Single Thiol Group Research Articles

Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe‐Functionalized Outer Surfaces

AbstractNanochannel technology has emerged as a powerful tool for label‐free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner walls of a nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel arrays with outer‐surface probes. The detection principle relies on the specific binding between the maleimide groups in outer‐surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation‐related diseases.

Detection of Unfolded Cellular Proteins Using Nanochannel Arrays with Probe-Functionalized Outer Surfaces.

Nanochannel technology has emerged as a powerful tool for label-free and highly sensitive detection of protein folding/unfolding status. However, utilizing the inner walls of a nanochannel array may cause multiple events even for proteins with the same conformation, posing challenges for accurate identification. Herein, we present a platform to detect unfolded proteins through electrical and optical signals using nanochannel arrays with outer-surface probes. The detection principle relies on the specific binding between the maleimide groups in outer-surface probes and the protein cysteine thiols that induce changes in the ionic current and fluorescence intensity responses of the nanochannel array. By taking advantage of this mechanism, the platform has the ability to differentiate folded and unfolded state of proteins based on the exposure of a single cysteine thiol group. The integration of these two signals enhances the reliability and sensitivity of the identification of unfolded protein states and enables the distinction between normal cells and Huntington's disease mutant cells. This study provides an effective approach for the precise analysis of proteins with distinct conformations and holds promise for facilitating the diagnoses of protein conformation-related diseases.

The lineage-specific, intrinsically disordered N-terminal extension of monothiol glutaredoxin 1 from trypanosomes contains a regulatory region

Glutaredoxins (Grx) are small proteins conserved throughout all the kingdoms of life that are engaged in a wide variety of biological processes and share a common thioredoxin-fold. Among them, class II Grx are redox-inactive proteins involved in iron-sulfur (FeS) metabolism. They contain a single thiol group in their active site and use low molecular mass thiols such as glutathione as ligand for binding FeS-clusters. In this study, we investigated molecular aspects of 1CGrx1 from the pathogenic parasite Trypanosoma brucei brucei, a mitochondrial class II Grx that fulfills an indispensable role in vivo. Mitochondrial 1CGrx1 from trypanosomes differs from orthologues in several features including the presence of a parasite-specific N-terminal extension (NTE) whose role has yet to be elucidated. Previously we have solved the structure of a truncated form of 1CGrx1 containing only the conserved glutaredoxin domain but lacking the NTE. Our aim here is to investigate the effect of the NTE on the conformation of the protein. We therefore solved the NMR structure of the full-length protein, which reveals subtle but significant differences with the structure of the NTE-less form. By means of different experimental approaches, the NTE proved to be intrinsically disordered and not involved in the non-redox dependent protein dimerization, as previously suggested. Interestingly, the portion comprising residues 65–76 of the NTE modulates the conformational dynamics of the glutathione-binding pocket, which may play a role in iron-sulfur cluster assembly and delivery. Furthermore, we disclosed that the class II-strictly conserved loop that precedes the active site is critical for stabilizing the protein structure. So far, this represents the first communication of a Grx containing an intrinsically disordered region that defines a new protein subgroup within class II Grx.

Comparative study on thiol drugs’ effect on tert-butyl hydroperoxide induced luminol chemiluminescence in human erythrocyte lysate and hemoglobin oxidation

The current studies have investigated the effect of heterocyclic drugs with the single thiol group (thiamazole, mercaptopurine) and dithiol aliphatic drugs (dimercaptosuccinic acid, dithiothreitol) under oxidative stress conditions, using tert-butyl hydroperoxide (t-BuOOH), in human erythrocyte lysate with the luminol-enhanced chemiluminescence technique. Knowing that oxidative processes induced by t-BuOOH are triggered by (oxy)hemoglobin (Hb), the effect of different thiol drugs (RSH) on isolated human Hb oxidation to methemoglobin (MHb) and hemichromes (HChr) was further considered. Three types of chemiluminescence curves, fitting to logistic-exponential model, have been revealed under influence of RSH. Structure of the data (MHb and HChr production, and free radical activity of RSH) in Principal Component Analysis visualization and kinetic profiles of chemiluminescence integrate information in terms of the diversity of RSH reaction mechanisms depending on the specific molecular context of the given thiol: aliphatic or aromatic nature as well as the number and position of the –SH groups in the molecule. The study conducted in presented in vitro systems indicates the potential role of thiol drugs mediated toxicity in an oxidative stress dependent mechanism.

