Transition Metal Binding Site Research Articles

Spectroscopic and computational investigations of Cobalt(II) binding to the innate immune protein human calprotectin.

Human calprotectin (CP) is an innate immune protein that participates in the metal-withholding response to infection by sequestering essential metal nutrients from invading microbial pathogens. CP is comprised of S100A8 (α subunit, 10.8kDa) and S100A9 (β subunit, 13.2kDa). Two transition-metal binding sites of CP form at the S100A8/S100A9 dimer interface. Site 1 is a His3Asp motif comprised of His83 and His87 from the S100A8 subunit and His20 and Asp30 from the S100A9 subunit. Site 2 is an unusual hexahistidine motif composed of S100A8 residues His17 and His27 and S100A9 residues His91, His95, His103, and His105. In the present study, the His3Asp and His6 sites of CP were further characterized by utilizing Co2+ as a spectroscopic probe. Magnetic circular dichroism spectroscopy was employed in conjunction with electron paramagnetic resonance spectroscopy and density functional theory computations to characterize the Co2+-bound S100A8(C42S)/S100A9(C3S) CP-Ser variant and six site variants that allowed the His3Asp and His6 sites to be further probed. Our results provide new insight into the metal-binding sites of CP-Ser and the effect of amino acid substitutions on the structure of site 2.

Study on metal binding capacity of the freshwater crab Sinopotamon henanense's recombinant copper specific binding metallothionein expressed in Escherichia coli.

The copper specific binding metallothionein (CuMT) is a type of cysteine-rich, metal-binding, small protein which plays an important role in Cu2+ metabolism in vertebrates. In this study, we investigated the metal tolerance and removing ability of recombinant strains harboring CuMT obtained in vivo from the freshwater crab Sinopotamon henanense (ShCuMT) in order to study its physiological functions and metal binding capacity. We performed a 3D modeling of ShCuMT and created its structural and functional models using the I-TASSER program. The shCumt gene was inserted into a pGEX-4t-1 vector and recombinant soluble ShCuMT was expressed in Escherichia coli. In addition, in order to characterize the tolerance and removing ability of heavy metals in E. coli with ShCuMT expression, the recombinant strains harboring ShCuMT were exposed to various concentrations of Cd2+, Cu2+ and Zn2+, respectively. The results showed that ShCuMT contains transition metal binding sites. In addition, E. coli cells expressing ShCuMT exhibited enhanced metal tolerance and higher removing ability of metal ions than control cells. However, compared with Cd2+ and Zn2+, E. coli cells expressing ShCuMT have stronger tolerance and higher removing ability of Cu2+. In general, ShCuMT contains multiple transition metal binding sites, and it could enhance tolerance and removing ability of metal ions. Therefore, ShCuMT can provide potential candidates for heavy metal bioremediation. This research on the metal binding properties of ShCuMT provides a scientific basis for bioremediation of heavy metal pollution by the recombinant strains.

Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4

Metal clusters are exploited by numerous metalloenzymes for catalysis, but it is not common to utilize a metal cluster for substrate transport across membrane. The recent crystal structure of a prototypic Zrt-/Irt-like protein (ZIP) metal transporter from Bordetella bronchiseptica (BbZIP) revealed an unprecedented binuclear metal center (BMC) within the transport pathway. Here, through a combination of bioinformatics, biochemical and structural approaches, we concluded that the two physically associated metal-binding sites in the BMC of human ZIP4(hZIP4) zinc transporter exert different functions: one conserved transition metal-binding site acts as the transport site essential for activity, whereas the variable metal-binding site is required for hZIP4's optimal activity presumably by serving as a secondary transport site and modulating the properties of the primary transport site. Sequential soaking experiments on BbZIP crystals clarified the process of metal release from the BMC to the bulky solvent. This work provides important insights into the transport mechanism of the ZIPs broadly involved in transition metal homeostasis and signaling, and also a paradigm on a novel function of metal cluster in metalloproteins.

Identification of a metallothionein gene in honey bee Apis mellifera and its expression profile in response to Cd, Cu and Pb exposure.

