Metal-protein Complexes Research Articles

Enhancing wound healing with zinc and silver nanocomposites synthesized with β-lactoglobulin: antimicrobial properties, collagen deposition, and systemic effects in a C57BL/6J mouse model

This study explores the potential of zinc and silver nanocomposites, synthesized with β-lactoglobulin, a whey protein, in promoting wound healing, using the C57BL/6J mouse model. Our research is distinct in its dual focus: assessing the antimicrobial efficacy of these nanocomposites and their impact on wound healing processes. The antimicrobial properties were investigated through minimum inhibitory concentration (MIC) assessments and colony-forming unit (CFU) tests, providing insights into their effectiveness against wound-associated microorganisms. Notably, the formulation's effective antibacterial concentration did not exhibit toxicity to mouse fibroblasts. A key aspect of our methodology involved the use of a stereoscopic microscope for detailed monitoring of the wound closure process. Additionally, the distribution and potential systemic effects of the zinc and silver ions were analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). This analysis was crucial in evaluating metal ion absorption through the wound site and estimating any toxic effects on the body. Our findings are particularly significant in the field of regenerative medicine. Transmission electron microscopy (TEM) revealed that the tested nanocomposites notably enhanced collagen deposition, a vital component in the wound healing process. Furthermore, a reduction in glycogen levels in hepatocytes was observed following treatment with these metal-protein dressings. This novel finding warrants further investigation. Overall, our findings highlight the diverse roles of zinc and silver nanocomposites in wound healing. This study not only contributes to our understanding of metal-protein complexes in tissue regeneration but also opens new avenues for research into the delivery mechanisms of such treatments for hard-to-heal wounds.

Mass spectrometry imaging of SOD1 protein-metal complexes in SOD1G93A transgenic mice implicates demetalation with pathology.

Amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motor neurons in the central nervous system (CNS). Mutations in the metalloenzyme SOD1 are associated with inherited forms of ALS and cause a toxic gain of function thought to be mediated by dimer destabilization and misfolding. SOD1 binds two Cu and two Zn ions in its homodimeric form. We have applied native ambient mass spectrometry imaging to visualize the spatial distributions of intact metal-bound SOD1G93A complexes in SOD1G93A transgenic mouse spinal cord and brain sections and evaluated them against disease pathology. The molecular specificity of our approach reveals that metal-deficient SOD1G93A species are abundant in CNS structures correlating with ALS pathology whereas fully metalated SOD1G93A species are homogenously distributed. Monomer abundance did not correlate with pathology. We also show that the dimer-destabilizing post-translational modification, glutathionylation, has limited influence on the spatial distribution of SOD1 dimers.

Metal ions in biomedically relevant macromolecular structures.

Understanding the functions of metal ions in biological systems is crucial for many aspects of research, including deciphering their roles in diseases and potential therapeutic use. Structural information about the molecular or atomic details of these interactions, generated by methods like X-ray crystallography, cryo-electron microscopy, or nucleic magnetic resonance, frequently provides details that no other method can. As with any experimental method, they have inherent limitations that sometimes lead to an erroneous interpretation. This manuscript highlights different aspects of structural data available for metal-protein complexes. We examine the quality of modeling metal ion binding sites across different structure determination methods, where different kinds of errors stem from, and how they can impact correct interpretations and conclusions.

Au Nanocluster Assisted Microstructural Reconstruction for Buried Interface Healing for Enhanced Perovskite Solar Cell Performance.

