Mammalian Metallothionein-3: New Functional and Structural Insights.

Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer's disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3.

Characterization of the role of metallothionein-3 in an animal model of Alzheimer's disease.

Among the dementias, Alzheimer's disease (AD) is the most commonly diagnosed, but there are still no effective drugs available for its treatment. It has been suggested that metallothionein-3 (MT-3) could be somehow involved in the etiology of AD, and in fact very promising results have been found in in vitro studies, but the role of MT-3 in vivo needs further analysis. In this study, we analyzed the role of MT-3 in a mouse model of AD, Tg2576 mice, which overexpress human Amyloid Precursor Protein (hAPP) with the Swedish mutation. MT-3 deficiency partially rescued the APP-induced mortality of females, and mildly affected APP-induced changes in behavior assessed in the hole-board and plus-maze tests in a gender-dependent manner. Amyloid plaque burden and/or hAPP expression were decreased in the cortex and hippocampus of MT-3-deficient females. Interestingly, exogenously administered Zn(7)MT-3 increased soluble Aß40 and Aß42 and amyloid plaques and gliosis, particularly in the cortex, and changed several behavioral traits (increased deambulation and exploration and decreased anxiety). These results highlight that the control of the endogenous production and/or action of MT-3 could represent a powerful therapeutic target in AD.

Rapid exchange of metal between Zn(7)-metallothionein-3 and amyloid-ß peptide promotes amyloid-related structural changes.

Metal ions, especially Zn(2+) and Cu(2+), are implemented in the neuropathogenesis of Alzheimer's disease (AD) by modulating the aggregation of amyloid-ß peptides (Aß). Also, Cu(2+) may promote AD neurotoxicity through production of reactive oxygen species (ROS). Impaired metal ion homeostasis is most likely the underlying cause of aberrant metal-Aß interaction. Thus, focusing on the body's natural protective mechanisms is an attractive therapeutic strategy for AD. The metalloprotein metallothionein-3 (MT-3) prevents Cu-Aß-mediated cytotoxicity by a Zn-Cu exchange that terminates ROS production. Key questions about the metal exchange mechanisms remain unanswered, e.g., whether an Aß-metal-MT-3 complex is formed. We studied the exchange of metal between Aß and Zn(7)-MT-3 by a combination of spectroscopy (absorption, fluorescence, thioflavin T assay, and nuclear magnetic resonance) and transmission electron microscopy. We found that the metal exchange occurs via free Cu(2+) and that an Aß-metal-MT-3 complex is not formed. This means that the metal exchange does not require specific recognition between Aß and Zn(7)-MT-3. Also, we found that the metal exchange caused amyloid-related structural and morphological changes in the resulting Zn-Aß aggregates. A detailed model of the metal exchange mechanism is presented. This model could potentially be important in developing therapeutics with metal-protein attenuating properties in AD.

The catalytic redox activity of prion protein-Cu(II) is controlled by metal exchange with the Zn(II) -thiolate clusters of Zn(7) metallothionein-3.

Silencing prion: Copper-catalyzed transformations of prion protein (PrP) lead to the production of reactive oxygen species (ROS), PrP oxidation, and cleavage and aggregation in transmissible spongiphorm encephalopathies. Zn(7) MT-3 efficiently targets Cu(II) bound in different coordination modes to PrP-Cu(II) . By an unusual redox-dependent metal-swap reaction, MT-3 modulates the catalytic redox properties of PrP-Cu(II) .

Chemistry and biology of mammalian metallothioneins.

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal-thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discussed.

Metallothionein and brain inflammation.

Since the seminal discoveries of Bert Vallee regarding zinc and metallothioneins (MTs) more than 50 years ago, thousands of studies have been published concerning this fascinating story. One of the most active areas of research is the involvement of these proteins in the inflammatory response in general, and in neuroinflammation in particular. We describe the general aspects of the inflammatory response, highlighting the essential role of the major cytokine interleukin-6, and review briefly the expression and function of MTs in the central nervous system in the context of neuroinflammation. Particular attention is paid to the Tg2576 Alzheimer disease mouse model and the preliminary results obtained in mice into which human Zn(7)MT-2A was injected, which suggest a reversal of the behavioral deficits while enhancing amyloid plaque load and gliosis.

The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets.

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21 Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M(8) (II)-MTC (M is Zn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn(6)-MTC and Cd(7)-MTC species from M(8) (II)-MTC after treatment with Chelex 100. The NMR characterization of Cd(7)-MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal-Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1-31) reveal a Cd(3)Cys(9) cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32-64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21 Cys are involved in the binding of eight to nine Cu(I) ions (Cu(8-9)-MTC). The subsequent generation of Cu(12)-MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MTs.

