Bioactive snail mucus-slime extract loaded chitosan scaffolds for hard tissue regeneration: the effect of mucoadhesive and antibacterial extracts on physical characteristics and bioactivity of chitosan matrix.

Biobased extracts comprise various bioactive components and they are widely used in tissue engineering applications to increase bioactivity as well as physical characteristics of biomaterials. Among animal sources, garden snailHelix aspersahas come into prominence with its antibacterial and regenerative extracts and show potential in tissue regeneration. Thus, in this study, bioactiveH. aspersaextracts (slime, mucus) were loaded in chitosan (CHI) matrix to fabricate porous scaffolds for hard tissue regeneration. Physical, chemical properties, antimicrobial activity was determined as well asin vitrobioactivity for bone and cartilage regeneration. Mucus and slime incorporation enhanced mechanical properties and biodegradation rate of CHI matrix. Scanning electron microscopy images showed that the average pore size of the scaffolds decreased with higher extract content. Mucus and slime extracts showed antimicrobial effect on two bacterial strains.In vitrocytotoxicity, osteogenic and chondrogenic activity of the scaffolds were evaluated with Saos-2 and SW1353 cell lines in terms of Alkaline phosphatase activity, biomineralization, GAG, COMP and hydroxyproline content. Cell viability results showed that extracts had a proliferative effect on Saos-2 and SW1353 cells when compared to the control group. Mucus and slime extract loading increased osteogenic and chondrogenic activity. Thus, the bioactive extract loaded CHI scaffolds showed potential for bone and cartilage regeneration with enhanced physical properties andin vitrobioactivity.

Comparative assessment of biomarker response to tissue metal concentrations in urban populations of the land snail Helix pomatia (Pulmonata: Helicidae).

The traffic pressure is increasing, resulting in the emission of atmospheric pollution. Soil organisms will need to respond to pollution stressors. Among them, land snails are valuable indicators of ecosystem disturbance. In this study, land snails Helix pomatia were sampled from three city localities with different traffic intensity. Oxidative stress biomarkers catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST) in the foot muscle (FM) and hepatopancreas (HP) tissue were determined. Also, five heavy metal (Cd, Cu, Ni, Pb, and Zn) concentrations were quantified in soil and tissue samples. According to the results, the highway induces the strongest contamination on the surrounding environment, with the highest metal concentrations measured in soil and snails. At the most polluted locality, only Cd exceeded some soil guidelines authorities that we referred to in this study. In addition, tissue Cd concentrations exceeded the United States Environmental Protection Agency (USEPA) value (1 mg kg-1) for soil invertebrate toxicity at all localities making it likely responsible for generating adverse effects in snails. Regarding HP, the CAT and GST are the most sensitive parameters that could be useful as oxidative stress biomarkers in snails exposed to the actual metals in the environment. On the other hand, in FM tissue, the most pronounced changes were recorded for GPX and GR. Based on tissue-specific enzyme responses, three urban populations were clearly separated. Therefore land snails are the promising candidates for quick field-based biomarker studies after showing a tissue-specific concentration-dependent induction of certain enzymes to heavy metals.

A multiresidue method for the analysis of pesticides, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls in snails used as environmental biomonitors.

This paper reports an optimized multiresidue extraction strategy based on the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) extraction procedure and on solid-phase microextraction (SPME) for the simultaneous screening of 120 pesticides, 16 polycyclic aromatic hydrocarbons, and 22 polychlorinated biphenyls from the terrestrial snail Helix aspersa. The optimized extraction method was based on QuEChERS using acetonitrile, followed by dispersive-Solid-phase extraction clean-up using primary secondary amine and octadecyl (C18) sorbents. The obtained extracts were analyzed by liquid chromatography coupled with tandem mass spectrometry and gas chromatography coupled with tandem mass spectrometry. This latest technique was preceded by a pre-concentration step using SPME with appropriate fibers. Afterwards, the method was validated for its linearity, sensitivity, recovery, and precision. Results showed high sensitivity, accuracy, and precision, with limits of detection and quantification lower than 20 ng g - 1 for most considered pollutants. Both inter and intra-day analyses revealed low relative standard deviation (%), which was lower than 20% for most targeted compounds. Moreover, the obtained regression coefficient (R2) was higher than 0.98 and the recoveries were higher than 60% for the majority of the assessed pollutants.

Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 µg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 µg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.

For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.

The Solution Structure and Dynamics of Cd-Metallothionein from Helix pomatia Reveal Optimization for Binding Cd over Zn.