The heterogeneity of perovskite film crystallization along the vertical direction leads to voids and traps at the buried interfaces, hampering both efficiency and stability of perovskite solar cells. Here, bovine serum albumin-functionalized Au nanoclusters (ABSA), combined with heavy gravity, high surface charge density, and strong interactions with the electron transport layer, are designed to reconstruct the buried interfaces for not only high-quality crystallization, but also improved carrier transfer. The ABSA macromolecules with amine function groups and larger surface charge density interact with the perovskite to improve the crystallinity, and gradually migrate towards the buried interface, healing the defective voids, hence suppressing surface recombination velocity from 3075 to 452 cm s-1 . The healed buried interface and the higher surface potential of ABSA-modified TiO2 lead to improved carrier extraction at the interface. The resulting solar cell attains a power conversion efficiency of 25.0% with negligible hysteresis and remarkable stability, maintaining 92.9% of their initial efficiency after 3200 h of exposure to the ambient atmosphere, they also exhibit better continuous irradiation stability compared to control devices. These findings provide a new metal-protein complex to eliminate the deleterious voids and defects at the buried interface for improved photovoltaic performance and stability.

Structural Characterization of Cu(I)/Zn(II)-metallothionein-3 by Ion Mobility Mass Spectrometry and Top-Down Mass Spectrometry.

Mammalian zinc metallothionein-3 (Zn7MT3) plays an important role in protecting against copper toxicity by scavenging free Cu(II) ions or removing Cu(II) bound to β-amyloid and α-synuclein. While previous studies reported that Zn7MT3 reacts with Cu(II) ions to form Cu(I)4Zn(II)4MT3ox containing two disulfides (ox), the precise localization of the metal ions and disulfides remained unclear. Here, we undertook comprehensive structural characterization of the metal-protein complexes formed by the reaction between Zn7MT3 and Cu(II) ions using native ion mobility mass spectrometry (IM-MS). The complex formation mechanism was found to involve the disassembly of Zn3S9 and Zn4S11 clusters from Zn7MT3 and reassembly into Cu(I)xZn(II)yMT3ox complexes rather than simply Zn(II)-to-Cu(I) exchange. At neutral pH, the β-domain was shown to be capable of binding up to six Cu(I) ions to form Cu(I)6Zn(II)4MT3ox, although the most predominant species was the Cu(I)4Zn(II)4MT3ox complex. Under acidic conditions, four Zn(II) ions dissociate, but the Cu(I)4-thiolate cluster remains stable, highlighting the MT3 role as a Cu(II) scavenger even at lower than the cytosolic pH. IM-derived collision cross sections (CCS) reveal that Cu(I)-to-Zn(II) swap in Zn7MT3 with concomitant disulfide formation induces structural compaction and a decrease in conformational heterogeneity. Collision-induced unfolding (CIU) experiments estimated that the native-like folded Cu(I)4Zn(II)4MT3ox conformation is more stable than Zn7MT3. Native top-down MS demonstrated that the Cu(I) ions are exclusively bound to the β-domain in the Cu(I)4Zn(II)4MT3ox complex as well as the two disulfides, serving as a steric constraint for the Cu(I)4-thiolate cluster. In conclusion, this study enhances our comprehension of the structure, stability, and dynamics of Cu(I)xZn(II)yMT3ox complexes.

Side chain inset of neurogenerative amino acids to metalloproteins: a therapeutic signature for huntingtin protein in Huntington's disease.

Huntington's disease is a dominant autosomal inherited neurodegenerative disease that results in progressive impairment, characterized by dementia, chorea, and behavioral and cognitive decline. The objective of this study was to investigate the potential activity of metalloproteins against the huntingtin protein using various insertion-based engineering computational methods. Metalloproteins, metal protein complexes involved in important biochemical and physiological processes, were explored as potential drug candidates for Huntington's disease. A total of 18 metalloproteins were selected as drug candidates and studied to assess their potential inhibitory effects on the huntingtin protein. The screening process was based on the lowest binding energy. The metalloprotein with the lowest docking score was chosen for side chain insertion of neurogenerative amino acids. The engineered metalloprotein was then evaluated based on physiochemical properties, allergenicity, toxicity, and surface accessibility. Cloning and expression analysis was performed to further investigate its potential as a therapeutic agent. The metalloprotein chosen for side chain insertion, cytochrome C oxidase, showed promising results. It was computed as a probable non-allergen and exhibited no toxic domains, indicating its non-toxic nature. Additionally, it demonstrated a strong binding affinity with the huntingtin protein, with a binding energy of -1,253.3 Kcal/mol. Metal-based proteins, when engineered with additional neurogenerative amino acids, hold potential as drug candidates for treating neurodegenerative diseases such as Huntington's disease. The successful development of these engineered metalloproteins could offer therapeutic advantages. Further testing, both in vitro and in vivo, is necessary to evaluate their efficacy and validate their potential activity as novel drugs for the treatment of neurodegenerative diseases.

