed conditions. Short- and long-term in vivo pharmacokinetics of the intravenously injected NPs were assessed by dynamic T2-weighted MRI and quantitative T2 mapping, revealing faster liver than spleen uptake, while no accumulation was observed in the kidneys. Finally, no histopathological changes were observed in any of the studied organs, including the liver, kidney, spleen, and lung, which provide further evidence of the biocompatibility of DyPO4 NPs and, therefore, their suitability as bioimaging probes.

Mechanical properties, corrosion, and biocompatibility of Mg-Zr-Sr-Dy alloys for biodegradable implant applications.

This study investigates the microstructure, mechanical properties, corrosion behavior, and biocompatibility of magnesium (Mg)-based Mg1Zr2SrxDy (x = 0, 1, 1.63, 2.08 wt %) alloys for biodegradable implant applications. The corrosion behavior of the Mg-based alloys has been evaluated in simulated body fluid using an electrochemical technique and hydrogen evolution. The biocompatibility of the Mg-based alloys has been assessed using SaSO2 cells. Results indicate that the addition of Dy to Mg-Zr-Sr alloy showed a positive impact on the corrosion behavior and significantly decreased the degradation rates of the alloys. The degradation rate of Mg1Zr2Sr1.0Dy decreased from 17.61 to 12.50 mm year-1 of Mg1Zr2Sr2.08Dy based on the hydrogen evolution. The ultimate compressive strength decreased from 270.90 MPa for Mg1Zr2Sr1Dy to 236.71 MPa for Mg1Zr2Sr2.08Dy. An increase in the addition of Dy to the Mg-based alloys resulted in an increase in the volume fraction of the Mg2 Dy phase, which mitigated the galvanic effect between the Mg17 Sr2 phase and the Mg matrix, and led to an increase in the corrosion resistance of the base alloy. The biocompatibility of the Mg-based alloys was enhanced with decreasing corrosion rates. Mg1Zr2Sr2.08Dy exhibited the lowest corrosion rate and the highest biocompatibility compared with the other Mg-based alloys. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2425-2434, 2018.

Synthesis and biological evaluation of a new dysprosium(III) complex containing 2,9-dimethyl 1,10-phenanthroline.

The binding of [Dy(dmp)2Cl3(OH2)], where dmp is 2,9-dimethyl 1,10-phenanthroline, with Fish salmon DNA (FS-DNA) is investigated by absorption and emission spectroscopy, quenching studies, salt dependent, and gel electrophoresis. The binding constant (Kb) of the interaction is calculated as (1.27 ± .05) × 105 M-1 from absorption spectral titration data. The Stern-Volmer constant (KSV), thermodynamic parameters involves ΔG°, ∆H°, and ∆S° are calculated by fluorescent data and Van't Hoff equation. The thermodynamic studies show that the reaction for the binding of the complex with FS-DNA is endothermic and entropically driven (ΔS° > 0, ΔH° > 0). The effect of the complex concentration on FS-DNA cleavage reactions is also investigated by gel electrophoresis. Furthermore, the Dy(III) complex has been screened for its antibacterial activity. The experimental results suggest that the Dy(III) complex binds significantly to FS-DNA by hydrophobic groove binding mode and the complex has more efficient antibacterial activity compared to its metal salt.

Dual responsive dysprosium-doped hydroxyapatite particles and toxicity reduction after functionalization with folic and glucuronic acids.

The development of probes for biomedical applications demands materials with low toxicity levels besides fluorescence or magnetic properties to be detected by confocal microscopes or MRI resonators. Several drug delivery systems or other biomedical materials prepared with hydroxyapatite have been proposed, however, toxicity effects might arise when the size of particles is nanometric. In this study, hydroxyapatite functionalized with glucuronic or folic acids presented lower oxidative stress, measured from lipoperoxides and nitric oxide indicators in rats than pure hydroxyapatite. In separated experiments, hydroxyapatite was doped with dysprosium cations by coprecipitation producing a single crystal phase with fluorescent properties easily visualized by confocal microscopy when excited at 488nm. These particles also presented the ability to modify the proton relaxation time in T1 maps collected by magnetic resonance imaging. These modified hydroxyapatite nanoparticles could be candidates to design bimodal probes with low toxicity.

