Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

锌_7与镉_7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白制备去金属金属硫蛋白、锌７与镉７金属硫蛋白．在不同ｐＨ条件下,比较后二者清除羟自由基能力;在ｐＨ６条件下,比较锌７－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理ｐＨ条件下锌７－金属硫蛋白清除羟自由基能力远强于镉７－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

脱金属硫蛋白与镉离子的络合作用及构象研究

用圆二色（ＣＤ）谱地研究兔肝脱金属硫属蛋白的两个亚型与Ｃｄ＾２＋的络合作用及对重组ＭＴ构象的影响。观测了ａｐｏ－ＭＴ垢巯基在空气和室温下的稳定性。在ＰＨ４．７１,镉重组ＭＴ１的ＣＤ谱特征峰在２５７ｎｍ（＋）,２３８ｎｍ（－）,２２６ｎｍ（＋）与镉诱导的天然ＭＴ１相同。在空气存在和ＰＨ７．９０的ＣＤ谱只有２４３ｎｍ（＋）一个峰。向两亚型分别加入７ｅｑＣｄ＾２＋测定ＣＤ谱随ＰＨ值的变化,发现在ＰＨ２．     

Folding pathway of apo-metallothionein induced by Zn2+, Cd2+ and Co2+.

Metal ion binding to the sulfhydryl groups of apometallothionein (apo-MT) causes both the formation of native metal-thiolate clusters and the folding of the polypeptide chain of each domain. Cd2+ and Zn2+ react with apo-MT to form metal-thiolate bonds in reactions that are complete within milliseconds and which are pH-dependent. Dual mixing experiments were conducted that involve the initial reaction of metal ion and apo-MT followed by mixing with 5,5'-N-dithio-bis(2-nitrobenzoate) or EDTA after 26 ms. They showed that structures had formed within the brief reaction period which were resistant to rapid reaction with reagents that interact with sulfhydryl groups or metal ions, respectively. It was concluded that native metallothionein domains had been constituted within this brief period. Apo-MT was also titrated with Co2+ to yield Co(n)-MT (n=1-7). Initially, Co2+ bound to independent, tetrahedral thiolate sites. Spectrophotometric analysis of the titration suggested that the independent Co(II) sites began to coalesce into clusters at n=4 (pH 7.2) or n=5 (pH 8.4). Back titration of free sulfhydryl groups (S) in Co(n)-MT (n=1-7) with iodoacetamide at pH 7.2 confirmed that clustering began at n=4. Upon conversion of these alkylated structures to the corresponding 113Cd2+ species 113Cd NMR spectroscopy established that the location of Co(II) in Co(n)-MT (n=1-3) was non-specific and that at n=4, the only observable structure was Co(II)4S11. The results suggest possible kinetic pathways of folding that are conceptually similar to those hypothesized for other small proteins.     

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.     

Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics-molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu6beta-MT with Cd4alpha-MT and Cu6alpha-MT with Cd3beta-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu+ ions preferentially coordinated to the beta domain in each case. Reaction of As3+ with the individual metal-free beta and alpha domains of MT resulted in three As3+ ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As3+ ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization-mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd2+ ions in two domains with stoichiometries of a novel Cd3S7 cluster and a beta-like Cd3S9 cluster.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Influence of chloride ligands on the structure of Zn- and Cd-metallothionein species

It is now commonly accepted that non-proteic ligands contribute to the structure and stability of metal-metallothionein (M-MT) species, although this contribution may differ substantially depending on the MT and the metal ions involved. Conversely, literature data are unconnected, lacking correlation studies between the contribution of inorganic ligands to the M-MT complexes and the corresponding CD and UV-vis fingerprints. To contribute towards filling this gap, we have analyzed the influence of chloride anions in the Zn- and Cd-MT complexes of mammalian MT1 and MT4 isoforms. Starting from the initial hypothesis that the shoulders appearing at 240nm in the UV-vis difference spectra during the Cd(II) titrations of Zn-MTs would be indicative of chloride participation in these metal-MT complexes, we can now propose that, while their absence definitely rules out these ligands being involved in metal coordination, their presence should not necessarily be attributed to the formation of metal-Cl bonds. Instead, we identified a global blue shift for the UV-vis difference spectral envelope as the most liable indication of chloride participation in the binding sites of the M-MT species. Following this criterion, we determined that chloride anions are bound to the Cd(7)-MT1 and Cd(4)-alphaMT1 complexes but not in the isostoichiometric Zn complexes, nor in the Zn- or Cd-complexes of the homologous MT4 peptides.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.     

