MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection

MitoTracker Green (MTG) is a mitochondrial-selective fluorescent label commonly used in confocal microscopy and flow cytometry. It is expected that this dye selectively accumulates in the mitochondrial matrix where it covalently binds to mitochondrial proteins by reacting with free thiol groups of cysteine residues. Here we demonstrate that MTG can be used as a protein labeling reagent that is compatible with a subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Although the MTG-labeled proteins and MTG do not seem to electrophoretically separate, an enhancement in fluorescence intensity of the product indicates that only proteins with free thiol groups are capable of reacting with MTG. In addition we propose that MTG is a partially selective label towards some mitochondrial proteins. This selectivity stems from the high MTG concentration in the mitochondrial matrix that favors alkylation of the available thiol groups in this subcellular compartment. To that effect we treated mitochondria-enriched fractions that had been prepared by differential centrifugation of an NS-1 cell lysate. This fraction was solubilized with an SDS-containing buffer and analyzed by CE-LIF. The presence of a band with fluorescence stronger than MTG alone also indicated the presence of an MTG-protein product. Confirming that MTG is labeling mitochondrial proteins was done by treating the solubilized mitochondrial fraction with 5-furoylquinoline-3-carboxaldehyde (FQ), a fluorogenic reagent that reacts with primary amino groups, and analysis by CE-LIF using two separate detection channels: 520 nm for MTG-labeled species and 635 nm for FQ-labeled species. In addition, these results indicate that MTG labels only a subset of proteins in the mitochondria-enriched fraction.     

Inhibition of alcohol dehydrogenases by thiol compounds

2-Mercaptoethanol is a strong inhibitor of LADH. The inhibitory effect is likely due to the binding of the SH group to the enzymatic zinc ion. Various thiol compounds do not inhibit YADH and it is suggested that the zinc atoms involved in the catalytic mechanism of LADH and YADH may have different structural arrangements and that these zinc atoms in YADH may not be blocked by thiol compounds. Thiol compounds also quench the enhanced fluorescence of LADH-NADH in a pH-dependent manner. At pH 9.2, the binding of coenzyme to LADH is replaced by 2-mercaptoethanol, whilst at pH 7.3, it further quenches the fluorescence of NADH-LADH. This quenching of fluorescence is likely attributed to a conformational change and energy transfer upon binding of 2-mercaptoethanol to the LADH-NADH complex. Complete reversal of the inhibitory effect of thiol compounds on LADH can be obtained by dialysis.     

The measurement of amino groups in proteins and peptides

A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5-200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30-50 times faster than with in-amino groups of model compounds. The in(420) of a trinitrophenylated thiol group was found to be 2250m(-1).cm(-1). The reaction with several amino acids, peptides and proteins is presented. The in(420) of a typical alpha-amino group was found to be 22000m(-1).cm(-1) and that of an in-amino group, 19200m(-1).cm(-1). Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.     

The role of nuclear serine proteases in the degradation of newly synthesized histones and ribosomal proteins

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.     

Protein thiol modifications visualized in vivo

Thiol-disulfide interconversions play a crucial role in the chemistry of biological systems. They participate in the major systems that control the cellular redox potential and prevent oxidative damage. In addition, thiol-disulfide exchange reactions serve as molecular switches in a growing number of redox-regulated proteins. We developed a differential thiol-trapping technique combined with two-dimensional gel analysis, which in combination with genetic studies, allowed us to obtain a snapshot of the in vivo thiol status of cellular proteins. We determined the redox potential of protein thiols in vivo, identified and dissected the in vivo substrate proteins of the major cellular thiol-disulfide oxidoreductases, and discovered proteins that undergo thiol modifications during oxidative stress. Under normal growth conditions most cytosolic proteins had reduced cysteines, confirming existing dogmas. Among the few partly oxidized cytosolic proteins that we detected were proteins that are known to form disulfide bond intermediates transiently during their catalytic cycle (e.g., dihydrolipoyl transacetylase and lipoamide dehydrogenase). Most proteins with highly oxidized thiols were periplasmic proteins and were found to be in vivo substrates of the disulfide-bond-forming protein DsbA. We discovered a substantial number of redox-sensitive cytoplasmic proteins, whose thiol groups were significantly oxidized in strains lacking thioredoxin A. These included detoxifying enzymes as well as many metabolic enzymes with active-site cysteines that were not known to be substrates for thioredoxin. H₂O₂-induced oxidative stress resulted in the specific oxidation of thiols of proteins involved in detoxification of H₂O₂ and of enzymes of cofactor and amino acid biosynthesis pathways such as thiolperoxidase, GTP-cyclohydrolase I, and the cobalamin-independent methionine synthase MetE. Remarkably, a number of these proteins were previously or are now shown to be redox regulated.     