Electron transport in asymmetric biphenyl molecular junctions: effects of conformation and molecule-electrode distance

On the basis of ab-initio calculations, we predict the effect of conformation and molecule-electrode distance on transport properties of asymmetric molecular junctions for different electrode materials M (M = Au, Ag, Cu, and Pt). The asymmetry in these junctions is created by connecting one end of the biphenyl molecule to conjugated double thiol (model A) and single thiol (model B) groups, while the other end to Cu atom. A variety of phenomena viz. rectification, negative differential resistance (NDR), switching has been observed that can be controlled by tailoring the interface state properties through molecular conformation and molecule-electrode distance for various M. These properties are further analyzed by calculating transmission spectra, molecular orbitals, and orbital energy. It is found that Cu electrode shows significantly enhanced rectifying performance with change in torsion angles, as well as with increase in molecule-electrode distances than Au and Ag electrodes. Moreover, Pt electrode manifests distinctive multifunctional behavior combining switch, diode, and NDR. Thus, the Pt electrode is suggested to be a good potential candidate for a novel multifunctional electronic device. Our findings are compared with available experimental and theoretical results.

Mixed disulfide formation at Cys141 leads to apparent unidirectional attenuation of Aspergillus niger NADP-glutamate dehydrogenase activity.

NADP-Glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoid 2-oxoglutarate saturation. Incubation with 2-hydroxyethyl disulfide (2-HED, the disulfide of 2-mercaptoethanol) resulted in preferential attenuation of AnGDH reductive amination (forward) activity but with a negligible effect on oxidative deamination (reverse) activity, when monitored in the described standard assay. Such a disulfide modified AnGDH displaying less than 1.0% forward reaction rate could be isolated after 2-HED treatment. This unique forward inhibited GDH form (FIGDH), resembling a hypothetical ‘one-way’ active enzyme, was characterized. Kinetics of 2-HED mediated inhibition and protein thiol titrations suggested that a single thiol group is modified in FIGDH. Two site-directed cysteine mutants, C141S and C415S, were constructed to identify the relevant thiol in FIGDH. The forward activity of C141S alone was insensitive to 2-HED, implicating Cys141 in FIGDH formation. It was observed that FIGDH displayed maximal reaction rate only after a pre-incubation with 2-oxoglutarate and NADPH. In addition, compared to the native enzyme, FIGDH showed a four fold increase in K0.5 for 2-oxoglutarate and a two fold increase in the Michaelis constants for ammonium and NADPH. With no change in the GDH reaction equilibrium constant, the FIGDH catalyzed rate of approach to equilibrium from reductive amination side was sluggish. Altered kinetic properties of FIGDH at least partly account for the observed apparent loss of forward activity when monitored under defined assay conditions. In sum, although Cys141 is catalytically not essential, its covalent modification provides a striking example of converting the biosynthetic AnGDH into a catabolic enzyme.

Effect of electrode materials on transport properties of asymmetric biphenyl molecular junctions

We analyze the effect of electrode materials Y (Y=Ag, Cu, and Pt) on electronic transport properties of asymmetric biphenyl molecular junctions using first-principles calculations. To introduce coupling asymmetry in these junctions, one end of the biphenyl molecule is terminated by conjugated double thiol (model A) and single thiol (model B) anchoring groups, while the other end is terminated by Cu atom. We reveal that, for Ag and Cu electrode, conjugated double thiol exhibit stronger rectifying performance in contrast to nonconjugated single thiol group. Further, when the molecule is connected to the Pt electrode through single thiol and Cu atom, multifunctional behavior with excellent rectifying performance and clear negative differential resistance (NDR) are observed for the first time. The present results will pave a new roadmap for designing functional molecular devices.

Ultrasound‒Mediated Synthesis of High‒Molecular Weight Polystyrene‒Grafted Silver Nanoparticles by Facile Ligand Exchange Reactions in Suspension

Ultrasound mediated facile ligand exchange method in suspension for the formation of polystyrene-grafted silver nanoparticles is reported. Amazingly, this method allows even grafting of very high molecular weight polystyrenes (up to 217 200 g mol(-1) ) having a single terminal thiol group at the chain end. Detailed studies are carried out to gain insights in the role of molecular weight of the ligands and the mechanism of the ligand exchange reactions. Key factors are determined to be the droplet formation by ultrasonification and low silver content, which enhances the availability of the terminal thiol end group significantly. The extraordinary compatibility of the ligand exchange method in particular regarding high molecular weights is attributed to hydrophilic orientation of the terminal thiol groups at the liquid-liquid interphase. This is proved conclusively by using an in situ method as a reference approach in which agglomeration occurs at considerably lower molecular weights due to the absence of preferred end group orientation within the polymer coil. In homogeneous phase only the chain length is found to be the crucial factor in stabilization of silver nanoparticles.