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Silver binding to rabbit liver metallothionein. Circular dichroism and emission study of silver-thiolate cluster formation with apometallothionein and the alpha and beta fragments

We report new spectroscopic properties for a range of silver-metallothionein species. The binding reactions that take place following addition of Ag+ to rabbit liver apoMT 2, and the apo alpha and -beta fragments have been studied using the techniques of circular dichroism (CD) and emission spectroscopy. Titrations carried out at 20 degrees C and 55 degrees C reveal for the first time the formation of a sequence of clusters (Ag6-MT, Ag12-MT and, finally, Ag18-MT) as Ag+ is added to rabbit apoMT 2. (The division of mammalian metallothioneins into two major subforms, MT 1 and MT 2, is based on differences in molecular charge, which results from differences in the sequence of amino acids that do not involve the cysteines.) It is proposed that the novel Ag18-MT complex forms with a structure that involves a well defined three-dimensional structure, in the same manner as that recently reported for the Hg18-MT complex (Cai, W. and Stillman, M. J., (1988) J. Am. Chem. Soc. 110, 7872-7873). Addition of silver in excess of 20 mol equivalents leads to the collapse of this structure. At the elevated temperatures, it is suggested that the protein can exert cooperativity so that completely filled domains are formed rather than mixtures of complexes. This contrasts with the kinetic product in which metals are bound across the peptide chain forming more random "cross-linked" regions in place of the cluster structure. CD spectra were recorded as Ag+ was added to the alpha and beta fragments formed from rabbit liver MT 1. The silver-containing fragments are less stable than the Ag-MT. The alpha and beta fragments exhibit CD spectral patterns indicative of stoichiometrically defined species. The presence of Ag3- alpha MT 1 and Ag6- alpha MT 1 is suggested by the spectral data obtained at 20 and 55 degrees C. Formation of Ag3- beta MT 1 is suggested by the spectral data recorded at 20 degrees C for the beta fragment. We also report that silver-containing metallothioneins are luminescent. Both the position of the band maximum in the 460-600 nm region and the emission intensity are strongly dependent on the stoichiometry of silver to protein. In the range of molar ratios for silver:MT of 1-12, bands at 465 and 520 nm intensify to a maximum for Ag10-MT 2. A band at 575 nm reaches a maximum for Ag16-MT 2. Analysis of the emission data suggests that Ag+ binds in a domain specific mechanism to apoMT 2.     

Fish and molluscan metallothioneins

Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a beta domain that binds three bivalent metal ions and an alpha domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Fourier transform IR and Fourier transform Raman spectroscopy studies of metallothionein-III: amide I band assignments and secondary structural comparison with metallothioneins-I and -II

The secondary structures of porcine brain Cu(4)Zn(3)-metallothionein (MT)-III and Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I from rabbit livers in the solid state are investigated by Fourier transform IR spectroscopy (FTIR) and Fourier transform Raman spectroscopy (FT-Raman). The Cu(4)Zn(3)MT-III contains 26-28% beta-turns and half-turns, 13-14% 3(10)-helices, 47-49% random coils, and 11-12% beta-extended chains. The structural comparison of porcine brain Cu(4)Zn(3)MT-III with rabbit liver Cd(5)Zn(2)MT-I (II) and Zn(7)MT-I shows that the contents of the random coil structure are obviously increased. The results indicate that the insert of an acidic hexapeptide in the alpha domain of Cu(4)Zn(3)MT-III possibly forms an alpha helix. However, because the bands assigned to the alpha-helix and random coil structures are overlapped in the spectra, the content of random coil structures in Cu(4)Zn(3)MT-III is therefore higher than those in Cd(5)Zn(2)MT-I, Cd(5)Zn(2)MT-II, and Zn(7)MT-I.     

Mercury(II) binding to metallothioneins. Variables governing the formation and structural features of the mammalian Hg-MT species.