Bromobimanes — fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Summary Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient.In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons—not stainable by the classical methods—which can be identified as peptidergic ones by the bromobimane technique.A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Cyst(e)ine residues of bovine white-matter proteolipid proteins. Role of disulphides in proteolipid conformation.

Cyst(e)ine residues of bovine white-matter proteolipid proteins were characterized in a highly purified preparation. From a total of 10.6 cyst(e)ine residues/molecule of protein, as determined by performic acid oxidation, 2.5-3 thiol groups were freely accessible to iodoacetamide, iodoacetic acid and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), when the proteins were solubilized in chloroform/methanol (C/M) (2:1, v/v). The presence of lipids had no effect on thiol-group exposure. One thiol group available to DTNB in C/M could not be detected when proteolipids were solubilized in the more polar solvent n-butanol. In a C/M solution of purified proteolipid proteins, SDS did not increase the number of reactive thiol groups, but the cleavage of one disulphide bridge made it possible to alkylate six more groups. C.d. and fluorescence studies showed that rupture of this disulphide bond changed the protein conformation, which was reflected in partial loss of helical structure and in a greater exposure to the solvent of at least one tryptophan residue. Cyst(e)ine residues were also characterized in the different components [PLP (principal proteolipid protein), DM20 and LMW (low-Mr proteins)] of the proteolipid preparation. Although the numbers of cyst(e)ine residues in PLP and DM20 were similar, in LMW fewer residues were alkylated under four different experimental conditions. The differences, however, are not simply related to differences in Mr.     

Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.     

In Vitro Induction of Starfish Oocyte Maturation by Cysteine Alkylesters

The effect of various disulfide-reducing agents including cysteine and its alkylesters on the induction of germinal vesicle breakdown (GVBD) in starfish ( Asterina pectinifera ) oocytes was investigated in vitro . Although cysteine did not induce GVBD, its alkylesters were effective. Cysteine alkylesters significantly mimicked the effect of 1-methyladenine (1-MeAde), the naturally occurring maturation-inducing hormone of starfish, on oocyte maturation. However, the effective concentrations and pH optimum for stimulation of oocyte maturation varied between 1-MeAde and the cysteine alkylesters. By comparing pKa values of the disulfide-reducing agents to pH of the medium, it is suggested that the redox potential of a disulfide-reducing agent is an important indicator its ability to induce oocyte maturation.
With the use of fluorescent probes for thiol groups, it was shown that the fluorescence in oocyte cortices increased within 5 min after administration of 1-MeAde. The fluorescence intensity in the cortices also increased after treatment with cysteine and its alkylesters, although the intensity was much stronger with the latter. Furthermore, both 1-MeAde and the disulfide-reducing agents were suggested to cause reduction of thiol groups within the plasma membrane as opposed to those on the external and internal surfaces. Thus, it is suggested that disulfide-reducing agents and 1-MeAde induce starfish oocyte maturation by changing the redox state of the thiol groups located within the oocyte plasma membrane.     

Disulfide bond formation between the active-site thiol and one of the several free thiol groups of chymopapain

