Binding properties of an orally active platinum anti-tumor drug JM216 with metallothionein in vivo

The abilities of the orally active platinum anti-tumor drug JM216 [trans-bis-acetato-cis-dichloroammine (cyclohexylamine) platinum (IV)] to induce the biosynthesis of metallothionein (MT) were investigated in rabbits given oral administrations or injections s. c. It is revealed that oral administration of JM216 can induce the MT synthesis in the liver but not in the kidney. The hepatic MT contained 7.11 ± 0.11 Zn and only little Pt or Cu. Injections of JM216 to rabbits can greatly elevate the MT levels in the liver, but increase the renal MT levels only slightly. The MT content as well as Pt concentration in the liver was much higher than that in the kidney. The metal stoichiometry in the purified renal MT was determined to be 4.41 ± 0.04 Zn, 0.36 ± 0.11 Pt and 2.50 ± 0.18 Cu per mole protein. The hepatic MT was still characterized as Zn₇MT. Both the treatment with oral administration and injections s.c. cannot lead to the increase of Pt content in the kidney. The oxidation state of platinum in the MT from the kidney was determined to be +2 by X-ray photoelectron spectroscopy. As compared with zinc compounds, JM216 was a very poor stimulator for MT biosynthesis in vivo. Pre-injections with Zn(NO₃)₂ significantly enhanced the MT levels as well as the Pt concentration compared with that resulting from injections with JM216 alone. Based on the experimental data, the role of MT in relation to its involvement in the metabolism and the mechanism of detoxification of Pt(IV) complexes are discussed.     

Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)₄(CysS)₁₁ cluster and a M(II)₃(CysS)₉ cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn₇MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn₇MT-1/Zn₇MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn₇MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn₇MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn₇MT-3 and the formation of the Pt–S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The ¹¹³Cd NMR studies of Pt₁ ¹¹³Cd₆MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn₇MT-3 were much faster than those of cisplatin and transplatin with Zn₇MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn₇MT-3 than Zn₇MT-2 in the acquired resistance to platinum-based drugs are discussed.     

Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-MT 1 and Zn₃-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–MT 1 and Zn₃-MT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag_x–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag_x–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the _xZn_y–MT (0y<7) species found, only M^I₄Zn₅–MT [(Zn₄)(₄Zn₁)] and M^I₇Zn₃–MT [(₃Zn₂)(₄Zn₁)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Comparison of the Structures of the Metal-thiolate Binding Site in Zn(II)-, Cd(II)-, and Hg(II)-Metallothioneins Using Molecular Modeling Techniques

Abstract

The first fully energy-minimized structures for a series of structurally related metal complexes of the important mammalian metal binding protein metallothionein are described. The structures were calculated based on structural information obtained from existing spectroscopic and crystallographic data, and minimized using molecular mechanics (MM2) techniques. A two domain structure, with stoichiometrics of M(II)₃?(S_cys)₉ and M(II)₄?(S_cys)₁₁ where M = zinc(II), cadmium(II), and mercury(II), was assembled and minimized. The resultant three-dimensional structure closely resembled that of rat liver Cd₅Zn₂?MT 1 obtained by analysis of x-ray diffraction data [A. H. Robbins, D. E. McRee, M. Williamson, S. A. Collett, N. H. Xuong, W. F. Furey, B. C. Wang and C. D. Stout, J. Mol. Biol. 221, 1269–1293 (1991)]. Minimized structures for Zn₇?MT, Cd₇?MT, and Hg₇?MT are reported. Deep crevices that expose the metal-thiolate clusters are seen in each structure. However, for the mercury-containing protein, much of the mercury-thiolate structure is visible and it is proposed that this provides access for extensive interaction between solvent water molecules and the mercury(II), resulting in the observed distortion away from tetrahedral geometry for Hg₇MT. Volume calculations are reported for the protein metallated with 7 Zn(II), Cd(II), or Hg(II). A series of structural changes calculated for the step-wise isomorphous replacement of Zn(II) by Cd(II) and Hg(II) in the Zn₄S₁₁ α domain are shown.     