Tuzaklamalı İyon Hareketliliği-Kütle Spektrometrisi Tekniği Kullanılarak İnsülinin Bakır ve Alkali Metal Komplekslerinin Konformasyonel Farklılıklarının Analizi

Molecular recognition, protein folding, and formation of supramolecular structures that occur at the molecular level of biological processes are based on noncovalent interactions. Interactions between metal atoms and proteins are based on noncovalent interactions that underlie the mechanisms involved in many cellular processes. The activities of enzymes are highly dependent on the interactions of such protein groups with cofactors, substrates, metal ions, and other proteins. The compositions and binding stoichiometry of protein-metal complexes can be determined with high accuracy performing mass spectrometry (MS) analysis. The conformational features of protein-metal complexes can be studied additionally using a mass spectrometer with ion mobility spectrometry (IMS) capability. This study focuses the monitoring the differences in the conformational changes of insulin protein during the formation of its complex with copper and alkali metals using trapped ion mobility spectrometry – time-of-flight (TIMS–TOF) mass spectrometer instrument. The compaction of the insulin structure by the formation of the insulin-copper complexes in the gas phase was determined with TIMS-TOF-MS analyses. However, no change was observed in the insulin structure with the addition of H, Na, and K atoms as adducts at the same analysis conditions.

Bioinspired metal/metal oxide nanoparticles: A road map to potential applications

Manufacturing of metal and metal oxide nanoparticles (M/MO NPs) in large quantities needed a strong reliable, sustainable, and eco-friendly protocol. Present work represents on biogenic approaches to fabricate green nanoparticles using green technology. The fabrications of M/MO NPs using natural bio-resources were engaged by means of alternative technique in place of conventional methods. These methods are naturally benign, straightforward, economical, and renewed technology; they does not content harmful chemicals, zero contaminants, and eco-friendly. The extracts from the biogenic resources are widely accepted owing to its capability to minimise and control the size and shape of metal and metal oxides NPs because of different structure directing agents, usually bioorganic phyto-chemicals. In this present review, we have summarized fabrication of different NPs like silver, gold, copper oxide, cobalt oxide, titanium oxide, cerium oxide, bismuth oxide, zinc oxide and nickel oxide nanoparticles using natural resources. The challenges, limiting factors and future directions of the bioinspired synthesis of metal/metal oxide NPs are also highlighted in this review. Moreover, biogenic materials has explored for further environmental remediation in terms of photocatalytic activity, elimination of organic waste, and antibacterial, antioxidant assay, and protein-metal complexes binding affinities by molecular docking.

Study on zinc ions binding to the individual casein fractions: αS1-, β- and κ-casein

The presented work is focused on the isotherm study on the Zn2+ ions binding to the individual casein fractions: αS1-, β- and κ-casein (αS1CN, βCN and κCN). The experimental isotherms were evaluated using Freundlich and Langmuir models. Subsequently, the stability of the obtained complexes in the respective Zn2+ ion concentrations (120, 350, 600 mg/L) was determined by carrying out zeta potential measurements. Capillary electrophoresis combined with inductively coupled plasma mass spectrometry (CE-ICP-MS) confirmed the occurring binding process. Additionally, physicochemical characteristics of the obtained metal-protein complexes was performed including scanning electron microscopy (SEM) in two modes (SE and Z-contrast) and the binding sites of caseins to Zn2+ ions were indicated using attenuated total reflectance infrared spectroscopy (FTIR-ATR) and Raman analysis as well as mass spectrometry technique (MALDI-TOF MS). Isothermal studies indicated a heterogeneously complex zinc ion adsorption process, and a stability study showed that the zeta potential is strongly related to the hydrophobicity, size and structure of the casein isoforms studied. Electron microscopy confirmed the modification of casein surfaces due to the addition of Zn2+ ions. Spectroscopic techniques indicated the interaction of zinc ions with polar amino acids of casein, such as glutamic acid (Glu) and aspartic acid (Asp), but also His, Cys. The influence of phosphate groups was also observed. Finally, the study culminated in a molecular docking study of the Zn2+ ion binding process, which confirmed the presence of the listed amino acids responsible for the binding process.