Chymopapain, a cysteine protease of papaya latex, has been purified with the use of fast protein liquid chromatography. Two homogeneous fractions were analyzed for thiol content and thiol reactivity. It was found that peak 1 and peak 2 contained two and three thiol groups, respectively, per mole of enzyme. This result is inconsistent with the general belief that chymopapain contains one essential and one nonessential thiol group and suggests that a significant portion of the thiol groups was oxidized in the previous preparations. Such an oxidation can account for some of the inconsistent results reported in the literature. An irreversibly oxidized nonessential thiol group may modify the catalytic function of chymopapain especially if it is close to the active site. That one thiol group resides indeed in the vicinity of the essential thiol group is clearly demonstrated by the biphasic reactions of chymopapain with disulfide compounds such as 2,2'-dipyridyl disulfide and 5,5'-dithiobis(2-nitrobenzoate). In the first step of these reactions a mixed disulfide is formed between the enzyme and the reactant, which is followed by a first-order, intramolecular reaction leading to the liberation of the second half of the disulfide compound. Furthermore, on addition of one Hg2+ ion, 2 mol of thiol group, one essential and one nonessential, disappears concomitantly. Formation of a disulfide bond between the catalytically competent thiol group and another free thiol group of chymopapain under physiological conditions may be of regulatory importance.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Endogenous peroxidase in mast cells localized with a semipermeable membrane technique

Synopsis Hamster mast cells have been found to give strong peroxidatic reactions at pH 5, 7.5 and 10 when sections of skeletal muscle are incubated for 2.5 h in the dark at room temperature on semipermeable membranes covering a gelled incubation medium consisting of 0.01% hydrogen peroxide, 5.5 mM diaminobenzidine and 1.36% agar dissolved in Universal buffer. The techniques is very efficient: with it, all mast cells react in marked contrast to the negative reaction they usually give with conventional techniques.The peroxidatic reactions are abolished if tissues are perfused beforehand with either aminotriazole or KCN but not if these inhibitors are incorporated in the gelled incubation medium. This and other evidence suggests that the mast cell reactions are not due to either catalase or haemoglobin adsorbed onto mast cell granules from lysed red blood cells.Skeletal muscle fibres do not exhibit any visible-peroxidase activity with the membrane technique.     

The integration of glutathione homeostasis and redox signaling

Formation of reactive oxygen species (ROS) is a common feature of abiotic and biotic stress reactions. ROS need to be detoxified to avoid deleterious reactions, but at the same time, the increased formation of ROS can also be exploited for redox signaling. Glutathione, as the most abundant low-molecular weight thiol in the cellular redox system, is used for both detoxification of ROS and transmission of redox signals. Detoxification of H(2)O(2) through the glutathione-ascorbate cycle leads to a transient change in the degree of oxidation of the cellular glutathione pool, and thus a change in the glutathione redox potential. The shift in the glutathione redox potential can be sensed by glutaredoxins (GRXs), small ubiquitous oxidoreductases, which reversibly transfer electrons between the glutathione redox buffer and thiol groups of target proteins. While very little is known about native GRX target proteins and their behavior in vivo, it is shown here that reduction-oxidation-sensitive GFP (roGFP), when expressed in plants, is an artificial target protein of GRXs. The specific interaction of roGFP with GRX results in continuous formation and release of the roGFP disulfide bridge depending on the actual redox potential of the cellular glutathione buffer. Ratiometric analysis of redox-dependent fluorescence allows dynamic imaging of the glutathione redox potential. It was hypothesized that a similar equilibration occurs between the glutathione buffer and native target proteins of GRXs. As a consequence, even minor deviations in the glutathione redox potential due to either depletion of reduced glutathione (GSH) or increasing oxidation can be exploited for fine tuning the activity of target proteins. The integration of the glutathione buffer with redox-active target proteins is a local reaction in specific subcellular compartments. This observation emphasizes the importance of subcellular compartmentalization in understanding the biology of the cellular redox system in plants.     

Bromobimanes--fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient. In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons--not stainable by the classical methods--which can be identified as peptidergic ones by the bromobimane technique. A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Protein thiols in spermatozoa and epididymal fluid of rats.

Thiol (SH) oxidation to disulphides (SS) is thought to be involved in sperm chromatin condensation and tail structure stabilization, which occur during maturation of spermatozoa. Previously developed procedures, using the fluorescent labelling agent monobromobimane (mBBr), enabled us to study the thiol-disulphide status of spermatozoa. Electrophoretic separation of labelled sperm proteins from the caput and cauda regions showed that during maturation thiol oxidation occurs in many protein fractions from the tail and that the magnitude of oxidation differs between proteins. Among the protein bands, one major band (MPB), probably a dense fibre constituent, is quantitatively prominent. N-Ethylmaleimide (NEM) or mBBr alkylation (of intact spermatozoa) changes the mobility of the caput MPB, but not that of the cauda MPB. The results indicated that the altered mobility of MPB is mainly due to a change in its shape, possibly resulting from the alkylation of a few critical SH groups. Epididymal fluid proteins contain both SH and SS. The thiol and disulphide content of the various epididymal proteins appears similar, although some diminution in fluorescence is seen in epididymal fluid proteins from the cauda region as compared with those from the caput region. The prominent changes in thiol status occur in the spermatozoa.     