Water-soluble oxoglaucine-Y(III), Dy(III) complexes: in vitro and in vivo anticancer activities by triggering DNA damage, leading to S phase arrest and apoptosis.

Complexes of yttrium(III) and dysprosium(III) with the traditional Chinese medicine active ingredient oxoglaucine (OG), namely [Y(OG)2(NO3)3]·CH3OH (1) and [Dy(OG)2(NO3)3]·H2O (2), were synthesized and characterized by elemental analysis, IR, ESI-MS, (1)H and (13)C NMR as well as single-crystal X-ray diffraction analysis. In vitro the complexes exhibited higher anticancer activity than the free ligand OG against the tested cancer cell lines. Among the tested cell lines, HepG2 is the most sensitive to the complexes. Complex 2 can trigger DNA damage in HepG2 cells, resulting in cell cycle arrest in the S phase and leading to cell apoptosis. The S phase cell-cycle arrest is caused via the ATM (ataxia-telangiectasia mutated)-Chk2-Cdc25A pathway. Chk2 is phosphorylated and activated in an ATM-dependent manner. It, in turn, phosphorylates Cdc25A phosphatise on serine124, causing the inactivation of Cdc25A in ubiquitin-mediated proteolytic degradation. The cyclin-Cdk complexes of the S phase could also be inhibited by limited supply of cyclins A and E. This irreversible cell cycle arrest process ultimately induces mitochondria-involved apoptotic cell death via the activation of Bcl-2 protein. Complex e2 ffectively inhibited tumour growth in the BEL-7402 xenograft mouse model and exhibited higher safety in vivo than cisplatin.

An increase of the energy coupling capacity of submitochondrial particles by lanthanides.

NADH and succinate oxidase activities of inside-out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Both inhibition by oligomycin and oligomycin plus lanthanides are completely relieved by an uncoupler. The respiratory control measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin-treated particles is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess EDTA. La3+ increases the extent of the aerobic energy-linked succinate-supported NAD+ reduction catalyzed by the oligomycin-treated submitochondrial particles. Lanthanides seem to be useful tools to increase the energy coupling capacity of the submitochondrial particles.

An increase in the energy coupling capacity of submitochondrial particles in the presence of lanthanides.

NADH and succinate oxidase activities of inside-out submitochondrial particles treated with excess oligomycin are inhibited by lanthanides (La3+ and Dy3+). Inhibition by both oligomycin and oligomycin plus lanthanides is completely relieved by an uncoupler. The respiratory control, measured as the stimulation of NADH or succinate oxidation caused by the addition of uncoupler to the oligomycin-treated particles, is thus increased in the presence of lanthanides. The coupling effect of lanthanides is completely prevented and rapidly reversed by excess of EDTA. La3+ increases the extent of the aerobic energy-linked succinate-supported NAD+ reduction catalyzed by the oligomycin-treated submitochondrial particles. Lanthanides seem to be a useful tool to increase the energy coupling capacity of the submitochondrial particles.

Rare earth elements as nonabsorbable fecal markers in studies of iron absorption.

The use of rare earth elements as nonabsorbable fecal markers for studies of iron absorption from sources labeled extrinsically with stable isotopes was evaluated. On 3 successive days 13 healthy fasting adults were given different stable isotopes of iron with samarium, ytterbium, or dysprosium. On day 1, three meals were given with 57Fe (1 mg per meal) plus samarium (0.33 mg per meal); on day 2, identical meals (taken with a calcium supplement to reduce iron bioavailability) were given with equivalent amounts of 58Fe-labeled iron and ytterbium; on day 3, a well-absorbed reference dose of 54Fe (3 mg) was given with 1 mg Dy. A complete fecal collection was carried out for 5-9 d and each stool was analyzed for rare earth elements by inductively coupled plasma-mass spectrometry and iron isotopes by thermal ionization quadrupole mass spectrometry. Mean recovery of rare earth elements was 101%, indicating that they are totally unabsorbed. The excretory pattern of the iron isotopes and the rare earth elements was very similar; the correlation coefficients between samarium and 57Fe, ytterbium and 58Fe, and dysprosium and 54Fe were 0.992, 0.989, and 0.988, respectively (P < 0.001). Iron absorption was calculated as the difference between isotope dose and fecal excretion. Mean (+/-SEM) iron absorption was 16.7 +/- 2.4%, 4.3 +/- 1.6%, and 40.3 +/- 3.1% on days 1-3, respectively. Predicted values estimated from the first 4 d of pooled feces, using the rare earth element recovery data to produce corrected figures for unabsorbed isotope, were in close agreement: 19.1 +/- 2.1%, 4.6 +/- 1.7%, and 40.8 +/- 3.1%, respectively (P < 0.001). With the diet of medium iron bioavailability and with the highly bioavailable reference dose it was possible to predict iron absorption accurately from only one or two stools, provided that they were sufficiently enriched with isotope and a rare earth element.

Effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors.

PURPOSE: To investigate the effects of texaphyrins on the oxygenation of EMT6 mouse mammary tumors in Balb/c Rw mice. Texaphyrins are synthetic, porphyrin-like molecules capable of stably coordinating lanthanide and nonlanthanide metals. Metallotexaphyrin compounds containing gadolinium (MGd), lutetium (MLu), europium (Eu-Tex), dysprosium (Dy-Tex), and manganese (Mn-Tex) were evaluated. METHODS: Tumor oxygenation was measured using an Eppendorf pO2 histograph when tumors, implanted intradermally in the rear dorsum, reached 150-200 mm3. Oxygen measurements were also made in the leg muscle of tumor-bearing mice, to determine whether changes in oxygenation occurred in nontumor tissue. RESULTS: Motexafin gadolinium (Xcytrin, MGd) seems to be an effective modulator of tumor oxygen tension. The mean of the median tumor pO2 6 hours after injection of MGd was 8.0 +/- 2.4 mm Hg. The control value was 1.5 +/- 0.4 mm Hg. The oxygen levels within EMT6 tumors were shifted significantly toward higher oxygen tensions 6-8 hours after i.v. injection of 40 micromol/kg MGd, thereby reducing the percentage of severely hypoxic readings (MGd, 6 hours: 44.6 +/- 4.3% <2.5 mm Hg; CONTROL: 69.4 +/- 3.0% <2.5 mm Hg). There was no significant change in the oxygenation of the leg muscle after MGd treatment. Eu-Tex and Mn-Tex increased the tumor oxygenation to a much lesser degree than MGd. MLu, Dy-Tex, and the vehicle (a 5% mannitol solution) did not modulate tumor oxygenation. CONCLUSIONS: MGd is an effective modulator of tumor oxygenation. The central metal composition of texaphyrin compounds is an important determinant of the effect of the texaphyrins on tumor oxygenation.

[Effect of metal oxides on the growth, hemolytic and serologic properties of Klebsiella pneumoniae]. / Vliianie oksidov metallov na rost, gemoliticheskie i serologicheskie svoistva Klebsiella pneumoniae.

Silicon, dysprosium, germanium, yttrium, iron, cobalt, samarium, lutecium oxides, as well as the mixture of 8 metal oxides, at a concentration of 20 g/l were found to produce a stimulating or inhibiting effect on the growth of K. pneumoniae strains 204 and K-9. Silicon, dysprosium, germanium and yttrium oxides were shown to stimulate the growth of K. pneumoniae strain 204. Iron, cobalt, samarium and lutecium oxides, as well as the mixtures of all oxides under study, inhibited the growth of this strain. Silicon, samarium and lutecium oxides produced no effect on the growth of K. pneumoniae strain K-9; at the same time germanium and yttrium oxides stimulated the growth of these bacteria, while dysprosium, iron, cobalt oxides, as well as the mixture of all oxides, inhibited their growth. The presence of metal oxides did not change the serological activity of the cultures of both strains growing old, i.e. by 24 hours of their growth. The addition of silicon, germanium and iron oxides to the culture medium increased the hemolytic activity of K. pneumoniae strain K-9 seven to ninefold in comparison with the control grown in a synthetic nutrient medium without metal oxides. The comparison of these two strains (K-9 and 204) revealed that K. pneumoniae strain K-9 possessed greater hemolytic activity.

Dy3+ and Nd3+ induced genetic mutation of bacillus alpha-amylase.

After treatment with DyCl(3) and NdCl(3), two strains of mutated bacilli showing increased alpha-amylase activity were isolated. The alpha-amylase genes were amplified, cloned, and sequenced. Sequencing revealed that there were either 11 or 14 bases altered in the two genes. These alterations took the form of base substitutions, and transversion was more common than transition. Based on these results, it was concluded that the rare earth compounds DyCl(3) and NdCl(3) were mutagens.