A facile and green ultrasonic-assisted synthesis of BSA conjugated silver nanoparticles

The formation and growth of hybrid nanoparticles of a protein BSA and silver by ultrasonic assistance were tracked by surface plasmon resonance signal of silver nanoparticles and light scattering. The hybrid nanoparticles were characterized by surface plasmon resonance spectra, light scattering, TEM, circular dichroism spectroscopy and zeta potential. Size along with the spherical shape of the nanoparticles could be controlled and nanoparticles with diameters ranging from 8 to 140nm could be obtained, depending upon the ultrasonication time (15–30min) and molar ratio of AgNO3/BSA (20–200). The role of single free thiol group in the reduction of silver ions was also investigated by using DTNB modified BSA and protein conjugated silver nanoparticles were formed even with thiol modified BSA. The growth and size of the nanoparticles were governed by ultrasonic assisted Ostwald ripening. BSA conjugated with silver nanoparticles showed changes in the secondary structure with an increase in the beta sheet structure to 33% as compared to 7% in native BSA as determined by CD spectra. Zeta potential measurements in the pH range of 2.0–12.0 demonstrated that the surface charges of the BSA conjugated silver nanoparticles were similar to that of native BSA suggesting that surface charges and overall three dimensional structure of BSA did not change much. This approach provides a strategy for completely green synthesis of hybrid nanoparticles consisting of a biological entity and an inorganic material. This is the first application of ultrasonic assistance in formation of such hybrid nanomaterials in aqueous media.

Engineering Surface Adhered Poly(vinyl alcohol) Physical Hydrogels as Enzymatic Microreactors

In this work, we characterize physical hydrogels based on poly(vinyl alcohol), PVA, as intelligent biointerfaces for surface-mediated drug delivery. Specifically, we assemble microstructured (μS) surface adhered hydrogels via noncryogenic gelation of PVA, namely polymer coagulation using sodium sulfate (Na(2)SO(4)). We present systematic investigation of concentrations of Na(2)SO(4) as a tool of control over assembly of μS PVA hydrogels and quantify polymer losses and retention within the hydrogels. For polymer quantification, we use custom-made PVA with single terminal thiol group in a form of mixed disulfide with Ellman's reagent which provides for a facile UV-vis assay of polymer content in coagulation baths, subsequent washes in physiological buffer, and within the hydrogel phase. Polymer coagulation using varied concentrations of sodium sulfate afforded biointerfaces with controlled elasticity for potential uses in investigating mechano-sensitive effects of mammalian cell culture. For surface mediated drug delivery, we propose a novel concept termed Substrate Mediated Enzyme Prodrug Therapy (SMEPT) and characterize μS PVA hydrogels as reservoirs for enzymatic cargo. Assembled functional interfaces are used as matrices for cell culture and delivery of anticancer drug achieved through administration of a benign prodrug, its conversion into an active therapeutic within the hydrogel phase, and subsequent internalization by adhered hepatic cells. Taken together, the presented data contribute significantly to the development of novel matrices for surface-mediated drug delivery and other biomedical applications.

Gas-Phase Peptide Sulfinyl Radical Ions: Formation and Unimolecular Dissociation

A variety of peptide sulfinyl radical (RSO•) ions with a well-defined radical site at the cysteine side chain were formed at atmospheric pressure (AP), sampled into a mass spectrometer, and investigated via collision-induced dissociation (CID). The radical ion formation was based on AP reactions between oxidative radicals and peptide ions containing single inter-chain disulfide bond or free thiol group generated from nanoelectrospray ionization (nanoESI). The radical induced reactions allowed large flexibility in forming peptide radical ions independent of ion polarity (protonated or deprotonated) or charge state (singly or multiply charged). More than 20 peptide sulfinyl radical ions in either positive or negative ion mode were subjected to low energy collisional activation on a triple-quadrupole/linear ion trap mass spectrometer. The competition between radical- and charge-directed fragmentation pathways was largely affected by the presence of mobile protons. For peptide sulfinyl radical ions with reduced proton mobility (i.e., singly protonated, containing basic amino acid residues), loss of 62 Da (CH(2)SO), a radical-initiated dissociation channel, was dominant. For systems with mobile protons, this channel was suppressed, while charge-directed amide bond cleavages were preferred. The polarity of charge was found to significantly alter the radical-initiated dissociation channels, which might be related to the difference in stability of the product ions in different ion charge polarities.