Metallothioneins are ubiquitous proteins important in metal homeostasis and detoxification. However, they have not previously been identified in honey bees or other Hymenoptera, where metallothioneins could be of ecophysiological and ecotoxicological significance. Better understanding of the molecular responses to stress induced by toxic metals could contribute to honey bee conservation. In addition, honey bee metallothionein could represent a biomarker for monitoring environmental quality. Here we identify and characterize a metallothionein gene in Apis mellifera (AmMT). AmMT is 1,680bp long and encodes a 48 amino acids protein with 15 cysteines and no aromatic residues. A metal response element upstream of the start codon, coupled with numerous cis-regulatory elements indicate the functional context of AmMT. Molecular modelling predicts several transition metal binding sites, and comparative phylogenetic analysis revealed five putative metallothionein proteins in three other hymenoptera species. AmMT was characterized by cloning the full-length coding sequence of the putative metallothionein. Recombinant AmMT was found to increase metal tolerance upon overexpression in Escherichia coli supplemented with Cd, Cu or Pb. Finally, in laboratory tests on honey bees, gene expression profiles showed a dose-dependant relationship between Cd, Cu and Pb concentrations present in food and AmMT expression, while field experiments showed induction of AmMT in bees from an industrial site compared to those from an urban area. These studies suggest that AmMT has metal binding properties in agreement with a possible role in metal homeostasis. Further functional and structural characterization of metallothionein in honey bees and other Hymenoptera are necessary.

Complex Nature of Protein Carbonylation Specificity After Metal-Catalyzed Oxidation.

Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood. In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry. Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45. Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.

Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173.

Background and AimsThe proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn2+ and Cu2+ concentration during hemodialysis.Methods and ResultsEpitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn2+ or Cu2+ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn2+ and Cu2+ concentrations after dialysis.ConclusionsOur study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.

Transmembrane Type-2-like Cu2+ Site in the P1B-3-type ATPase CopB: Implications for Metal Selectivity

Metal selectivity in P1B-type ATPase transporters is determined by conserved amino acid residues in their transmembrane helices responsible for metal binding and transport across the cellular membrane. The Cu(2+)-selective CopB from Archaeoglobus fulgidus has been investigated to explore the coordination chemistry of the transition metal binding sites in P1B-3-type ATPases. Electronic absorption, electron paramagnetic resonance, and X-ray absorption spectroscopic studies indicate the presence of a high-affinity transmembrane Type-2-like Cu(2+) center in which a single cupric ion is coordinated in a distorted square pyramidal geometry by mixed nitrogen/oxygen and sulfur ligands.

Discovering the electronic circuit diagram of life: structural relationships among transition metal binding sites in oxidoreductases

Oxidoreductases play a central role in catalysing enzymatic electron-transfer reactions across the tree of life. To first order, the equilibrium thermodynamic properties of these proteins are governed by protein folds associated with specific transition metals and ligands at the active site. A global analysis of holoenzyme structures and functions suggests that there are fewer than approximately 500 fundamental oxidoreductases, which can be further clustered into 35 unique groups. These catalysts evolved in prokaryotes early in the Earth's history and are largely responsible for the emergence of non-equilibrium biogeochemical cycles on the planet's surface. Although the evolutionary history of the amino acid sequences in the oxidoreductases is very difficult to reconstruct due to gene duplication and horizontal gene transfer, the evolution of the folds in the catalytic sites can potentially be used to infer the history of these enzymes. Using a novel, yet simple analysis of the secondary structures associated with the ligands in oxidoreductases, we developed a structural phylogeny of these enzymes. The results of this 'composome' analysis suggest an early split from a basal set of a small group of proteins dominated by loop structures into two families of oxidoreductases, one dominated by α-helices and the second by β-sheets. The structural evolutionary patterns in both clades trace redox gradients and increased hydrogen bond energy in the active sites. The overall pattern suggests that the evolution of the oxidoreductases led to decreased entropy in the transition metal folds over approximately 2.5 billion years, allowing the enzymes to use increasingly oxidized substrates with high specificity.