Metallothioneins (MTs) are cysteine-rich polypeptides that are naturally found coordinated to monovalent and/or divalent transition metal ions. Three metallothionein isoforms from the Roman snail Helix pomatia are known. They differ in their physiological metal load and in their specificity for transition metal ions such as Cd2+ (HpCdMT isoform) and Cu+ (HpCuMT isoform) or in the absence of a defined metal specificity (HpCd/CuMT isoform). We have determined the solution structure of the Cd-specific isoform (HpCdMT) by nuclear magnetic resonance spectroscopy using recombinant isotopically labeled protein loaded with Zn2+ or Cd2+. Both structures display two-domain architectures, where each domain comprises a characteristic three-metal cluster similar to that observed in the ß-domains of vertebrate MTs. The polypeptide backbone is well-structured over the entire sequence, including the interdomain linker. Interestingly, the two domains display mutual contacts, as observed before for the metallothionein of the snail Littorina littorea, to which both N- and C-terminal domains are highly similar. Increasing the length of the linker motionally decouples both domains and removes mutual contacts between them without having a strong effect on the stability of the individual domains. The structures of Cd6- and Zn6-HpCdMT are nearly identical. However, 15N relaxation, in particular 15N R2 rates, is accelerated for many residues of Zn6-HpCdMT but not for Cd6-HpCdMT, revealing the presence of conformational exchange effects. We suggest that this snail MT isoform is evolutionarily optimized for binding Cd rather than Zn.

HelixComplex snail mucus exhibits pro-survival, proliferative and pro-migration effects on mammalian fibroblasts.

Snail mucus is a mixture of active substances commonly thought to have healthy properties for the treatment of skin disorders. Although snail mucus is an ingredient of several cosmetic and para-pharmaceutic products, a comprehensive characterization of chemical composition and biological effects is still missing. Crude purified extracts from Helix aspersa muller mucus (HelixComplex) were prepared and, after chemical characterization, tested on in vitro experimental models. Differently from what expected, HelixComplex was characterized by the presence of small amounts of glycolic acid and allantoin. By using different in vitro assays on fibroblast cultures, we found that HelixComplex lacked of cytotoxicity, protected cells from apoptosis (p < 0.05) and, importantly, was able to significantly induce cell proliferation and migration through direct and indirect mechanisms. These effects were associated to morphological changes, cytoskeleton re-organization and release of cytokines. In conclusion, our findings suggest that snail mucus biological effects are attributable to cell proliferation and migration, and pave the way for further investigating snail mucus potential as therapeutic agent.

Seasonal distributions of heavy metal concentrations in different snail (Helix pomatia) tissues from an urban environment in Serbia.

The aim of this study was to evaluate the differences between concentrations of heavy metals (cadmium, copper, zinc, iron, and manganese) in terrestrial gastropods, Helix pomatia, collected in four different environment locations in Pancevo city, Serbia. Metal concentrations in the foot and in the digestive gland were measured. Heavy metals cadmium, copper, zinc, iron, and manganese were measured in triplicate by atomic absorption spectrophotometry. Also, this study investigated correlations between the trace element content in the soft tissues and different seasons (summer and fall). The results showed that heavy metal concentrations of the measured elements in both snail tissues were significantly higher in polluted sites when compared to the background levels in the city. In digestive gland tissues, the concentration of heavy metals was higher compared with that in foot tissues. The bioaccumulation (heavy metal concentrations in soft tissues) of heavy metals (Cd, Cu, Zn, Fe, and Mn) in snails, Helix pomatia, has been assessed and related to seasons and sites. Heavy metal accumulation in the soft tissues of snails could provide a powerful monitoring tool for the assessment of environmental heavy metal pollution.

Assay for galactose-deficient IgA1 enables mechanistic studies with primary cells from IgA nephropathy patients.

AIMS: IgA nephropathy, the most common primary glomerulonephritis worldwide, is characterized by glomerular deposition of galactose-deficient IgA1 and elevated serum levels of this IgA1 glycoform. Current ELISA methods lack sensitivity to assess galactose deficiency using small amounts of IgA1, which limits studies in primary cells due to modest IgA1 production in isolated peripheral-blood lymphocytes. METHODS: Lectin from Helix pomatia was conjugated to biotin or acridinium ester and used in ELISA to detect galactose deficiency of IgA1 using small amounts of IgA1. RESULTS: Lectin conjugated to acridinium had an approximately a log-fold increased sensitivity compared with biotin-labeled lectin. CONCLUSIONS: The new method of using lectin from Helix pomatia conjugated to acridinium increased assay sensitivity, allowing future mechanistic studies with cultured primary cells.