Characterization of the cadmium complex of peptide 49–61: a putative nucleation center for cadmium-induced folding in rabbit liver metallothionein IIA

 The synthetic peptide fragment containing residues 49–61 of rabbit liver metallothionein II (MT-II) (Ac-Ile-Cys-Lys-Gly-Ala-Ser-Asp-Lys-Cys-Ser-Cys-Cys-Ala-COOH), which includes the only sequential four cysteines bound to the same metal ion in Cd₇MT, forms a stable, monomeric Cd-peptide complex with 1 : 1 stoichiometry (Cd:peptide) via Cd-thiolate interactions. This represents the first synthesis of a single metal-binding site of MT independent of the domains. The ¹¹¹Cd NMR chemical shift at 716 ppm indicates that the ¹¹¹Cd²⁺ in the metal site is terminally coordinated to four side-chain thiolates of the cysteine residues. The pH of half dissociation for this Cd-peptide derivative, ∼3.3, demonstrates an affinity similar to that for Cd₇MT. Molecular mechanics calculations show that the thermodynamically most stable folding for this isolated Cd²⁺ center has the same counterclockwise chirality (Λ or S) observed in the native holo-protein. These properties are consistent with its proposed role as a nucleation center for cadmium-induced protein folding. However, the kinetic reactivity of the CdS₄ structure toward 5,5′-dithiobis(5-nitrobenzoate) (DTNB) and EDTA is greatly increased compared to the complete cluster (α-domain or holo-protein). The rate law for the reaction with DTNB is rate=(k _uf +k _1,f +k _2,f [DTNB])[peptide], where k _uf=0.15 s^–1, k _1,f=2.59×10^–3s^–1, and k _2,f=0.88 M^–1s^–1. The ultrafast step (uf), observable only by stopped-flow measurement, is unprecedented for mammalian (M₇MT) and crustacean (M₆MT) holo-proteins or the isolated domains. The accommodation of other metal ions by the peptide indicates a rich coordination chemistry, including stoichiometries of M-peptide for Hg²⁺, Cd²⁺, and Zn²⁺, M₂-peptide for Hg²⁺ and Au⁺, and (Et₃PAu)₂-peptide. Received: 9 December 1998 / Accepted: 20 May 1999     

Can Satraplatin be hydrated before the reduction process occurs? The DFT computational study

Hydration reactions of two anticancer Pt(IV) complexes JM149 and JM216 (Satraplatin) were studied computationally together with the hydration of the Pt(II) complex JM118, which is a product of the Satraplatin reduction. Thermodynamic and kinetic parameters of the reactions were determined at the B3LYP/6-311++G(2df.2pd)//B3LYP/6-31 + G(d)) level of theory. The water solution was modeled using the COSMO implicit solvation model, with cavities constructed using Klamt’s atomic radii. It was found that hydration of the Pt(IV) complexes is an endergonic/endothermic reaction. It follows the (pseudo)associative mechanism is substantially slower (k?≈?10^-11 s^?1) than the corresponding reaction of Pt(II) analogues ((k?≈?10^-5 s^?1). Such a low value of the reaction constant signifies that the hydration of JM149 and Satraplatin is with high probability a kinetically forbidden reaction. Similarly to JM149 and Satraplatin, the hydration of JM118 is an endothermic/endoergic reaction. On the other hand, the kinetic parameters are similar to those of cisplatin Zimmermann et al. (J Mol Model 17:2385–2393, 2011), allowing the hydration reaction to occur at physiological conditions. These results suggest that in order to become active Satraplatin has to be first reduced to JM118, which may be subsequently hydrated to yield the active species.

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

 The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn₇MT and Cd₇MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn₇MT and pyridylazoresorcinol for Cd₇MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn₇MT concentrations. The complex NEM concentration dependence of each phase, k _f, obs=k ^f ₁+k ^f ₂ [NEM] and k _s, obs=k ^s ₁+k ^s ₂ [NEM], demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested. The reactions of Cd₇MT are less complete than those of Zn₇MT and occur more slowly. ¹¹¹Cd-NMR studies of the partially alkylated ¹¹¹Cd₇MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions, and rules out the possibility of cooperative reactions at either cluster. Received: 5 August 1996 / Accepted: 24 October 1996     

Metal stoichiometry of isolated and arsenic substituted metallothionein: PIXE and ESI-MS study

The stoichiometric analysis of the metal induced Metallothionein (MT) is pertinent for understanding the metal-MT interactions. Despite innumerable publications on MT, the literature addressing these aspects is limited. To bridge this gap, PIXE and ESI-MS analysis of the commercial rabbit liver MT1 (an isoform of MT), zinc induced isolated rat liver MT1, apo and Arsenic substituted rabbit liver MT1 have been carried out. These techniques in combination provide information about number and the signature of all the metal ions bound to MT. By using ESI-MS in the rabbit MT1, ions of Zn _n MT1 (n = 0, 1, 4, 5, 6, 7) whereas, in rat MT1, the Zn₁MT1 and Zn₅MT1 ions are observed. PIXE analysis shows that some copper along with zinc is also present in the rabbit as well as rat MT1 which could not be assessed with ESI-MS. During As metallation reaction with rabbit MT1, with increase in arsenic concentration, the amount of arsenic bound to MT1 also increases, though not proportionally. The presence of both Zn and Cu in MT1 on Zn supplementation can be related to the role of MT in Zn and Cu homeostasis. Further, the presence of partially metallated MT1 suggests that MT1 may donate fractional amount of metal from it’s fully metallated form to other proteins where Zn acts as a cofactor.     

