Proton Relaxation Enhancement Research Articles

Application of Community Detection Algorithm to Investigate the Correlation between Imaging Biomarkers of Tumor Metabolism, Hypoxia, Cellularity, and Perfusion for Precision Radiotherapy in Head and Neck Squamous Cell Carcinomas.

Simple SummaryIntegration of multimodality imaging (MMI) methods in head and neck squamous cell carcinomas (HNSCC) provides complementary information of the tumor and its microenvironment. Quantitative positron emission tomography (PET)/computed tomography (CT), DW- and DCE-MRI provide the functional information of tumor tissue based on metabolic process, diffusion of water molecules, and enhancement of water proton relaxation with a contrast agent, respectively. The present study aimed to investigate correlations at pre-treatment between quantitative imaging metrics derived from FDG-PET/CT(SUL), FMISO-PET/CT (K1, k3, TBR, and DV), DW-MRI (ADC, IVIM [D, D*, and f]), and FXR DCE-MRI [Ktrans, ve, and τi]) using a community detection algorithm (CDA) based on the “spin-glass model” and Spearman rank analysis in patients with HNSCC. Correlations between MMI-derived quantitative metrics evaluated using a CDA in addition to the Spearman analysis in a larger population may enable the identification of potential biomarkers for prognostication and management of patients with HNSCC.The present study aimed to investigate the correlation at pre-treatment (TX) between quantitative metrics derived from multimodality imaging (MMI), including 18F-FDG-PET/CT, 18F-FMISO-PET/CT, DW- and DCE-MRI, using a community detection algorithm (CDA) in head and neck squamous cell carcinoma (HNSCC) patients. Twenty-three HNSCC patients with 27 metastatic lymph nodes underwent a total of 69 MMI exams at pre-TX. Correlations among quantitative metrics derived from FDG-PET/CT (SUL), FMSIO-PET/CT (K1, k3, TBR, and DV), DW-MRI (ADC, IVIM [D, D*, and f]), and FXR DCE-MRI [Ktrans, ve, and τi]) were investigated using the CDA based on a “spin-glass model” coupled with the Spearman’s rank, ρ, analysis. Mean MRI T2 weighted tumor volumes and SULmean values were moderately positively correlated (ρ = 0.48, p = 0.01). ADC and D exhibited a moderate negative correlation with SULmean (ρ ≤ −0.42, p < 0.03 for both). K1 and Ktrans were positively correlated (ρ = 0.48, p = 0.01). In contrast, Ktrans and k3max were negatively correlated (ρ = −0.41, p = 0.03). CDA revealed four communities for 16 metrics interconnected with 33 edges in the network. DV, Ktrans, and K1 had 8, 7, and 6 edges in the network, respectively. After validation in a larger population, the CDA approach may aid in identifying useful biomarkers for developing individual patient care in HNSCC.

A Novel Class of 1 H-MRI Contrast Agents Based on the Relaxation Enhancement Induced on Water Protons by 14 N-Containing Imidazole Moieties.

This study reports the development of a completely new class of MRI contrast agents, displaying remarkable relaxation effects in the absence of paramagnetic metal ions. Their detection requires the acquisition of images at variable magnetic field strength as provided by fast field cycling imaging scanners. They contain poly-histidine chains (poly-His), whose imidazole groups generate 14 N-quadrupolar-peaks that cause a relaxation enhancement of water protons at a frequency (1.38±0.3 MHz) that is readily detectable from the frequencies associated with endogenous proteins. The poly-His quadrupolar peaks are detectable only when the polymer is in a solid-like form, that is, at pH>6.6. Above this value, their intensity is pH dependent and can be used to report on the occurring pH changes. On this basis, the poly-His moieties were conjugated to biocompatible polymers, such as polylactic and glycolic acid, in order to form stable nanoparticles able to encapsulate structured water in their core. FFC images were acquired to assess their contrast-generating ability.