Helix aspersa maxima mucus exhibits antimelanogenic and antitumoral effects against melanoma cells.

Snail secretion is currently revolutionizing the world of cosmetics and human skin care. The efficacy of snail secretion in wounds healing has been proven both in vitro and by clinical studies. However, the potential anti-tumor effect of snail secretion was poorly investigated. In this report, our in vitro study showed that Helix aspersa maxima species snail slime (SS) could not only treat melanogenesis but also endowed with anti-tumoral activity against human melanoma cells. Indeed, SS reduced melanin content and tyrosinase activity on B16F10 cells with IC50 values of 288 µg/mL and 286 µg/mL, respectively, without altering cell viability. This effect was also observed, at a lesser extent, on human melanoma IGR-39 and SK-MEL-28 cell lines. On another hand, SS specifically inhibited the viability of IGR-39 and SK-MEL-28 cells associated to an apoptotic effect highlighted by PARP cleavage. It is worth to note that SS did not affect the viability of B16F10 cells and non tumorigenic HaCaT cells. Interestingly, this extract was found to inhibit migration and invasion of both human melanoma cells through reducing the expression of Matrix metalloproteinase MMP2. Snail slime also exerted a high inhibitory effect on IGR-39 cell adhesion through blocking the function of α2ß1 (45%), αvß3 (38%) integrins and by reducing the expression levels of αv and ß1 integrins. The presented results shed light on the potential anti-melanoma effect of SS and support its use against skin diseases.

Adsorption/desorption of biomacromolecules involved in catalytic hydrogen evolution.

Previously, it has been shown that proteins and some polysaccharides (PSs) catalyse hydrogen evolution, producing electrochemical signals on mercury electrodes. The catalytic hydrogen evolution reaction (CHER) of the above-mentioned biomacromolecules was studied by voltammetric and chronopotentiometric stripping (CPS) methods. To obtain more information about electrode processes involving CHER, here we used protein such as BSA, and chitosan as a PS; in addition, we investigated dextran as a control PS not involved in CHER. We studied biomacromolecules by phase-sensitive alternating current (AC) voltammetry. Using phase-in AC voltammetry, for CHER-involved biomacromolecules we observed a CHER peak at highly negative potentials, similar to that observed with other voltammetric and CPS methods. On the other hand, by means of the adsorption/desorption processes studied in phase-out AC voltammetry, we uncovered a sharp and narrow decrease of capacitive current in the potential range of the CHER peak, denominated as the tensammetric minimum. This minimum was closely related to the CHER peak, as demonstrated by similar dependences on specific conditions affecting the CHER peak such as buffer capacity and pH. A tensammetric minimum was not observed for dextran. Our results suggest specific organization of biopolymer layers at negative potentials observed only in biomacromolecules involved in CHER.

N-glycan structures of ß-HlH subunit of Helix lucorum hemocyanin.

The carbohydrate structures of molluscan hemocyanins have recently received particular interest due to their specific monosaccharide composition, as well as their immunostimulatory properties and application in clinical studies. For the first time, we investigated N-glycans of the structural subunit ß-HlH of hemocyanin isolated from Helix lucorum. In total, 32 different glycans were enzymatically liberated and characterized by tandem mass spectrometry using a Q-Trap mass spectrometer. Our study revealed a highly heterogeneous mixture of glycans with composition Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1. The oligosaccharide chains are mostly modified at the inner core by ß1-2-linked xylose to ß-mannose, by α1-6-fucosylation of the innermost GlcNAc residue (the Asn-bound GlcNAc), and by methylation. The glycans of ß-HlH mainly contain a terminal MeHex residue; in some cases even two, three or four of these residues occur. Several carbohydrate chains in ß-HlH are core-fucosylated without Xyl and also possess a high degree of methylation. This study shows the presence of mono- and bi-antennary N-glycans as well as hybrid type structures with or without core-fucosylation.

Cadmium Accumulation and Pathological Alterations in the Midgut Gland of Terrestrial Snail Helix pomatia L. from a Zinc Smelter Area: Role of Soil pH.