Straightforward thiol-mediated protein labelling with DTPA: Synthesis of a highly active 111In-annexin A5-DTPA tracer

BackgroundAnnexin A5 (anxA5) has been found useful for molecular imaging of apoptosis and other biological processes.MethodsHere, we report an optimised two-step synthesis of annexin A5-diethylene triamine pentaacetic acid (DTPA) (anxA5-DTPA) for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a single purification step. The use of a recombinant annexin A5 (cys-anxA5) with a single thiol group allowed regionally specific coupling, without affecting the binding domain of cys-anxA5.ResultsThe metal complexing capacity of anxA5-DTPA was investigated by labelling with 111In3+ and Eu3+. Binding of modified anxA5-DTPA to apoptotic cells was tested in competition experiments with a fluorescent anxA5 derivative (anxA5-FITC) using flow cytometry and compared with that of wildtype anxA5 or non-binding anxA5-DTPA (M1234-anxA5-DTPA). The binding affinity to apoptotic cells of the anxA5-DTPA conjugate does not differ from that of wildtype anxA5.ConclusionsThis two-step synthesis of annexin A5-DTPA resulted in biologically active anxA5-DTPA, which can be labelled with radionuclides for use in SPECT and PET imaging.

Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein

Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimer’s disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.

Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold

The synthesis of multithiolated DNA molecules that can be used to produce self-assembled monolayers of single-stranded DNA oligonucleotides on gold substrates is described. Generation 3 polyamidoamine (PAMAM) dendrimers were conjugated to DNA oligomers and functionalized with ~30 protected thiol groups. The protected thiol groups-thioacetate groups-allowed the dendrimer-DNA constructs to be stored in a buffer solution for at least 2 months before deprotection without any observable decrease in their ability to assemble into functional layers. The monolayers formed using these multithiolated DNA probe strands demonstrate target capture efficiencies comparable to those of analogous monolayers assembled with DNA functionalized with single thiol groups. A functional advantage of using dendrimer headgroups is the resistance to probe strand loss in prolonged exposure to buffer solutions at a high temperature (95 °C).

On the interaction of bovine serum albumin with ionic surfactants: Temperature induced EPR changes of a maleimide nitroxide reflect local protein dynamics and probe solvent accessibility

The interaction of bovine serum albumin (BSA) with the ionic surfactants sodium dodecylsulfate (SDS, anionic), cetyltrimethylammonium chloride (CTAC, cationic) and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS, zwitterionic) was studied by electron paramagnetic resonance (EPR) spectroscopy of spin label covalently bound to the single free thiol group of the protein. EPR spectra simulation allows to monitor the protein dynamics at the labeling site and to estimate the changes in standard Gibbs free energy, enthalpy and entropy for transferring the nitroxide side chain from the more motionally restricted to the less restricted component. Whereas SDS and CTAC showed similar increases in the dynamics of the protein backbone for all measured concentrations, HPS presented a smaller effect at concentrations above 1.5 mM. At 10 mM of surfactants and 0.15 mM BSA, the standard Gibbs free energy change was consistent with protein backbone conformations more expanded and exposed to the solvent as compared to the native protein, but with a less pronounced effect for HPS. In the presence of the surfactants, the enthalpy change, related to the energy required to dissociate the nitroxide side chain from the protein, was greater, suggesting a lower water activity. The nitroxide side chain also detected a higher viscosity environment in the vicinity of the paramagnetic probe induced by the addition of the surfactants. The results suggest that the surfactant–BSA interaction, at higher surfactant concentration, is affected by the affinities of the surfactant to its own micelles and micelle-like aggregates. Complementary DLS data suggests that the temperature induced changes monitored by the nitroxide probe reflects local changes in the vicinity of the single thiol group of Cys-34 BSA residue.