Detailed insight into chromium species released from failed CoCrMo implants: Ex vivo periprosthetic tissues study.

This unique study provides information on Cr species and their distribution in periprosthetic tissues of patients with metal-on-polyethylene joint implants. Co-Cr-Mo alloy has been widely used in joint replacement and represents a source of metal derived species. In the case of chromium, previous studies on periprosthetic tissues revealed mainly Cr(III) distribution, whereas the potential release of carcinogenic Cr(VI) species has been still a subject of debate. Here, an analytical approach utilizing speciation and fractionation was developed to analyze periprosthetic tissue samples collected from wide range of patients with failed total hip or knee replacements. The results reveal that Cr(III) is mainly released in the form of insoluble CrPO4 and Cr2 O3 particles. The highest Cr contents were found in periprosthetic tissues of patients suffering from aseptic loosening and having more Cr-based implants in the body. Cr species penetrated tissue layers, but their levels decreased with the distance from an implant. The detailed speciation/fractionation study carried out using the set of consecutive periprosthetic tissues of a patient with extensive metallosis showed the presence of trace amounts of free Cr(III), nanoparticles, and metal-protein complexes, but the majority of Cr still occurred in CrPO4 form. Carcinogenic Cr(VI) species were not detected. Up to date, there is no published human tissue study focused on the detailed speciation of both soluble and insoluble Cr-based species in the context of failing total hip and knee replacements.

Learning to Identify Physiological and Adventitious Metal-Binding Sites in the Three-Dimensional Structures of Proteins by Following the Hints of a Deep Neural Network.

Thirty-eight percent of protein structures in the Protein Data Bank contain at least one metal ion. However, not all these metal sites are biologically relevant. Cations present as impurities during sample preparation or in the crystallization buffer can cause the formation of protein–metal complexes that do not exist in vivo. We implemented a deep learning approach to build a classifier able to distinguish between physiological and adventitious zinc-binding sites in the 3D structures of metalloproteins. We trained the classifier using manually annotated sites extracted from the MetalPDB database. Using a 10-fold cross validation procedure, the classifier achieved an accuracy of about 90%. The same neural classifier could predict the physiological relevance of non-heme mononuclear iron sites with an accuracy of nearly 80%, suggesting that the rules learned on zinc sites have general relevance. By quantifying the relative importance of the features describing the input zinc sites from the network perspective and by analyzing the characteristics of the MetalPDB datasets, we inferred some common principles. Physiological sites present a low solvent accessibility of the aminoacids forming coordination bonds with the metal ion (the metal ligands), a relatively large number of residues in the metal environment (≥20), and a distinct pattern of conservation of Cys and His residues in the site. Adventitious sites, on the other hand, tend to have a low number of donor atoms from the polypeptide chain (often one or two). These observations support the evaluation of the physiological relevance of novel metal-binding sites in protein structures.

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co2+ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe2+, as it is customarily done, Co2+ is most often used in experiments because Fe2+ is extremely unstable owing to the fast oxidation reaction Fe2+ → Fe3+. Protein stability is monitored following the conformational changes induced by Co2+ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co2+ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.