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity.

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.

Effects of Zn(2+), Ca(2+), and Mg(2+) on the structure of Zn(7)metallothionein-3: evidence for an additional zinc binding site.

Human metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain, where it plays an important role in the homeostasis of the essential metal ions Cu(+) and Zn(2+). Like other mammalian metallothioneins (MT-1 and -2), the protein contains a M(II)(3)(CysS)(9) and a M(II)(4)(CysS)(11) cluster localized in two independent protein domains linked by a flexible hinge region. However, there is a substantially increased number of acidic residues in MT-3 (11 residues) compared with MT-2 (four residues) which may act as binding ligands for additional metal ions. In this study, the binding of Zn(2+), Ca(2+), and Mg(2+) to human Zn(7)MT-3 and its mutant lacking an acidic hexapeptide insert, Zn(7)MT-3(Delta55-60), was investigated and compared with the binding of Zn(7)MT-2. By using spectroscopic and spectrometric techniques, we demonstrate that one additional Zn(2+) binds with an apparent binding constant (K(app)) of approximately 100 microM to Zn(7)MT-3 and Zn(7)MT-3(Delta55-60), but not to Zn(7)MT-2. The changes in spectroscopic features of metal-thiolate clusters and gel filtration behavior reveal that the formation of Zn(8)MT-3 is immediate and is accompanied by a decrease in the Stokes radius (R(s)). The changes in the R(s) suggest a mutual approach of both protein domains. The fast binding of Zn(2+) is followed by a slow time-dependent protein dimerization. The binding of Zn(2+) to Zn(7)MT-3 is specific as in the presence of Ca(2+) and Mg(2+) only an alteration of the R(s) of Zn(7)MT-3 at substantially higher concentrations was observed. The significance of these findings for the biological role of MT-3 is 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 alpha-domain and the beta-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 beta-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.

Interaction of metallothionein-2 with platinum-modified 5'-guanosine monophosphate and DNA.

Human metallothioneins (MTs), a family of cysteine- and metal-rich metalloproteins, play an important role in the acquired resistance to platinum drugs. MTs occur in the cytosol and the nucleus of the cells and sequester platinum drugs through interaction with their zinc-thiolate clusters. Herein, we investigate the ability of human Zn 7MT-2 to form DNA-Pt-MT cross-links using the cisplatin- and transplatin-modified plasmid DNA pSP73. Immunochemical analysis of MT-2 showed that the monofunctional platinum-DNA adducts formed DNA- cis/ trans-Pt-MT cross-links and that platinated MT-2 was released from the DNA- trans-Pt-MT cross-links with time. The DNA- cis/ trans-Pt-MT cross-links were also formed in the presence of 2 mM glutathione, a strong S-donor ligand. Independently, we used 5'-guanosine monophosphate (5'-GMP) platinated at the N7 position as a model of monofunctional platinum-DNA adducts. Comparison of reaction kinetics revealed that the formation of ternary complexes between Zn 7MT-2 and cis-Pt-GMP was faster than that of the trans isomer. The analysis of the reaction products with time showed that while the formation of ternary GMP- trans-Pt-MT complex(es) is accompanied by 5'-GMP release, a stable ternary GMP- cis-Pt-MT complex is formed. In the latter complex, a fast initial formation of two Pt-S bonds was followed by a slow formation of an additional Pt-S bond yielding an unusual Pt(II)S 3N coordination with N7-GMP as the only N-donor ligand. The ejection of negligible zinc from the zinc-thiolate clusters implies the initial formation of Zn-(mu-SCys)-Pt bridges involving the terminal thiolate ligands. The biological implications of these studies are discussed.

Implications on zinc binding to S100A2.

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.

Structure of the mammalian NOS regulator dimethylarginine dimethylaminohydrolase: A basis for the design of specific inhibitors.

Dimethylarginine dimethylaminohydrolase (DDAH) is involved in the regulation of nitric oxide synthase (NOS) by metabolizing the free endogenous arginine derivatives N(omega)-methyl-L-arginine (MMA) and N(omega),N(omega)-dimethyl-L-arginine (ADMA), which are competitive inhibitors of NOS. Here, we present high-resolution crystal structures of DDAH isoform 1 (DDAH-1) isolated from bovine brain in complex with different inhibitors, including S-nitroso-L-homocysteine and Zn2+, a regulator of this mammalian enzyme. The structure of DDAH-1 consists of a propeller-like fold similar to other arginine-modifying enzymes and a flexible loop, which adopts different conformations and acts as a lid at the entrance of the active site. The orientation and interaction mode of inhibitors in the active site give insight into the regulation and the molecular mechanism of the enzyme. The presented structures provide a basis for the structure-based development of specific DDAH-1 inhibitors that might be useful in the therapeutic treatment of NOS dysfunction-related diseases.