A Novel Class of 1H‐MRI Contrast Agents Based on the Relaxation Enhancement Induced on Water Protons by 14N‐Containing Imidazole Moieties

AbstractThis study reports the development of a completely new class of MRI contrast agents, displaying remarkable relaxation effects in the absence of paramagnetic metal ions. Their detection requires the acquisition of images at variable magnetic field strength as provided by fast field cycling imaging scanners. They contain poly‐histidine chains (poly‐His), whose imidazole groups generate 14N‐quadrupolar‐peaks that cause a relaxation enhancement of water protons at a frequency (1.38±0.3 MHz) that is readily detectable from the frequencies associated with endogenous proteins. The poly‐His quadrupolar peaks are detectable only when the polymer is in a solid‐like form, that is, at pH&gt;6.6. Above this value, their intensity is pH dependent and can be used to report on the occurring pH changes. On this basis, the poly‐His moieties were conjugated to biocompatible polymers, such as polylactic and glycolic acid, in order to form stable nanoparticles able to encapsulate structured water in their core. FFC images were acquired to assess their contrast‐generating ability.

Relaxometric Studies of Gd-Chelate Conjugated on the Surface of Differently Shaped Gold Nanoparticles.

Nowadays, magnetic resonance imaging (MRI) is one of the key, noninvasive modalities to detect and stage cancer which benefits from contrast agents (CA) to differentiate healthy from tumor tissue. An innovative class of MRI CAs is represented by Gd-loaded gold nanoparticles. The size, shape and chemical functionalization of Gd-loaded gold nanoparticles appear to affect the observed relaxation enhancement of water protons in their suspensions. The herein reported results shed more light on the determinants of the relaxation enhancement brought by Gd-loaded concave cube gold nanoparticles (CCGNPs). It has been found that, in the case of nanoparticles endowed with concave surfaces, the relaxivity is remarkably higher compared to the corresponding spherical (i.e., convex) gold nanoparticles (SPhGNPs). The main determinant for the observed relaxation enhancement is represented by the occurrence of a large contribution from second sphere water molecules which can be exploited in the design of high-efficiency MRI CA.

Surface manganese substitution in magnetite nanocrystals enhances T1 contrast ability by increasing electron spin relaxation.

Magnetite nanoparticles, with good biocompatibility and favorable magnetic properties, have the potential to be the best candidate for non-gadolinium MRI contrast agents. However, they usually show low T1 contrast ability, largely because Fe(ii) ions have a short electronic relaxation time and also have a small number of unpaired electrons with inefficient proton relaxation enhancement. Herein, we report a novel strategy to increase the T1 contrast ability of magnetite nanoparticles, through substituting the undesirable Fe(ii) ions with Mn(ii) ions. Mn(ii) ions have a longer electronic relaxation time (10-8 s) and more unpaired electrons (5 unpaired electrons). We successfully construct diverse-shaped manganese ferrite nanoparticles with abundant magnetic ions, Mn(ii) and Fe(iii), exposed on the surface. These manganese ferrite nanoparticles exhibit remarkably higher longitudinal relaxivity than their parent iron oxide nanoparticles. We demonstrate that the increase in T1 relaxivity is attributed to the extended electronic relaxation time and the increased number of unpaired electrons on the surface of the nanoparticles by controlling surface features, particularly by adjusting the substitution degree of Mn(ii) ions and in situ coating. This study provides an insightful strategy to improve the T1 contrast ability of iron oxide nanoparticles, which is urgently needed for developing high-performance non-gadolinium T1 contrast agents for imaging and diagnosis of disease.

Compact Micellization: A Strategy for Ultrahigh T1 Magnetic Resonance Contrast with Gadolinium-Based Nanocrystals.

Paramagnetic gadolinium (Gd(3+))-based nanocrystals (NCs) with a large number of confined gadolinium ions can be expected to heavily enhance the longitudinal (T1) relaxation of water protons compared to clinical gadolinium complexes with only a single paramagnetic center. However, paramagnetic Gd(3+)-NCs reported to date show only a modest T1 relaxivity of ∼10 mM(-1) s(-1) per Gd(3+) at 1.5 T, only about 3-times higher than clinical Gd(3+) complexes. Here we demonstrate a strategy that achieves ultrahigh T1 relaxivity that is about 25-times higher than clinical Gd(3+) complexes by controlling the proximity of water protons to a paramagnetic NC surface. Using NaGdF4 NCs (∼3 nm) coated with PEG-ylated phospholipid (DSPE-PEG) micelles, we show that the distance of water protons to the NCs surface can be tuned by controlling the NC-micelle sizes. Increasing the ratio of DSPE-PEG to NCs during micellization decreases the size of NC-micelles, enhancing the proximity of water to the NC surface. Using this strategy, we have achieved compact NC-micelles (hydrodynamic diameter, HD ∼ 5 nm) with ultrahigh T1 relaxivity of ∼80 mM(-1) s(-1) per Gd(3+) at 1.41 T. The findings reported here demonstrate a nanostructured Gd(3+)-contrast agent (CA) that simultaneously achieves an ultrahigh T1 relaxivity approaching theoretical predictions, extremely compact size (HD < 5 nm), and a biocompatible surface. Our results show the hitherto unknown ultrahigh T1 relaxation enhancement of water protons in close proximity to a colloidal gadolinium-NC surface that is achievable by precise control of their surface structure.