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition.

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.

[K-strophanthin-beta complexing with calcium, magnesium and dysprosium ions]. / Kompleksoobrazovanie k-strofantina-beta s ionami kal'tsiia, magniia i disproziia.

Nuclear magnetic resonance, microcalorimetry and the use of the ion-selecting electrode evidenced that k-strophanthine-beta forms complexes with the calcium, magnesium and disprosium ions. Changes in the position of the k-strophanthine-beta carbohydrate parts and aglycones signals bear witness to their participation in the complexing.

Double-quantum-filtered 23Na NMR study of intracellular sodium in the perfused liver.

We acquired double-quantum-filtered 23Na NMR spectra from perfused liver, using a range of tau values from 0.2 to 24 ms, where tau is the separation between the first and second pi/2 pulses in the radio-frequency pulse sequence. For each tau value we compared the amplitude of the double-quantum-filtered 23Na NMR signal acquired from intracellular sodium ions when the liver was perfused with buffer containing the "shift reagent" Dy(PPP)2 to the amplitude of the total double-quantum-filtered 23Na NMR signal acquired when the liver was perfused with buffer containing no Dy(PPP)2. For tau < or = 4 ms, the average ratio of the two amplitudes was 0.98 +/- 0.03 (mean +/- SEM). For tau > or = 8 ms, the average ratio was significantly less than 1. These results demonstrate that double-quantum-filtered 23Na NMR signals acquired from perfused liver using short tau values arise almost exclusively from intracellular sodium ions, but double-quantum-filtered 23Na NMR signals acquired from perfused liver using long tau values contain contributions from both intracellular and extracellular sodium ions. This conclusion suggests that multiple-quantum-filtered 23Na NMR spectroscopy will be useful in studying intracellular sodium levels in the perfused liver, and possibly in the intact liver in vivo.

Dynamic NMR measurement of volume regulatory changes in Amphiuma RBC Na+ content.

23Na nuclear magnetic resonance (NMR) and conventional chemical methods were employed to measure Na+ fluxes in Amphiuma red blood cells (RBC) during volume regulation. Paramagnetic shift reagents [dysprosium triethylenetetraminehexaacetic acid (DyTTHA) and dysprosium tripolyphosphate (Dy(TPP)2)] were used to alter extracellular Na+ magnetic resonance. Data are presented describing 23Na resonance dependence on shift reagent, sodium and calcium concentration. We confirmed that the shift reagents neither enter the cells nor alter intracellular Na+, K+, and Cl-concentrations under control conditions when extracellular calcium was maintained greater than 0.5 mM. We also confirmed that the shift reagent complexes chelate calcium [Dy(TPP)2 much more so than DyTTHA] and that their toxic effects could be alleviated by adjusting calcium in the cell's suspension medium to control levels. In parallel experiments, where volume-activated Na+ fluxes ranged from 0.3 to 3 mmol Na+/kg dry cell solid (DCS) x minute in cells containing from 30 to 150 mmol Na+/kg DCS, changes in intracellular sodium measured by 32Na NMR were within 4% of those measured by conventional destructive methods. Finally, we present data that are consistent with the interpretation that 6 mmol Na+/kg DCS plus 16% of intracellular Na+ is NMR invisible.

Topological characterization of Escherichia coli DMSO reductase by electron paramagnetic resonance spectroscopy of an engineered [3Fe-4S] cluster.

We have applied the technique of distance estimations using the exogenous paramagnetic probe dysprosium (III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase (DmsABC) in situ in cytoplasmic membrane and whole cell preparations. The electron transfer subunit (DmsB) of this enzyme contains four [4Fe-4S] clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes. We have utilized a mutant of DmsABC in which one of the [4Fe-4S] clusters of DmsB has been changed to a [3Fe-4S] cluster [Rothery, R. A., & Weiner, J. H. (1991) Biochemistry 30, 8296-8305]. This mutant (DmsB-C102S) has a single magnetically isolated EPR visible [3Fe-4S] cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe. We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant. DyEDTA enhances the spin relaxation of the [3Fe-4S] signal in everted membrane vesicles. It has a smaller effect on the spin relaxation of the [3Fe-4S] signal in whole cell preparations. We conclude that the [3Fe-4S] cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane. We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.