Carbosilane dendrons as stabilizing agents for the formation of gold nanoparticles

The synthesis and characterization of two new carbosilane dendrons functionalized in the focal point with a single thiol group is described. These molecules were used as stabilizing agents for the formation of gold nanoparticles. These materials were obtained by the reduction of hydrogen tetrachloroaurate phase-transferred into toluene in the presence of the carbosilane dendrons. The gold nanoparticle-cored dendrons (NCDs) were characterized by TEM, XPS, AFM, IR, UV, and NMR techniques. Average particle diameter ranged from 2.5 to 2.6 nm with narrow size distribution and remarkable stability; in particular, the elimination of the protecting shells to give naked gold, after NCDs deposition on a support, is more difficult using carbosilane dendrons than in the case of conventional dodecanethiol-protected gold nanoparticles.

Cyclopentenone prostaglandin-induced unfolding and aggregation of the Parkinson disease-associated UCH-L1.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) has been implicated in Parkinson's disease (PD) and is present in neurofibrillary tangles or Lewy bodies. However, the molecular basis for UCH-L1s involvement in proteinacious fibril formation is still elusive, especially in regard to the pathogenicity of the I93M mutation. Here we show that modification of UCH-L1 by cyclopentenone prostaglandins causes unfolding and aggregation. A single thiol group on Cys152 reacts with the alpha,beta-unsaturated carbonyl center in the cyclopentenone ring of prostaglandins, resulting in a covalent adduct. We also show that the PD-associated I93M mutant of UCH-L1 is well-folded, structurally similar to the wild-type protein, and aggregates upon conjugation by cyclopentenone prostaglandins. Our findings suggest a possible mechanistic link between UCH-L1 modification by cyclopentenone prostaglandins and the etiology of neurodegeneration.

The influence of a single thiol group on the electronic and optical properties of the smallest diamondoid adamantane

At the nanoscale, the surface becomes pivotal for the properties of semiconductors due to an increased surface-to-bulk ratio. Surface functionalization is a means to include semiconductor nanocrystals into devices. In this comprehensive experimental study we determine in detail the effect of a single thiol functional group on the electronic and optical properties of the hydrogen-passivated nanodiamond adamantane. We find that the optical properties of the diamondoid are strongly affected due to a drastic change in the occupied states. Compared to adamantane, the optical gap in adamantane-1-thiol is lowered by approximately 0.6 eV and UV luminescence is quenched. The lowest unoccupied states remain delocalized at the cluster surface leaving the diamondoid's negative electron affinity intact.

S-Nitrosylated Human Serum Albumin-mediated Cytoprotective Activity Is Enhanced by Fatty Acid Binding

Binding of oleate to S-nitrosylated human serum albumin (SNO-HSA) enhances its cytoprotective effect on liver cells in a rat ischemia/reperfusion model. It enhances the antiapoptotic effect of SNO-HSA on HepG2 cells exposed to anti-Fas antibody. To identify some of the reasons for the increased cytoprotective effects, additional experiments were performed with glutathione and HepG2 cells. As indicated by 5,5′-dithiobis-2-nitrobenzoic acid binding, the addition of oleate increased the accessibility of the single thiol group of albumin. Binding of increasing amounts of oleate resulted in increasing and more rapid S-transnitrosation of glutathione. Likewise, binding of oleate, or of a mixture of endogenous fatty acids, improved S-denitrosation of SNO-HSA by HepG2 cells. Oleate also enhanced S-transnitrosation by HepG2 cells, as detected by intracellular fluorescence of diaminofluorescein-FM. All of the S-transnitrosation caused by oleate binding was blocked by filipin III. Oleate also increased, in a dose-dependent manner, the binding of SNO-HSA labeled with fluorescein isothiocyanate to the surface of the hepatocytes. A model in two parts was worked out for S-transnitrosation, which does not involve low molecular weight thiols. Fatty acid binding facilitates S-denitrosation of SNO-HSA, increases its binding to HepG2 cells and greatly increases S-transnitrosation by hepatocytes in a way that is sensitive to filipin III. A small nitric oxide transfer takes place in a slow system, which is unaffected by fatty acid binding to SNO-HSA and not influenced by filipin III. Thus, fatty acids could be a novel type of mediator for S-transnitrosation.

Evaluation of Maleimide Derivative of DOTA for Site-Specific Labeling of Recombinant Affibody Molecules

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.