Diffusion-Enhanced Luminescence Resonance Energy Transfer in the Cytoplasm of Live Bacterial Cells

We previously found that a 17-amino acid lanthanide-binding tag (LBT) expressed as a fusion with a cytoplasmic protein in E. coli takes up Tb3+ in live cells (Biochemistry 50, 6789, 2011). The protein is called DAL after its three domains: dihydrofolate reductase, ankyrin repeats, and LBT. We expressed DAL with a C-terminal 6X-His-tag to create a transition metal binding site for an acceptor of luminescence resonance energy transfer (LRET) from the Tb3+ donor on the LBT. Cu2+ added outside the cells appeared to be transported into the cytoplasm, where it quenched the luminescence of DAL-His-tag similarly to purified DAL-His-tag in free solution. When we expressed DAL lacking a His-tag, we also observed Cu2+ quenching of luminescence, and removal of the highly charged ankyrin domains did not alter Cu2+ quenching. The quenching mechanism is likely to be LRET, because Cu+, which lacks spectral overlap with Tb3+, does not quench DAL luminescence. We conclude that the energy transfer probably occurs by a diffusion-enchanced mechanism. In the small volume of a bacterial cell, freely diffusing donors and acceptors are likely to come in contact during the excited state lifetime of Tb3+ (2 msec). This suggests a possible method for measuring association-dissociation equilibria of large macromolecuar complexes in live bacterial cells. Protein monomers labeled with LRET acceptors (e.g. GFP or FlAsH-tags) can quench DAL donors by diffusional LRET. When associated into a few large assemblies, the acceptors will have much slower diffusion and their locations will no longer be distributed throughout the cytoplasm. We expect that after large assemblies form, diffusion-enhanced LRET will greatly decrease. We plan to test this on E. coli assemblies such as the fiber-forming enzyme CTP synthase, and the ethanolamine utilization microcompartment.

New Insights into CFTR as GSH Transporter

The cystic fibrosis transmembrane regulator (CFTR) chloride channel is an important member of the ATP-binding cassette superfamily because dysfunction or low expression of CFTR was found in patients with human cystic fibrosis. Further studies show low mitochondrial GSH levels in the CFTR-knockout mouse lung. Because impaired mitochondrial oxidative metabolism plays a critical role in lung diseases, CFTR has been proposed to be expressed on the mitochondria to promote GSH transportation across mitochondria to protect the lung from oxidative damage. However, it has not been well-established that CFTR functions as GSH transporter under the physiological conditions. Here, patch clamp studies demonstrate that only outward GSH currents across inside-out membrane patches were found with WT CFTR while both inward and outward GSH currents were observed with the constitutively active K190C/K978C CFTR construct with a high open probability. Thus, asymmetric GSH permeation may be state-dependent. Further studies show that the constitutively active CFTR mutant T338C/K190C/K978C was completely inhibited by extracellular Cu2+ and inhibition was fractionally reversed by 10mM intracellular membrane-impermeant GSH and completely by intracellular membrane-permeant TPEN. In contrast, extracellular Cu2+ failed to suppress the activity of the CFTR mutant K190C/K978C. The application of 10mM GSH or TPEN to the intracellular side of this construct did not increase the channel activity, either. These results suggest that native CFTR may function as GSH transport under the physiological condition and designed transition metal binding sites at the channel pore can be used to define its GSH permeability.

The Molecular Basis for Calprotectin Function in Nutritional Immunity

Background: The S100A8/S100A9 heterodimer calprotectin (CP) is expressed abundantly in neutrophils and plays a key role in innate immunity. In tissue abscesses, CP sequesters the transition metals manganese (Mn) and zinc (Zn) which serves to inhibit microbial growth and contributes to the control of pathogens. This activity is often referred to as nutritional immunity. We have shown previously that CP contains two transition metal binding sites, both of which have high affinity for Zn, however only one of which exhibits high affinity for Mn. Methods: Here we report an in-depth characterization of the biophysical and biochemical properties of CP-Mn and CP-Zn complexes using a combination of isothermal titration calorimetry (ITC), mutagenesis, and microbiology experiments. Atomic level details into the structural basis for function are provided by a high-resolution crystal structure of CP with bound Mn. Results: The crystal structure of CP with bound Mn has been determined to 1.8 A. ITC experiments show a stoichiometry of two Zn ions with dissociation constants of 1.4 nM and 5.6 nM, whereas only one Mn ion binds with high affinity (dissociation constant 1.3 nM). Mutants of CP with inactivated metal binding sites have been designed and characterized physically and functionally. Conclusions: CP binds two Zn ions, but only one Mn ion, with high affinity. The x-ray crystal structure reveals a single octahedrally coordinated Mn ion at the S100A8/S100A9 subunit interface that also involves the C-terminal tail of S100A9. The chelation of Mn by six histidine residues is unique and has not been observed in other Mn binding proteins. The biochemical and biophysical characterization of wild type and mutant CP variants that inhibit Mn binding show that sequestration of Mn is required for the antimicrobial activity of CP. The distinctive features of the Mn binding site explain why CP exhibits broad-spectrum anti-microbial activity via a nutritional immunity mechanism.