Kinetic studies of the reactions of some metal reconstituted metallothioneins with the electrophilic disulfide 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)

Rabbit liver Pt₇MT, Zn₇MT, Bi₇MT were reconstituted and the kinetic studies of the reactions with electrophilic disulphide 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) were investigated to explore the possible mechanism of metals release from metallothioneins. It is revealed that the Pt₇MT, Zn₇MT react with DTNB biphasically, yielding a four-term rate law: Rate = k _1f [MT]+k _2f [DTNB][MT]+k _1s [MT]+k _2s [DTNB][MT]. Zn₇MT reacts with disulphide DTNB more rapidly and has significantly greater observed rate constants k _s, k _f than Pt₇MT. The kinetic data for Bi₇MT indicate that the reaction is monophasic and the rate law is proved to be: Rate = k ₁ [MT]+k ₂ [DTNB][MT]. The observed pseudo-first order rate constants for above MTs are insensitive to pH value. Based on the available experimental data, the different kinetic behaviors of MTs reactions with electrophilic disulphide DTNB and a possible mechanism to release the coordinated metal ions are discussed.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Metal binding in metallothioneins: Competition for cadmium and zinc between chelex-100 and metal binding sites in metallothionein

A study of the use of the metal chelation properties of Chelex-100 in metal binding reactions of metallothionein (MT), is described. The stoichiometric ratios of bound metals in MT were determined at several stages during a titration in which the Zn(II) in Zn₇MT was displaced by Cd(II), by using Chelex-100 to sequester the free zinc. The stoichiometric ratios provide convincing supporting evidence that the complicated circular dichroism spectral properties observed during the titration arise because the incoming cadmium is distributed across both domains in the protein. It is shown that Chelex-100 does not sequester zinc or cadmium directly from the metallothionein binding sites. Use of Chelex-100 over the temperature range −20 to 65 °C is demonstrated. The chelation capacity of Chelex-100 (in terms of μ metal ion/mg resin) has been determined for a range of elements important in metal toxicology, including: cadmium (33 μ), zinc (22 μ), copper (19 μ), silver (38 μ), lead (40 μ) and mercury (40 μ).     

Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

ZnuA is the periplasmic Zn²⁺-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn²⁺-bound, and Co²⁺-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn²⁺ with Co²⁺ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn²⁺ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn²⁺ (estimated K _d < 20 nM), Co²⁺, Ni²⁺, Cu²⁺, Cu⁺, and Cd²⁺, but not Mn²⁺. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn²⁺ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Native Copper- and Zinc- Binding Protein Metallothionein Blocks Copper-Mediated Aβ Aggregation and Toxicity in Rat Cortical Neurons

Background

A major pathological hallmark of AD is the deposition of insoluble extracellular β-amyloid (Aβ) plaques. There are compelling data suggesting that Aβ aggregation is catalysed by reaction with the metals zinc and copper.

Methodology/Principal Findings

We now report that the major human-expressed metallothionein (MT) subtype, MT-2A, is capable of preventing the in vitro copper-mediated aggregation of Aβ_1–40 and Aβ_1–42. This action of MT-2A appears to involve a metal-swap between Zn₇MT-2A and Cu(II)-Aβ, since neither Cu₁₀MT-2A or carboxymethylated MT-2A blocked Cu(II)-Aβ aggregation. Furthermore, Zn₇MT-2A blocked Cu(II)-Aβ induced changes in ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons.

Conclusions/Significance

These results indicate that MTs of the type represented by MT-2A are capable of protecting against Aβ aggregation and toxicity. Given the recent interest in metal-chelation therapies for AD that remove metal from Aβ leaving a metal-free Aβ that can readily bind metals again, we believe that MT-2A might represent a different therapeutic approach as the metal exchange between MT and Aβ leaves the Aβ in a Zn-bound, relatively inert form.     

Evolution of 1, 3, 5-trisubstituted bipyrazole scaffold based platinum(II) complexes as a biological active agent