Ischemic preconditioning enhances fatty acid-dependent mitochondrial uncoupling

This study tests the hypothesis that ischemic preconditioning (IP) changes fatty acid (FA)-dependent uncoupling between mitochondrial respiration and oxidative phosphorylation. We found that IP does not alter mitochondrial membrane integrity or FA levels, but enhances membrane potential decreases when FA are present, in an ATP-sensitive manner. FA hydroperoxides had equal effects in control and preconditioned mitochondria, and GTP did not abrogate the IP effect, suggesting uncoupling proteins were not involved. Conversely, thiol reductants and atractyloside, which inhibits the adenine nucleotide translocator, eliminated the differences in responses to FA. Together, our results suggest that IP leads to thiol oxidation and activation of the adenine nucleotide translocator, resulting in enhanced FA transport and mild mitochondrial uncoupling.     

A method of immobilization on the solid support of complex and simple enzymes retaining their activity

Immobilization of native proteins, retaining their activity, on the solid support is often crucial for a variety of biochemical assays involving protein-protein interactions. In this study we describe a technique which allows binding of both complex (protein kinase CK2) and simple (calf intestine alkaline phosphatase, CIP) enzymes to the solid support without denaturization of the proteins. This method is based on the covalent cross-linking of the enzymes to the bifunctional resin, containing the secondary amino and thiol groups, in a coupling reaction with the imidoester dimethyl pimelimidate hydrochloride. Both enzymes in their bound form were active in the specific biochemical assays. We also found that the CK2 and CIP resins did not change their activity for at least 3 months, and the quality of these resins were not affected by high salts or reducing agents. Thus, this method can be recommended for general use to generate active enzymes coupled to the solid support.     

The soluble carnitine palmitoyltransferase from bovine liver. A comparison with the enzymes from peroxisomes and from the mitochondrial inner membrane.

The properties of two carnitine acyltransferases (CPT) purified from bovine liver are compared to confirm that they are different proteins. The soluble CPT and the inner CPT from mitochondria differ in subunit Mr, native Mr, pI and reactivity with thiol reagents. All eight free thiol groups in soluble CPT react with 5,5'-dithiobis-(2-nitrobenzoate) in the absence of any unfolding reagent, and activity is gradually lost. The inner CPT activity is completely stable in the presence of 5,5'-dithiobis-(2-nitrobenzoate), and only one thiol group per molecule of subunit is modified in the native enzyme. Antisera to each enzyme inhibit that enzyme, but do not cross-react. CPT activity in subcellular fractions can now be identified by titration with these antibodies. The soluble CPT from bovine liver is probably peroxisomal in origin, but, although antigenically similar, it differs from the peroxisomal carnitine octanoyltransferase found in rat and mouse liver in its specificity for the longer-chain acyl-CoA substrates.     

Diaminobenzidine Induces Fluorescence in Nervous Tissue and Provides Intrinsic Counterstaining of Sections Prepared for Peroxidase Histochemistry

3,3'-Diaminobenzidine (DAB) is widely used as a chromogen for visualization of horseradish peroxidase activity in neuroanatomical tracing experiments and in immunohistochemistry. The product of the enzymatically catalyzed oxidation of DAB by hydrogen peroxide is brown and nonfluorescent. In frozen sections of formaldehyde fixed rat and mouse brain that had been exposed to DAB either alone or with hydrogen peroxide, we observed strong greenish fluorescence in myelinated nerve fibers and in the somata of some neurons. This fluorescence was not associated with brown coloration and was not due to endogenous peroxidase activity. Extractions, blocking reactions, and other histochemical tests indicate that the fluorescence resulted from the combination of DAB with aldehyde groups that were formed by oxidation of unsaturated linkages in lipids. DAB induced fluorescence provides a simple and useful demonstration of background anatomy in sections that also contain specifically localized deposits of peroxidase activity.