Binding motifs of silver in prion octarepeat model peptides: a joint ion mobility, IR and UV spectroscopies, and theoretical approach

Transition metal-ion complexation is essential to the function and structural stability of many proteins. We studied silver complexation with the octarepeat motif ProHisGlyGlyGlyTrpGlyGln of the prion protein, which shows competitive sites for metal chelation including amide, indole and imidazole groups. This octapeptide is known as a favourable transition metal binding site in prion protein. We used ion mobility spectrometry (IMS), infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory calculations (DFT) to identify the binding motifs of a silver cation on HisGlyGlyGlyTrp peptide as well as on peptide subsequences. Ultra-violet photodissociation (UVPD) and collision induced dissociation mass spectrometry together with the time-dependent density functional method was then exploited to study the influence of binding sites on optical properties and on the ground and excited states reactivity of the peptide. We show that the metal cation is bound to the π-system of the indole group and a nitrogen atom of the imidazole group and that charge transfers from the indole group to the silver cation occur in excited electronic states.

Nutrient Metal Sequestration by Calprotectin Inhibits Bacterial Superoxide Defense, Enhancing Neutrophil Killing of Staphylococcus aureus

By sequestering manganese and zinc, the neutrophil protein calprotectin plays a crucial role in host defense against bacterial and fungal pathogens. However, the essential processes disrupted by calprotectin remain unknown. We report that calprotectin enhances the sensitivity of Staphylococcus aureus to superoxide through inhibition of manganese-dependent bacterial superoxide defenses, thereby increasing superoxide levels within the bacterial cell. Superoxide dismutase activity is required for full virulence in a systemic model of S.aureus infection, and disruption of staphylococcal superoxide defenses by calprotectin augments the antimicrobial activity of neutrophils promoting invivo clearance. Calprotectin mutated in two transition metal binding sites and therefore defective in binding manganese and zinc does not inhibit microbial growth, unequivocally linking the antimicrobial properties of calprotectin to metal chelation. These results suggest that calprotectin contributes to host defense by rendering bacterial pathogens more sensitive to host immune effectors and reducing bacterial growth.

Metalloproteins and the pyrite-based origin of life: a critical assessment.

We critically examine the proposal by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that putative transition metal binding sites in protein components of the translation machinery of hyperthermophiles provide evidence of a direct relationship with the FeS clusters of pyrite and thus indicate an autotrophic origin of life in volcanic environments. Analysis of completely sequenced cellular genomes of Bacteria, Archaea and Eucarya does not support the suggestion by Wächtershäuser (Prokaryotes 1:275-283, 2006a, Philos Trans R Soc Lond B Biol Sci 361: 787-1808, 2006b) that aminoacyl-tRNA synthetases and ribosomal proteins bear sequence signatures typical of strong covalent metal bonding whose absence in mesophilic species reveals a process of adaptation towards less extreme environments.

ANTI-INFECTIVE PROTECTIVE PROPERTIES OF S100 CALGRANULINS.