With the aim of extending our knowledge on the reaction pathways of Zn-metallothionein (MT) and apo-MT species in the presence of Hg(II), we monitored the titration of Zn7-MT, Zn4-alphaMT and Zn3-betaMT proteins, at pH 7 and 3, with either HgCl2 or Hg(ClO4)2 by CD and UV-vis spectroscopy. Detailed analysis of the optical data revealed that standard variables, such as the pH of the solution, the binding ability of the counter-ion (chloride or perchlorate), and the time elapsed between subsequent additions of Hg(II) to the protein, play a determinant role in the stoichiometry, stereochemistry and degree of folding of the Hg-MT species. Despite the fact that the effect of these variables is unquestionable, it is difficult to generalize. Overall, it can be concluded that the reaction conditions [pH, time elapsed between subsequent additions of Hg(II) to the protein] affect the structural properties more substantially than the stoichiometry of the Hg-MT species, and that the role of the counter-ion becomes particularly apparent on the structure of overloaded Hg-MT.     

金属硫蛋白及其结构域突变体的紫外和圆二色光谱学研究

采用 p GEX- 4T- 1融合表达载体高效表达所得的金属硫蛋白及其结构域突变体 ,包括 α结构域 ,β结构域 ,α- KKS- α和 β- KKS- β( KKS为金属硫蛋白结构域之间的天然连接区氨基酸 ) ,经纯化和纯度鉴定后 ,利用紫外和圆二色光谱进行结构研究 .在脱金属的上述蛋白中 ,固定 p H为中性 ,改变加入 Cd2 + 的比例 ,或固定 Cd2 + 浓度 ,逐渐调节 p H至中性 ,观察紫外和圆二色光谱中镉硫金属簇吸收峰的形成 .研究结果表明 :镉硫金属簇的形成依赖于加入金属的比例和 p H值 ,所有蛋白均于p H3.1 5以上开始形成明显的吸收峰 .紫外图谱中的吸收峰位于 2 54nm附近 ,但在圆二色图谱中不同蛋白形成的峰的位置不同 ,MT,α结构域和 α- KKS- α在 2 2 5nm和 2 58nm处有吸收 ,β结构域在 2 60 nm处有吸收 ,而 β- KKS- β在 2 4 5nm处有吸收 ;向 α结构域 ,β结构域 ,MT,α- KKS- α和 β-KKS- β中分别加入 4eq,3eq,7eq,8eq和 6eq( eq:equivalent,当量 )的 Cd2 +时 ,吸收峰可达到最大值 .同时发现 α结构域的吸收峰强于 β结构域 ,而且双结构域突变体的镉硫金属簇则明显强于相应的单结构域突变体 ,这表明吸收峰的强弱与金属结合力的大小相关 ,而且结构域之间存在相互作用 ,从而影响与金属的结合 .     

锌7与镉7－金属硫蛋白清除羟自由基的比较

分离及纯化兔肝金属硫蛋白.制备去金属金属硫蛋白、锌₇与镉₇金属硫蛋白．在不同pH条件下,比较后二者清除羟自由基能力;在pH6条件下,比较锌₇－金属硫蛋白与有关蛋白和无机锌盐清除羟自由基效果．结论是在近生理pH条件下锌₇－金属硫蛋白清除羟自由基能力远强于镉₇－金属硫蛋白．金属硫蛋白清除羟自由基的能力主要来源于蛋白中处于还原态的流基．     

Structural properties of metal-free apometallothioneins

The metalated forms of metallothionein are well studied (particularly Zn-MT, Cu-MT and Cd-MT), but almost nothing is known about the chemical and structural properties of apometallothioneins despite their importance in initial metalation and subsequent demetalation. Electrospray ionization mass spectrometry was used to provide a detailed view of the structural properties of the metal-free protein. Mass spectra of Zn(7)-MT and apo-MT at pH 7 exhibit the same charge state distribution, indicating that apo-MT is tightly folded like the metallated protein, whereas apo-MT at pH 3 exhibits a charge state spectrum associated with unfolding or denaturation. Benzoquinone was used to modify the cysteines in the β-MT (9Bq), and α-MT (11Bq) fragments, and the full βα-MT (20Bq) protein. ESI-MS showed that the overall volume and, therefore, the extent of folding for the modified proteins is similar to that of Zn-MT. Molecular modeling using MM3-MD methods provided the volume of each modified protein. The volumes of the partially modified proteins follow the same trend as the charge states, showing that ESI-MS is an excellent method with which to follow small changes in protein folding as a function of applied chemical stress. The data suggest that the structure of apo-βα-MT is more organized than previously considered.