High relaxivity Gd(III)-DNA gold nanostars: investigation of shape effects on proton relaxation.

Gadolinium(III) nanoconjugate contrast agents (CAs) have distinct advantages over their small-molecule counterparts in magnetic resonance imaging. In addition to increased Gd(III) payload, a significant improvement in proton relaxation efficiency, or relaxivity (r1), is often observed. In this work, we describe the synthesis and characterization of a nanoconjugate CA created by covalent attachment of Gd(III) to thiolated DNA (Gd(III)-DNA), followed by surface conjugation onto gold nanostars (DNA-Gd@stars). These conjugates exhibit remarkable r1 with values up to 98 mM(-1) s(-1). Additionally, DNA-Gd@stars show efficient Gd(III) delivery and biocompatibility in vitro and generate significant contrast enhancement when imaged at 7 T. Using nuclear magnetic relaxation dispersion analysis, we attribute the high performance of the DNA-Gd@stars to an increased contribution of second-sphere relaxivity compared to that of spherical CA equivalents (DNA-Gd@spheres). Importantly, the surface of the gold nanostar contains Gd(III)-DNA in regions of positive, negative, and neutral curvature. We hypothesize that the proton relaxation enhancement observed results from the presence of a unique hydrophilic environment produced by Gd(III)-DNA in these regions, which allows second-sphere water molecules to remain adjacent to Gd(III) ions for up to 10 times longer than diffusion. These results establish that particle shape and second-sphere relaxivity are important considerations in the design of Gd(III) nanoconjugate CAs.

Specific and nonspecific interactions in ultraweak protein-protein associations revealed by solvent paramagnetic relaxation enhancements.

Weak and transient protein–protein interactions underlie numerous biological processes. However, the location of the interaction sites of the specific complexes and the effect of transient, nonspecific protein–protein interactions often remain elusive. We have investigated the weak self-association of human growth hormone (hGH, KD = 0.90 ± 0.03 mM) at neutral pH by the paramagnetic relaxation enhancement (PRE) of the amide protons induced by the soluble paramagnetic relaxation agent, gadodiamide (Gd(DTPA-BMA)). Primarily, it was found that the PREs are in agreement with the general Hwang-Freed model for relaxation by translational diffusion (J. Chem. Phys.1975, 63, 4017–4025), only if crowding effects on the diffusion in the protein solution are taken into account. Second, by measuring the PREs of the amide protons at increasing hGH concentrations and a constant concentration of the relaxation agent, it is shown that a distinction can be made between residues that are affected only by transient, nonspecific protein–protein interactions and residues that are involved in specific protein–protein associations. Thus, the PREs of the former residues increase linearly with the hGH concentration in the entire concentration range because of a reduction of the diffusion caused by the transient, nonspecific protein–protein interactions, while the PREs of the latter residues increase only at the lower hGH concentrations but decrease at the higher concentrations because of specific protein–protein associations that impede the access of gadodiamide to the residues of the interaction surface. Finally, it is found that the ultraweak aggregation of hGH involves several interaction sites that are located in patches covering a large part of the protein surface.

NMR Relaxation Enhancement of Water Protons by Gd-Doped Boron Nitride Nanotubes

The longitudinal NMR relaxation rate (R1) of water protons was measured on aqueous suspensions of Gd@BNNTs (i.e., boron nitride nanotubes doped with 0.4% w/w of Gd) coated with glycol-chitosan in the 0.01–30.0 MHz Larmor frequency range, by using a fast field-cycling relaxometer. The dispersion curve of the relaxivity (r1) relative to Gd, determined exploiting R1 data from Gd@BNNT and BNNT suspensions, shows a logarithmic dependence on frequency which is characteristic of a relaxation enhancement dominated by an outer sphere mechanism governed by two-dimensional diffusion of water in proximity of Gd3+ ions on the BNNT surface. Quantitative information on water diffusion and affinity for the surface was obtained by analyzing the r1 NMRD curves in terms of a model for two-dimensional diffusion. The effects of the nanotube assembly in water and of the coating hydrophilicity on the interactions between water molecules and Gd3+ ions governing the proton relaxation enhancement at the basis of contrast in magnet...

