Fluorescein Mercuric Acetate Research Articles

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.

Unwinding of DNA induced by fluorescein mercuric acetate.

AbstractFluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. This unwinding process can be followed by measuring absorption changes of this reagent. For untreated calf thymus DNA, the initial rate was very slow, and the shape of the kinetic curve was sigmoidal. When double‐strand breaks of DNA were produced by DNase II treatment or sonication, the initial rate increased and the sigmoidal character disappeared. The initial rate was shown to be proportional to the concentration of helix ends. From this relation the rate of unwinding was estimated to be 2.0 base pairs/sec at 1.0 × 10−5M fluorescein mercuric acetate and 25°C. DNase I treatment, which produces single‐strand breaks and a smaller number of double‐strand breaks, also increased the initial rate. However, this increase was due only to the double‐strand breaks, that is, single‐strand breaks had no significant effect on the initial rate. Also, uv irradiation increased the initial rate linearly with uv dose, at least up to 2 × 105 erg/mm2, suggesting that this increase is due to photoproducts other than usual pyrimidine dimers. We discuss the usefulness of this kinetic method in structural studies of DNA.

Inhibition of the nad-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents

1. 1. The NAD-linked glutamate dehydrogenase (EC 1.4.1.2) partially purified from epimastigotes of Trypanosoma cruzi was strongly inhibited by the sulfhydryl reagents fluorescein mercuric acetate (FMA), p-chloromercuribenzoate ( p-CMB), 5,5′ dithiobis (2-nitrobenzoate) (DTNB), N-ethylmaleimide (NEM), o-iodosobenzoate (IBz) and iodoacetamide (IAm). 2. 2. The [I] 50 values (concentration of inhibitor for 50% inhibition) were 0.12, 1, 20, 80 μM, 1.2 and 25 mM, respectively, and the inhibition was nearly complete. Iodoacetate was practically ineffective. 3. 3. The inhibition by p-CMB or FMA, and to some extent that by DTNB, but not that by NEM or IBz, could be partially reversed by addition of β-mercaptoethanol. 4. 4. The enzyme partially modified by preincubation with p-CMB or IBz presented the same apparent K m values for α-oxoglutarate, NADH and NH 4Cl, with a decreased apparent V max. 5. 5. The results suggest that one or more sulfhydryl groups, at or near the active site, are required for the activity of this glutamate dehydrogenase, which seems to be the most sensitive to thiol reagents among the similar enzymes studied so far.

Isolation of elastic tissue microfibrils derived from cultured cells of calf ligamentum nuchae.

Cells cultured from calf ligamentum nuchae produce extracellular microfibrils identical to those of intact elastic tissue as determined by ultrastructural appearance, degradative enzyme susceptibilities and amino acid composition. A method to isolate large amounts of the microfibrillar component using fluorescein mercuric acetate is described. The cultures do not appear to synthesize the amorphous component, elastin, in that radioactive desmosine and isodesmosine were not detected after incubating cultures with labeled lysine nor was elastin seen ultrastructurally.

Fluorescence studies of the sodium and potassium transport adenosine triphosphatase labeled with fluorescein mercuric acetate and anthroylouabain.

Fluorescence studies of the sodium and potassium transport adenosine triphosphatase labeled with fluorescein mercuric acetate and anthroylouabain.

Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1

The region of plasmid NR1 concerned with resistance to Hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColE1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColE1, cells containing the plasmid was as sensitive to Hg2+ and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColE1, cells with the hybrid plasmid were hypersensitive to Hg2+ and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).

Fluorescein mercuric acetate as a probe of the dynamic structure of double-helical DNA

Fluorescein mercuric acetate causes the unwinding of DNA and binds to the separated bases. The kinetics of this unwinding process were studied using both untreated DNA and sonicated DNA at various pH values (6.8–9.3) and Na + concentrations (10–250 mM). The unwinding process is explained by assuming a nucleation in the middle of DNA (as a function of time) as well as at the helix ends (immediately after addition of this reagent) and the subsequent growth of the nuclei. The frequency of the nucleation in the middle of DNA appears to be markedly affected by pH and Na + concentration. In contrast, the reaction rate of this reagent with heat-denatured DNA was almost insensitive to these environmental variables. The growth rate of the unwinding nuclei in double-stranded DNA also appears insensitive. The most important implication of this study is that in the low pH range (6.8–7.5) the reactivity of thermally-induced locally open regions in the middle of double-helical DNA toward this reagent appears much higher than that of heat-denatured DNA. Since this reagent is negatively charged, these findings are discussed in view of its electrostatic interaction with the locally open regions.