The calgranulins are a subgroup of proteins in the S100 family (calgranulin A, S100A8; calgranulin B, S100A9 and calgranulin C, S100A12) that provide protective anti-infective and anti-inflammatory functions for the mammalian host. In this review, we discuss the structure-function relationships whereby S100A8 and S100A9, and for comparison, S100A12, provide intra- and extracellular protection during the complex interplay between infection and inflammation and how the calgranulins are regulated to optimally protect the host. Ideally located to support epithelial barrier function, calprotectin, a complex of S100A8/S100A9, is expressed in squamous mucosal keratinocytes and innate immune cells present at mucosal surfaces. The calgranulins are also abundantly produced in neutrophils and monocytes, whereas expression is induced in epidermal keratinocytes, gastrointestinal epithelial cells and fibroblasts during inflammation. The calgranulins show species-specific expression and function. For example, S100A8 is chemotactic in rodents but not in humans. In humans, S100A12 appears to serve as a functional chemotactic homolog to murine S100A8. Transition metal-binding and oxidation sites within calgranulins are able to create structural changes that may orchestrate new protective functions or binding targets. The calgranulins thus appear to adopt a variety of roles to protect the host. In addition to serving as a leukocyte chemoattractant, protective functions include oxidant scavenging, antimicrobial activity, and chemokine-like activities. Each function may reflect the concentration of the calgranulin, post-transcriptional modifications, oligomeric forms, and the proximal intracellular or extracellular environments. Calprotectin and the calgranulins are remarkable as multifunctional proteins dedicated to protecting the intra- and extracellular environments during infection and inflammation.

Prediction of 3D metal binding sites from translated gene sequences based on remote‐homology templates

Database-scale analysis was performed to determine whether structural models, based on remote homologues, are effective in predicting 3D transition metal binding sites in proteins directly from translated gene sequences. The extent by which side chain modeling alone reduces sensitivity and selectivity is shown to be <10%. Surprisingly, selectivity was not dependent on the level of sequence homology between template and target, or on the presence of a metal ion in the structural template. Applying a modification of the CHED algorithm (Babor et al., Proteins 2008;70:208-217) and machine learning filters, a selectivity of approximately 90% was achieved for protein sequences using unrelated structural templates over a sequence identity range of 18-100%. Below approximately 18% identity, the number of analyzable target-template pairs and predictability of metal binding sites falls off sharply. A full third of structural templates were found to have target partners only in the remote homology range of 18-30%. In this range, nonmetal-binding templates are calculated to be the majority and serve to predict with 50% sensitivity at the geometric level. Overall, sensitivity at the geometric level for targets having templates in the 18-30% sequence identity range is 73%, with an average of one false positive site per true site. Protein sequences described as "unknown" in the UniProt database and composed largely of unidentified genome project sequences were studied and metal binding sites predicted. A web server for prediction of metal binding sites from protein sequence is provided.

Quantification of single‐stranded nucleic acid and oligonucleotide interactions with metal ions by affinity capillary electrophoresis – Part II

The interactions between tetranucleotides or heptanucleotides and inorganic cations have been measured by affinity CZE. The variation in migration behavior with increasing concentrations of divalent cations (Ca(2+), Mg(2+) and Ni(2+)) in the running buffer was investigated and quantified by the calculation of binding constants for mononuclear and multinuclear interactions. In addition to these fundamental studies of binding equilibria, the effect of sequence and the position of the guanine transition metal-binding site in the oligonucleotide have been investigated.

MetalDetector: a web server for predicting metal-binding sites and disulfide bridges in proteins from sequence

The web server MetalDetector classifies histidine residues in proteins into one of two states (free or metal bound) and cysteines into one of three states (free, metal bound or disulfide bridged). A decision tree integrates predictions from two previously developed methods (DISULFIND and Metal Ligand Predictor). Cross-validated performance assessment indicates that our server predicts disulfide bonding state at 88.6% precision and 85.1% recall, while it identifies cysteines and histidines in transition metal-binding sites at 79.9% precision and 76.8% recall, and at 60.8% precision and 40.7% recall, respectively. Freely available at http://metaldetector.dsi.unifi.it. Details and data can be found at http://metaldetector.dsi.unifi.it/help.php.

Prediction of transition metal‐binding sites from apo protein structures

Metal ions are crucial for protein function. They participate in enzyme catalysis, play regulatory roles, and help maintain protein structure. Current tools for predicting metal-protein interactions are based on proteins crystallized with their metal ions present (holo forms). However, a majority of resolved structures are free of metal ions (apo forms). Moreover, metal binding is a dynamic process, often involving conformational rearrangement of the binding pocket. Thus, effective predictions need to be based on the structure of the apo state. Here, we report an approach that identifies transition metal-binding sites in apo forms with a resulting selectivity >95%. Applying the approach to apo forms in the Protein Data Bank and structural genomics initiative identifies a large number of previously unknown, putative metal-binding sites, and their amino acid residues, in some cases providing a first clue to the function of the protein.

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/μg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.