In Vivo Imaging of Nitric Oxide by Magnetic Resonance Imaging Techniques

Nitric oxide (NO) biosensors are novel tools for real‐time bioimaging of tissue oxygen changes and physiological monitoring of tissue vasculature. Nitric oxide behavior further enhances its role in mapping signal transduction at the molecular level. Spectrometric electron paramagnetic resonance (EPR) and fluorometric imaging are well known techniques with the potential for in vivo bioimaging of NO. In tissues, NO is a specific target of nitrosyl compounds for chemical reaction, which provides a unique opportunity for application of newly identified NO biosensors. However, the accuracy and sensitivity of NO biosensors still need to be improved. Another potential magnetic resonance technique based on short term NO effects on proton relaxation enhancement is magnetic resonance imaging (MRI), and some NO biosensors may be used as potent imaging contrast agents for measurement of tumor size by MRI combined with fluorescent imaging. The present review provides supporting information regarding the possible use of nitrosyl compounds as NO biosensors in MRI and fluorescent bioimaging showing their measurement limitations and quantitative accuracy. These new approaches open a perspective regarding bioimaging of NO and the in vivo elucidation of NO effects by magnetic resonance techniques.

Gd3+ complexes conjugated to Pittsburgh compound B: potential MRI markers of β-amyloid plaques

In an effort towards the visualization of β-amyloid (Aβ) plaques by T1-weighted magnetic resonance imaging for detection of Alzheimer's disease, we report the synthesis and characterization of stable, noncharged Gd(3+) complexes of three different 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid monoamide derivatives conjugated to Pittsburgh compound B, a well-established marker of Aβ plaques. The ligands L1, L2, and L3 differ in the nature and size of the spacer linking the macrocyclic chelator and the Pittsburgh compound B targeting moiety, which affects their lipophilicity, the octanol-water partition coefficients of the complexes ranging from -0.15 to 0.32. Given their amphiphilic behavior, the complexes form micelles in aqueous solution (critical micellar concentration 1.00-1.49 mM). The parameters determining the relaxivity, including the water exchange rate and the rotational correlation times, were assessed for the monomeric and the micellar form by a combined (17)O NMR and (1)H nuclear magnetic relaxation dispersion (NMRD) study. They are largely influenced by the aggregation state and the hydrophobic character of the linkers. The analysis of the rotational dynamics for the aggregated state in terms of local and global motions using the Lipari-Szabo approach indicates highly flexible, large aggregates. On binding of the complexes to human serum albumin or to the amyloid peptide Aβ1-40 in solution, they undergo a fourfold and a twofold relaxivity increase, respectively (40 MHz). Proton relaxation enhancement studies confirmed moderate interaction of Gd(L1) and Gd(L3) with human serum albumin, with KA values ranging between 250 and 910 M(-1).

Nitroxide Radicals@US‐Tubes: New Spin Labels for Biomedical Applications

AbstractThe encapsulation of nitroxide radicals within ultrashort (ca. 50 nm) single‐walled carbon nanotubes (US‐tubes) is achieved. Tempo‐ and Iodo‐Tempo@US‐tubes are characterized by thermogravimetric analysis (TGA), X‐ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Electron paramagnetic resonance (EPR) spectra display characteristic signals due to the detection of the spin probes within the US‐tubes. Longitudinal proton relaxivities (r1) of both nitroxide@US‐tubes samples are 7 to 13 times greater than the free nitroxide radicals in solution, giving relaxivities comparable to the clinical contrast agent (CA) Magnevist. In addition, transverse proton relaxivities (r2) show unprecedented proton relaxation enhancement in comparison to any other reported nitroxide radical‐based system or the clinically approved T2 CA, Resovist, under the same conditions. T2‐weighted magnetic resonance imaging (MRI) phantom images show that the encapsulation of nitroxide radicals within the US‐tubes produces good contrast enhancement due to their high r2 relaxivities. The nitroxide radicals@US‐tube agents are a new promising class of spin probes for MRI and electronic paramagnetic resonance imaging (EPRI) labeling, tracking, and diagnosis.