Abstract

Square planar mononuclear platinum(II) complexes having general formula [Pt(Lⁿ)Cl₂], (where, Lⁿ?=?L^1–4) were synthesized with neutral bidentate heterocyclic 1,3,5-trisubstituted bipyrazole based ligands. The synthesized compounds were characterized by physicochemical method such as TGA, molar conductance, micro-elemental analysis and magnetic moment, and spectroscopic method such as, FT-IR, UV–vis, ¹H NMR, ¹³C NMR and mass spectrometry. Biological applications of the compounds were carried out using in vitro brine shrimp lethality bioassay, in vitro antimicrobial study against five different pathogens, and cellular level cytotoxicity against Schizosaccharomyces pombe (S. Pombe) cells. Pt(II) complexes were tested for DNA interaction activities using electronic absorption titration, viscosity measurements study, fluorescence quenching technique and molecular docking assay. Binding constants (K_b) of ligands and complexes were observed in the range of 0.23–1.07?×?10⁵?M^?1 and 0.51–3.13?×?10⁵?M^?1, respectively. Pt(II) complexes (I–IV) display an excellent binding tendency to biomolecule (DNA) and possess comparatively high binding constant (K_b) values than the ligands. The DNA binding study indicate partial intercalative mode of binding in complex-DNA. The gel electrophoresis activity was carried out to examine DNA nuclease property of pUC19 plasmid DNA.     

Reaction of the zinc sensor FluoZin-3 with Zn7-metallothionein: Inquiry into the existence of a proposed weak binding site

It has been reported that Zn₇-metallothionein (MT), contains one weak binding site for Zn²⁺. To test this conclusion, rabbit liver MT isolated at pH 7 was reacted with chelating agents of modest affinity for Zn²⁺. Contrary to the previous study, no evidence was found for Zn²⁺ stoichiometrically bound to the protein with an apparent stability constant of about 10⁸. Indeed, stability constant measurements based upon competition between Zn₇-MT and ligands of known stability with Zn²⁺ showed that all of the protein bound Zn²⁺ displayed the same stability constant at pH 7.4 and 25 °C of (1.7 ± 0.6) × 10¹¹. Brief reaction of Zn₇-MT with strong acid converted it into MT^* and upon reneutralization into Zn₇-MT^*, which demonstrated reactivity of about 1 Zn²⁺/mol MT with competing ligands. Acid titration of Zn₇-MT to pH 2 or below rapidly resulted in the formation of Zn₇-MT^* that displayed biphasic titration with base, revealing the rebinding of lower affinity Zn²⁺ between pH 5 and 7. Since MT is commonly acidified during preparation, care must be taken to document which form of the protein is present in subsequent experiments at pH 7.     

Isolation and characterization of metallothionein from guinea pig liver

The amino acid composition, and the absorption, circular dichroism (CD) and magnetic circular dichroism spectra of a metalloprotein induced in the livers of guinea pigs by the injection of CdCl₂ are reported. The amino acid composition of this protein closely resembles that of rat liver metallothionein (MT). We show that this protein has spectroscopic properties that closely follow the behaviour previously reported for several other cadmium-containing metallothioneins in its spectral response to changes in pH, and to the addition of cadmium and copper(I). Dramatic changes are observed in the CD spectrum during the addition of copper(I); it is suggested that these changes are the result of the formation of a mixed Cu(I)/Cd(II) cluster that forms in the α domain once the β domain has been saturated with Cu(I). These results are of particular importance in the characterization of this protein as belonging to the metallothionein class of proteins, as spectral changes of this type are directly related to the displacement of Cd²⁺ and Zn²⁺ from the two, thiolatecluster binding sites that are amongst the unique properties of mammalian metallothioneins. It is demonstrated that the CD spectrum provides a sensitive indicator of the presence of these special metal binding sites by indicating changes in the binding geometry and stoichiometry in response to an incoming metal. These results indicate that the guinea pig liver metallothionein induced by injections of CdCl₂ uses the same α and β type of clusters for cadmium binding as rat liver Cd, Zn-MT, even though there are minor differences in the amino acid composition between the guinea pig and rat liver proteins.     

Synthesis,Physico-Chemical investigations of Co(II), Ni(II) and Cu(II) complexes and their in vitro microbial,cytotoxic, DNA cleavage studies

A series of metal complexes of cobalt(II), nickel(II), and copper(II) have been synthesized with newly derived biologically active ligands. These ligands were synthesized by the condensation of 2-amino-4-phenyl-1,3-thiazole with 8-formyl-7-hydroxy- 4-methylcoumarin. The probable structure of the complexes has been proposed on the basis of analytical and spectroscopic data (IR, UV-Vis, ESR, FAB-mass, and thermoanalytical). Electrochemical study of the complexes is also reported. Elemental analysis of the complexes confined them to stoichiometry of the type ML₂.2H₂O [M = Co(II), Ni(II), and Cu(II)]. The Schiff base and its metal(II) complexes have been screened for their antibacterial (Escherichia coli, Staphylococcus aureus, Staphylococcus pyogenes, and Pseudomonas aeruginosa) and antifungal activities (Aspergillus niger, Aspergillus flavus, and Cladosporium) by the MIC method. The brine shrimp bioassay was carried out to study their in vitro cytotoxic properties, and also the Schiff base and its metal(II) complexes have been studied for DNA cleavage.