Specific reactions of S-nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetase.

S-Transnitrosation is an important bioregulatory process whereby NO(+) equivalents are transferred between S-nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R-S-N(O(-))-S-R' and it has been proposed that products different from S-nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N-thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S-nitroso-l-homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S-nitrosothiols S-nitroso-l-cysteine (CysNO) and S-nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N-thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N-thiosulfoximide was formed. The reasons likely responsible for the absence of the N-thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC(50) value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed.

Reaction of Zn7metallothionein with cis- and trans-[Pt(N-donor)2Cl2] anticancer complexes: trans-Pt(II) complexes retain their N-donor ligands.

Intrinsic and acquired resistance are major drawbacks of platinum-based cancer therapy. The protein superfamily of cysteine- and ZnII-rich proteins, metallothioneins (MT), efficiently inactivate these antitumor drugs because of the strong reactivity of platinum compounds with S-donor molecules. In this study the reactions of human Zn7MT-2 with twelve cis/trans-[Pt(N-donor)2Cl2] compounds and [Pt(dien)Cl]Cl, including new generation drugs, were investigated and the products characterized. A comparison of reaction kinetics revealed that trans-PtII compounds react faster with Zn7MT-2 than cis-PtII compounds. The characterization of the products showed that while all ligands in cis-PtII compounds were replaced by cysteine thiolates, trans-PtII compounds retained their N-donor ligands, thus remaining in a potentially active form. These results provide an increased understanding of the role of MT in the acquired resistance to platinum-based anticancer drugs.

Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein.

Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

Cysteine and glutathione trigger the Cu-Zn swap between Cu(ii)-amyloid-ß4-16 peptide and Zn7-metallothionein-3.

Cysteine and glutathione are able to reduce Cu(ii) coordinated to the peptide amyloidß4-16, and shuttle the resulting Cu(i) to partially replace Zn(ii) in the protein Zn7-metallothionein-3. The released Zn(ii) in turn binds to amyloid-ß4-16. Thus cysteine and glutathione are modulators of Cu/Zn-distribution between metallothionein-3 and amyloid-ß4-16.

Sodium and Potassium Ions in Proteins and Enzyme Catalysis.

The group I alkali metal ions Na(+) and K(+) are ubiquitous components of biological fluids that surround biological macromolecules. They play important roles other than being nonspecific ionic buffering agents or mediators of solute exchange and transport. Molecular evolution and regulated high intracellular and extracellular M(+) concentrations led to incorporation of selective Na(+) and K(+) binding sites into enzymes to stabilize catalytic intermediates or to provide optimal positioning of substrates. The mechanism of M(+) activation, as derived from kinetic studies along with structural analysis, has led to the classification of cofactor-like (type I) or allosteric effector (type II) activated enzymes. In the type I mechanism substrate anchoring to the enzyme active site is mediated by M(+), often acting in tandem with a divalent cation like Mg(2+), Mn(2+) or Zn(2+). In the allosteric type II mechanism, M(+) binding enhances enzyme activity through conformational transitions triggered upon binding to a distant site. In this chapter, following the discussion of the coordination chemistry of Na(+) and K(+) ions and the structural features responsible for the metal binding site selectivity in M(+)-activated enzymes, well-defined examples of M(+)-activated enzymes are used to illustrate the structural basis for type I and type II activation by Na(+) and K(+).

Detection of neuronal growth inhibitory factor (metallothionein-3) in polyacrylamide gels and by Western blot analysis.

Neuronal growth inhibitory factor (GIF) is a small cysteine-rich metal binding protein downregulated in Alzheimer's disease. The protein belongs to the superfamily of metallothioneins (MTs) and was classified as MT-3. Although first identified as a brain specific protein, several reports now indicate a substantially broader expression pattern. However, currently available detection methods for MT-3 show low sensitivity in gel electrophoresis and Western blot. We have developed a fast and sensitive method for MT-3 detection in SDS-PAGE (detection limit approximately 10 ng) and Western blot (detection limit approximately 1 ng). The method is based on the chemical modification of cysteine residues with the dye monobromobimane and an improved blotting protocol.