The role of diffusion in ferritin-induced relaxation enhancement of protons

The influence of proton diffusion on nuclear magnetic resonance (NMR) relaxation was investigated in the presence of horse spleen ferritin at 7T. Binary mixtures of water and glycerol were used to control diffusion within the range of 0.6–2.0×10−9m2/s, which was confirmed by pulsed gradient techniques. The effect of chemical exchange by hydrolysis between water and glycerol on relaxation was characterized with Carr–Purcell–Meiboom–Gill (CPMG) dispersion experiments. The relaxation rate enhancement of the protons due to ferritin was found to be inversely proportional to the diffusion coefficient. The enhancement increased by a factor of 3.6 over the range of diffusion coefficients, while the hydroxyl proton concentration decreased by a factor of 1.3. This result is in disagreement with the proton exchange dephasing model, which is independent of diffusion but predicts an inverse dependence on the hydroxyl concentration. Our data indicate that the role of diffusion dominates and must be considered when relaxation rates are used to determine iron concentration in vivo.

Mesoporous silica-coated hollow manganese oxide nanoparticles as positive T1 contrast agents for labeling and MRI tracking of adipose-derived mesenchymal stem cells.

Mesoporous silica-coated hollow manganese oxide (HMnO@mSiO2) nanoparticles were developed as a novel T1 magnetic resonance imaging (MRI) contrast agent. We hypothesized that the mesoporous structure of the nanoparticle shell enables optimal access of water molecules to the magnetic core, and consequently, an effective longitudinal (R1) relaxation enhancement of water protons, which value was measured to be 0.99 (mM−1s−1) at 11.7 T. Adipose-derived mesenchymal stem cells (MSCs) were efficiently labeled using electroporation, with much shorter T1 values as compared to direct incubation without electroporation, which was also evidenced by signal enhancement on T1-weighted MR images in vitro. Intracranial grafting of HMnO@mSiO2-labeled MSCs enabled serial MR monitoring of cell transplants over 14 days. These novel nanoparticles may extend the arsenal of currently available nanoparticle MR contrast agents by providing positive contrast on T1-weighted images at high magnetic field strengths.

Paramagnetic Relaxation Assisted Docking of a Small Indole Compound in the HIV-1 gp41 Hydrophobic Pocket

The hydrophobic pocket contained within the gp41 coiled coil is an important target for small molecules designed to inhibit HIV-1 fusion. While various screening experiments have identified molecules purported to bind in this pocket, few have confirmed details of the interaction, instead relying on computational docking to predict the binding mode. This is made more challenging by the fact that residues lining the hydrophobic pocket are highly flexible, as is typical for a protein-protein interaction site, limiting the predictive power of computational tools. In this study, we report on an NMR method to define the binding mode of 1-5i, a compound in a series of newly developed indole inhibitors. We show that paramagnetic relaxation enhancement of ligand protons due to an MTSL group positioned close to the binding pocket could be applied quantitatively to distinguish between more than 30 different computational poses, selecting a single pose that agreed with the NMR data. In this pose, important hydrophobic and polar contacts occur with pocket lysine, tryptophan, and glutamine residues, including putative hydrogen bonds between the ligand carboxylate and the lysine ε-amino group. A study of the ligand orientation suggests directions for optimization.

Supramolecular protamine/Gd-loaded liposomes adducts as relaxometric protease responsive probes

A new approach to enzyme-responsive MRI agents based on the use of liposomes loaded with a high number of paramagnetic metal complexes (Gd-HPDO3A) is presented. It relies on the disruption of low relaxivity aggregates formed by liposomes and a macromolecular substrate that is selectively cleaved by the enzyme of interest. The interaction of anionic liposomes composed of POPC:CHOL:DPGS and the cationic protein protamine yields a poorly soluble supramolecular assembly endowed with a low relaxivity. The action of the serine protease trypsin causes the digestion of protamine and the consequent de-assembly of the supramolecular adduct. The process is accompanied by an overall relaxation enhancement of solvent water protons as consequence of the dissolution of the aggregated liposomes. The observed increase of relaxivity is linearly dependent on the enzyme concentration. An illustrative example of the possible use of the herein presented responsive agent has been reported. It consists of the entrapment of the supramolecular assembly in alginate microcapsules that have often been used as envelopes for in vivo applications of stem cells and pancreatic islets. The change in the observed longitudinal relaxation rate R(1) (leading to an hyperintense signal in the corresponding MR images) may act as a sensor of the protease activity in the biological environment in which the capsules is located.