Electronic properties and surface potential evaluations at the protein nano-biofilm/oxide interface: Impact on corrosion and biodegradation

The formation of a protein nano-biofilm, which exhibits a special electronic behavior, on the surface of metals or oxide biomaterials considerably influences the crucial subsequent interactions, particularly the corrosion and biodegradation processes. This study discusses the impact of electrical surface potential (ESP) of a single or nano-biofilm of albumin protein on the electrochemical interactions and electronic property evolutions (e.g., charge carriers, space charge capacitance (SCC), and band bending) occurring on the surface oxide of CoCrMo implants. Scanning Kelvin probe force microscopy (SKPFM) results indicated that ESP or surface charge distribution on a single or nano-biofilm of the albumin protein is lower than that of a CoCrMo complex oxide layer, which hinders the charge transfer at the protein/electrolyte interface. Using a complementary approach, which involved performing Mott-Schottky analysis at the electrolyte/protein/oxide interface, it was revealed that the albumin protein significantly increases the SCC magnitude and number of n-type charge carrier owing to increased band bending at the SCC/protein interface; this facilitated the acceleration of metal ion release and metal-protein complex formation. The nanoscale SKPFM and electrochemical analyses performed in this study provide a better understanding of the role of protein molecules in corrosion/biodegradation of metallic biomaterials at the protein nano-biofilm/oxide interface.

Modular Evolution and Population Variability of Oikopleura dioica Metallothioneins.

Chordate Oikopleura dioica probably is the fastest evolving metazoan reported so far, and thereby, a suitable system in which to explore the limits of evolutionary processes. For this reason, and in order to gain new insights on the evolution of protein modularity, we have investigated the organization, function and evolution of multi-modular metallothionein (MT) proteins in O. dioica. MTs are a heterogeneous group of modular proteins defined by their cysteine (C)-rich domains, which confer the capacity of coordinating different transition metal ions. O. dioica has two MTs, a bi-modular OdiMT1 consisting of two domains (t-12C and 12C), and a multi-modular OdiMT2 with six t-12C/12C repeats. By means of mass spectrometry and spectroscopy of metal-protein complexes, we have shown that the 12C domain is able to autonomously bind four divalent metal ions, although the t-12C/12C pair –as it is found in OdiMT1– is the optimized unit for divalent metal binding. We have also shown a direct relationship between the number of the t-12C/12C repeats and the metal-binding capacity of the MTs, which means a stepwise mode of functional and structural evolution for OdiMT2. Finally, after analyzing four different O. dioica populations worldwide distributed, we have detected several OdiMT2 variants with changes in their number of t-12C/12C domain repeats. This finding reveals that the number of repeats fluctuates between current O. dioica populations, which provides a new perspective on the evolution of domain repeat proteins.

PHYLOGENETIC AFFINITY OF RAT AND SOME MAMMALIAN SPECIES METALLOTHIONEIN GENES

Metallothioneins ( are low molecular weight proteins, which have ability to bind bothmono and divalent metals and to form metal protein complexes. In mammals, MT are presentedby four isoforms (MT 1, MT 2, MT 3 and MT 4), which are encoded by the family of metallotioneingenes. The purpose of the present study was to determine the p hylogenetic affinity of different ratRattus norvegicus MT genes with other mammalian spe cies, as well as to determine the degree ofsimilarity between genes of MT isoforms within s pecies Rattus norvegicus. Therefore nucleotidesequences of MT genes were aligned, the percentage of identity and the number of gaps wascounted and a phylogenetic analysis was carrie d out. For genes of all Rattus norvegicus MTisoforms, significant similarity with other ma mmalian species Mus musculus Homo s ap iens Susscrofa was shown. The coding sequences ( of genes of Rattus norvegicus MT 1, MT 2A, MT 3and MT 4 isoforms have a significant similarity among themselves. The MT genes are hig hlyconservative, both at the interspecific and intraspecific levels, which is probably associated withtheir biological functions.