The purpose of this study was to determine whether cadmium (Cd) accumulation and toxicity in the midgut gland of Helix pomatia snails living in a Cd-contaminated area were related to soil pH. Toxic responses in the midgut gland (i.e., increased vacuolization and lipid peroxidation) occurred in H. pomatia snails exhibiting the highest Cd levels in the gland (265-274 µg/g dry wt) and living on acidic soil (pH 5.3-5.5), while no toxicity was observed in snails accumulating less Cd (90 µg/g) and ranging on neutral soil (pH 7.0), despite the fact that total soil Cd was similar in the two cases. The accumulation of Cd in the gland was directly related to the water extractable Cd in soil, which in turn correlated inversely with soil pH, indicating that this factor had a significant effect on tissue Cd. It appeared further that the occurrence of Cd toxicity was associated with low levels of metallothionein in the gland of snails ranging on acidic soil.

Helix aspersa gelatin as an emulsifier and emulsion stabilizer: functional properties and effects on pancreatic lipolysis.

Emulsions are widely used in food and pharmaceutical applications for the encapsulation, solubilization, entrapment, and controlled delivery of active ingredients. In order to fulfill the increasing demand for clean label excipients, natural polymers could be used to replace the potentially irritative synthetic surfactants used in emulsion formulation. In the present study, we have studied the properties of oil-in-water emulsions prepared with land snail gelatin (LSG) as the sole emulsifying agent, extracted and described for the first time. LSG was evaluated in terms of proximate composition, oil and water holding capacity, emulsifying and foaming properties, color and amino acid composition. Emulsions of trioctanoylglycerol (TC8) and olive oil were made at different gelatin/oil ratios and changes in droplet-size distribution were determined. The superior emulsifying properties of LSG, the susceptibility of gelatin protein emulsions increasing flocculation on storage, and the coalescence of gelatin emulsions following centrifugation were demonstrated. Furthermore, the effect of LSG on the activity of turkey pancreatic lipase (TPL) was evaluated through the pH-stat methodology with TC8 and olive oil emulsions. The LSG affected the TPL activity in a concentration-dependent way. Our results showed that LSG, comparably to gum arabic, increases the pancreatic lipase activity and improves its stability at the oil-water interface.

Antimicrobial Activity of Molluscan Hemocyanins from Helix and Rapana Snails.

For the first time the antimicrobial activities of hemocyanins from the molluscs Rapana venosa (RvH) and Helix aspersa (HaH) have been tested. From the hemolymph of the garden snail H. aspersa one structural subunit (ßc-HaH ) and eight functional units (FUs, ßc-HaH-a to ßc-HaH-h) were isolated, and their N-terminal sequences and molecular weights, ranging between 45 and 65 kDa, determined. The antimicrobial test of the hemocyanins against different bacteria showed that only two FUs from Rapana, RvH1-b and RvH1-e, exhibit a low inhibition effect against Staphylococcus aureus. In contrast and surprisingly, the structural subunit ßc-HaH of H. aspersa not only shows strong antimicrobial activities against S. aureus and the likewise Gram-positive Streptococcus epidermidis, but also against the Gram-negative bacterium Escherichia coli. We suggest that this subunit therefore has the potential to become a substitute for the commonly used antibiotics against which bacterial resistance has gradually been developed.

Measuring Faecal Epi-Androsterone as an Indicator of Gonadal Activity in Spotted Hyenas (Crocuta crocuta).

Enzyme immunoassays (EIA) that measure faecal testosterone metabolites (fTM) are useful tools to monitor gonadal activity. The aim of this study was to validate an "in-house" epiandrosterone EIA to monitor fTM in spotted hyenas. FTM were characterised in a male and a female hyena that each received an injection of 3H-testosterone. High-performance liquid chromatography (HPLC) analyses revealed a cluster of highly polar enzyme-hydrolysable hormone metabolite conjugates. We performed hydrolysis using ß-glucuronidase to deconjugate metabolites and improve sensitivity of the assay. Because ß-glucuronidase from Helix pomatia has been reported to bias testosterone measurements in some species, we compared the enzymatic activity of the commonly used ß-glucuronidase extracted from H. pomatia with the same enzyme from Escherichia coli. Our results showed that ß-glucuronidases from both sources produced similar results from spotted hyena faeces. We therefore hydrolysed samples with H. pomatia enzymes. HPLC analyses also demonstrated that following hydrolysis the epiandrosterone EIA measured significant amounts of immunoreactive metabolites corresponding to radiolabelled metabolites in both sexes. Additionally, HPLC and GC-MS analyses confirmed the presence of epiandrosterone in faeces of spotted hyenas. The biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM levels in response to a testosterone injection within 16 h, (2) no biological responsiveness to an adrenocorticotropic hormone (ACTH) injection and (3) significant differences in fTM levels between juvenile males and adult immigrant males in a free-ranging wild population. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor gonadal activity in spotted hyenas.

Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.

In vitro antiproliferative effect of Helix aspersa hemocyanin on multiple malignant cell lines.

As an extension of our studies on the antitumour properties of various hemocyanins, we sought to compare the antiproliferative effects of hemocyanins derived from two snail species: Helix lucorum (HIH) and Helix aspersa (HaH). This is the first report on the antitumour effects of HaH. We hypothesized that HaH has antitumour effects not only against bladder cancer, as previously shown with other hemocyanins, but also on other cancer cell lines. The antiproliferative properties of the mentioned hemocyanins were investigated in vitro on the following human cell lines: bladder cancer (CAL-29 and T-24), ovarian cancer (FraWü), acute monocytic leukemia (THP-1), prostate cancer (DU-145), glioma cancer (LN-18), and Burkitt's lymphoma (Daudi). The properties of HaH were compared to those of HlH, keyhole limpet hemocyanin (KLH), and two positive controls (doxorubicin and mitomycin C). An antiproliferative effect of the total molecule and one structural subunit of HaH, betac-HaH, against both bladder cancer cell lines, T-24 and CAL-29, was observed. The cytotoxic effect of HaH ranged between 15% and 60% among the other tested cell lines. The endotoxin contamination did not affect the efficacy of HaH. Therefore, HlH and HaH could be appropriate for more detailed investigations of their use as antitumour agents for the studied cancers.

Cantareus aspersus metallothionein metal binding abilities: the unspecific CaCd/CuMT isoform provides hints about the metal preference determinants in metallothioneins.

In Proteomics, gene/protein families including both specialized and non-specialized paralogs are an invaluable tool to study the evolution of structure/function relationships in proteins. Metallothioneins (MTs) of the pulmonate gastropod molluscs (snails) offer one of the best materials to study the metal-binding specificity of proteins, because they consist of a polymorphic system that includes members with extremely distinct metal preferences but with a high protein sequence similarity. Cantareus aspersus was the first snail where three paralogous MTs were isolated: the highly specific cadmium (CaCdMT) and copper (CaCuMT) isoforms, and an unspecific CaCd/CuMT isoform, so called because it was natively isolated as a mixed Cd and Cu complex. In this work, we have thoroughly analyzed the Zn(2+)-, Cd(2+)- and Cu(+)-binding abilities of these three CaMTs by means of the spectroscopic and spectrometric characterization of the respective recombinant, as well as in vitro-substituted, metal-complexes. The comparison with the orthologous HpMTs and the study of the isoform-determinant residues allow correlating the protein sequence variability with the coordination capabilities of these MTs. Surprisingly, the CaCuMT isoform exhibits a stronger Cu-thionein character than the HpCuMT ortholog, and the CaCd/CuMT isoform could be defined as a non-optimized Cu-thionein, which has not attained any defined functional differentiation in the framework of the snail MT gene/protein family.

Cognate and noncognate metal ion coordination in metal-specific metallothioneins: the Helix pomatia system as a model.

The Helix pomatia metallothionein (MT) system, namely, its two highly specific forms, HpCdMT and HpCuMT, has offered once again an optimum model to study metal-protein specificity. The present work investigates the most unexplored aspect of the coordination behavior of MT polypeptides with respect to either cognate or noncognate metal ions, as opposed to the standard studies of cognate metal ion coordination. To this end, we analyzed the in vivo synthesis of the corresponding complexes with their noncognate metals, and we performed a detailed spectroscopic and spectrometric study of the Zn(2+)/Cd(2+) and Zn(2+)/Cu(+) in vitro replacement reactions on the initial Zn-HpMT species. An HpCuMTAla site-directed mutant, exhibiting differential Cu(+)-binding abilities in vivo, was also included in this study. We demonstrate that when an MT binds its cognate metal, it yields well-folded complexes of limited stoichiometry, representative of minimal-energy conformations. In contrast, the incorporation of noncognate metal ions is better attributed to an unspecific reaction of cysteinic thiolate groups with metal ions, which is dependent on their concentration in the surrounding milieu, where no minimal-energy structure is reached, and otherwise, the MT peptide acts as a multidentate ligand that will bind metal ions until its capacity has been saturated. Additionally, we suggest that previous binding of an MT polypeptide with its noncognate metal ion (e.g., binding of Zn(2+) to the HpCuMT isoform) may preclude the correct folding of the complex with its cognate metal ion.