High proton relaxivity for gadolinium oxide nanoparticles

Nanosized materials of gadolinium oxide can provide high-contrast enhancement in magnetic resonance imaging (MRI). The objective of the present study was to investigate proton relaxation enhancement by ultrasmall (5 to 10 nm) Gd(2)O(3) nanocrystals. Gd(2)O(3) nanocrystals were synthesized by a colloidal method and capped with diethylene glycol (DEG). The oxidation state of Gd(2)O(3) was confirmed by X-ray photoelectron spectroscopy. Proton relaxation times were measured with a 1.5-T MRI scanner. The measurements were performed in aqueous solutions and cell culture medium (RPMI). Results showed a considerable relaxivity increase for the Gd(2)O(3)-DEG particles compared to Gd-DTPA. Both T (1) and T (2) relaxivities in the presence of Gd(2)O(3)-DEG particles were approximately twice the corresponding values for Gd-DTPA in aqueous solution and even larger in RPMI. Higher signal intensity at low concentrations was predicted for the nanoparticle solutions, using experimental data to simulate a T(1)-weighted spin echo sequence. The study indicates the possibility of obtaining at least doubled relaxivity compared to Gd-DTPA using Gd(2)O(3)-DEG nanocrystals as contrast agent. The high T (1) relaxation rate at low concentrations of Gd(2)O(3) nanoparticles is very promising for future studies of contrast agents based on gadolinium-containing nanocrystals.

Effect of the intracellular localization of a Gd‐based imaging probe on the relaxation enhancement of water protons

Gd-HPDO3A has been internalized into rat hepatocarcinoma cells in the cytoplasm (by electroporation) or in intracellular vesicles (by pinocytosis), respectively. In the former case, the observed relaxation rates are likely dependent upon the amount of internalized paramagnetic complex, whereas in the latter case the relaxation enhancement is "quenched" to a plateau value (about 3 s(-1)) when the entrapped amount of Gd-chelate is higher than 1 x 10(10) Gd/cell. The observed behavior has been accounted in terms of a theoretical treatment based on equations formally derived by Labadie et al. (J Magn Reson B 1994;105:99-102). On this basis, entrapment into intracellular vesicles has been treated as a three-site water exchange (extracellular/cytoplasm/vesicle compartments), whereas the cell pellets containing the paramagnetic agent spread out in the cytoplasm can be analyzed by a two-site exchange system.

Nuclear spin relaxation study of aqueous raffinose solution in the presence of a gadolinium contrast agent

Paramagnetic enhancement of nuclear spin-lattice relaxation rates (PREs) was measured in aqueous solution of the trisaccharide raffinose in the presence of a gadolinium(III) complex, GdDTPA-BMA, used as a magnetic resonance imaging contrast agent. The relaxation enhancement of aqueous protons was measured over a broad range of magnetic fields, using field-cycling apparatus in addition to conventional spectrometers. The nuclear magnetic relaxation dispersion profile thus obtained was interpreted with a recently developed model, allowing for both inner- and outer-sphere relaxation. The relaxation enhancement for the carbon-13 nuclei in raffinose was studied under high-resolution conditions at three magnetic fields, whereas the sugar proton PRE was measured at two fields. The PRE of the sugar nuclei could be interpreted in a consistent way, assuming that it was caused by the outer-sphere mechanism. The electron spin relaxation was found to be a less important source of modulation of the electron-nuclear dipole-dipole interaction than the mutual translational diffusion.

Identification of protein surfaces by NMR measurements with a pramagnetic Gd(III) chelate.

Gd-diethylenetriamine pentaacetic acid-bismethylamide, Gd(DTPA-BMA), is shown to be a reagent suitable for the identification of protein surfaces. Compared to the conventionally used spin-label TEMPOL, Gd(DTPA-BMA) is a stronger relaxation agent, requiring lesser concentrations to achieve the same paramagnetic relaxation enhancement of solvent-exposed protein protons. It is also less hydrophobic and therefore less prone to specific binding to proteins. Relaxation enhancements predicted by a second-sphere interaction model correlated with experimental data recorded with ubiquitin, while the correlation with corresponding data recorded with TEMPOL was poor.