Semi-Automated Determination of Heavy Metals in Autopsy Tissue Using Robot-Assisted Sample Preparation and ICP-MS

The endoprosthetic care of hip and knee joints introduces multiple materials into the human body. Metal containing implant surfaces release degradation products such as particulate wear and corrosion debris, metal-protein complexes, free metallic ions, inorganic metal salts or oxides. Depending on the material composition of the prostheses, a systemic exposure occurs and may result in increasing metal concentrations in body fluids and tissues especially in the case of malfunctions of the arthroplasty components. High concentrations of Cr, Co, Ni, Ti and Al affect multiple organs such as thyroid, heart, lung and cranial nerves and may lead to metallosis, intoxications, poly-neuropathy, retinopathy, cardiomyopathy and the formation of localized pseudo tumors. The determination of the concentration of metals in body fluids and tissues can be used for predicting failure of hip or knee replacements to prevent subsequent severe intoxications. A semi-automated robot-assisted measurement system is presented for the determination of heavy metals in human tissue samples using inductively coupled plasma mass spectrometry (ICP-MS). The manual and automated measurement processes were similarly validated using certified reference material and the results are compared and discussed. The automation system was successfully applied in the determination of heavy metals in human tissue; the first results are presented.

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein-metal complexes and 10 protein-protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein-metal complexes offers insight into the metal-binding sites.

Mass Spectrometry-Based Structural Analysis of Cysteine-Rich Metal-Binding Sites in Proteins with MetaOdysseus R Software.

Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal–protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.

Characterizing the general chelating affinity of serum protein fetuin for lanthanides.

Fetuin is an abundant blood protein that participates in multiple biological processes, including the transport and regulation of calcium. Fetuin is also known to have a high affinity for uranium (as the uranyl dioxo cation) and plutonium, thus it has been suggested as one of the main endogenous chelating biomolecules involved in the transport of actinides following an internal uptake event. Nevertheless, no direct measurements of its affinity for f-elements beside these two actinides have been reported. Here, we investigate the interaction between fetuin and trivalent lanthanides, such as samarium, europium, terbium, and dysprosium, by mass spectrometry and fluorescence spectroscopy. Mass spectrometry results indicated that fetuin has four metal binding sites for the metal ions studied. Upon formation, the metal-protein complexes showed luminescence emission as a result of antenna sensitization of the metal ions, whose photophysics were characterized and exploited to perform direct spectrofluorimetric titrations. Furthermore, the thermodynamic constants were calculated for all complexes, confirming the formation of stable complexes with log [Formula: see text] values between 26 and 27. In characterizing the affinity of the serum protein fetuin for several f-elements, this study expands upon the initial findings focused on uranyl and plutonium, and contributes to a better understanding of the internal distribution and deposition of lanthanides, potentially representative of trivalent actinides.

Screening of potential uranium protein targets in fish ovaries after chronic waterborne exposure: Differences and similarities between roach and zebrafish

Concentration of uranium (U), a naturally encountered radioactive element in earth's crust, can be enhanced in freshwater ecosystems (μg.L−1 – mg.L−1) due to various anthropogenic activities. The consequent aquatic organism exposure to U leads to its accumulation in all organs, particularly in the gonad, and in subcellular fractions (mainly the cytosol); then it is known to affect fish at several biological levels, and more particularly, at a reproduction endpoint, with a decrease in the total number of eggs, spawn events and larvae survival. The understanding of U reprotoxicity requires the fine knowledge of its speciation at molecular level, i.e., its interaction with cytosolic biomolecules. In this study, we focus on the U-protein interactions in gonads. A non-denaturating extraction protocol combined with size exclusion chromatography (SEC) allowed the separation of metal-protein complexes in ovaries of U-contaminated wild roaches before their elemental detection (ICP MS). This enables unprecedented information to be obtained about U distribution in ovaries of autochthonous fish, Rutilus rutilus, which is different in some points from that obtained in the model species, Danio rerio under controlled laboratory conditions at a similar concentration level. Finally, the ability to transpose results from model to autochthonous fish was briefly discussed.