Fluorescence labeling of the human erythrocyte anion transport system. Subunit structure studied with energy transfer

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419–2427). The vesicles have a functional anion transport system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescence titrations show a maximum labeling stoichiometry of 1.3 ± 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu 2+ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion

Absorption, Fluorescence, and Linear Dichroism Spectra of Fluorescein Mercuric Acetate (FMA) bound to F-actin

Two kinds of F-actin were prepared; one binds magnesium at the unique divalent cation binding site of actin protomer and the other binds calcium at this site. They were designated as F(Mg)-actin and F(Ca)-actin, respectively. The binding of fluorescein mercuric acetate (FMA) to F(Mg)-actin and F(Ca)-actin was studied spectroscopically. The absorption and fluorescence spectra of bound FMA differed slightly but distinctly between F(Mg)-actin and F(Ca)-actin. Moreover, FMA bound to F(Mg)-actin showed linear dichroism in the presence of 2 mM MgCl2 (or 2mM CaCl2) in the solvent, while the dichroism was abolished by the removal of divalent cations from the solvent. In contrast, FMA bound to F(Ca)-actin did not show any appreciable linear dichroism irrespective of the presence (or absence) of divalent cations in the solvent. These results suggest that the structure of F-actin is characteristically regulated by divalent cations in a dual mode.

Separation and characterization of venom components in Loxosceles reclusa—II. Protease enzyme activity

A protease from brown recluse spider venom hydrolyzes the amide linkage of amino acids containing aliphatic, aromatic, or basic side chains. The mol. wt of the enzyme was 29,600 by SDS electrophoresis. The K m for l-leucyl-β-naphthylamide is 3·75 × 10 -4 M and the activation energy was 49,000 cal/mole. The pH profile indicates that the enzyme has a narrow active region centered at pH 7·2. Spider venom protease is inhibited irreversibly by ethylenediamine tetraacetic acid, N-ethylmaleimide and fluorescein mercuric acetate.

Characterization of the membrane fraction isolated by the fluorescein mercuric acetate technique of Barland and Schroeder

Using scanning electron microscopy we have demonstrated that the membrane fraction isolated by the fluorescein mercuric acetate technique of Barland and Schroeder (Barland, P. and Schroeder, E.A. (1975) J. Cell Biol. 45, 662–668) represents a topologically distinct membrane which circumscribes the cell nucleus. Our data suggest that not all the cells within a nonsynchronized cell population release a membrane fraction after treatment according to the technique of Barland and Schroeder, but rather that the efficiency of membrane release achieved using this preparative technique is dependent on the morphology of individual cells. Our work has also demonstrated that the peptide composition of the membrane fraction isolated by the technique of Barland and Schroeder differs from the peptide composition of the plasma membrane-enriched fraction isolated by the technique of Brunette and Till (Brunette, D.M. and Till, J.E. (1971) J. Membrane Biol. 5, 215–224). This difference in peptide composition is particularly noticeable among the higher molecular weight proteins, glycoproteins and iodineateable membrane components. The data which we have accumulated suggest that the compositional differences noted between the two membrane isolates do not result from differential extraction of membrane components during the ZnCl 2-fluorescein mercuric acetate treatments required in the isolation technique originally described by Barland and Schroeder. However, our data do clearly demonstrate that the membrane isolation technique of Barland and Schroeder cannot be used to study the general composition of the plasma membrane.

Indirect amplification method for determining mercury by direct-current polarography. Application to organomercury compounds

An amplification method for the determination of 0.5–70 ppm (2.5 × 10 −6 to 3.5 × 10 −4 M) of Hg(II) is described. Hg(II) is reacted with a slight excess of KI, and the excess iodide is oxidized by bromine water and measured polarographically as iodate with sixfold amplification. Alternatively, the iodate formed is reacted to liberate iodine which is then reduced to iodide, and again oxidized to yield six iodate ions for every iodide ion originally present; 2 Microdetermination of Mercury in Organomercurial Compounds Compound Sample (mg) Hg (%) SD (%) Calcd Found Error Phenylmercuric Acetate 4.682 59.57 60.04 +0.47 0.68 3.690 59.08 −0.49 Fluorescein-mercuric acetate 3.000 47.22 47.09 −0.13 0.64 3.325 47.21 −0.01 4.173 46.33 −0.89 Mercurochrome 6.884 26.72 25.73 −0.99 0.59 5.427 26.12 −0.60 3.180 26.74 +0.02 4.903 26.50 −0.22 4.542 26.88 +0.16 polarographic reduction requires 36 electrons. Oxidation of the excess iodide with periodate yields four iodate ions for every iodide ion and therefore allows 24-fold amplification. Microdetermination of mercury in organomercurials is achieved using the sixfold method following closed flask combustion: the average percentage error for 10 determinations is ±0.40 and the time required for one sample run is 45 min.

Lactose repressor protein modified with fluorescein mercuric acetate.

The reaction of lac repressor protein with fluorescein mercuric acetate has been studied. A maximum of 1.5 cysteine residues/monomer was modified with this reagent; the presence of ligands (inducer, anti-inducer, nonspecific DNA) did not affect ‘the reaction. Modification of the cysteines increased the affinity of the protein for inducer, but did not affect binding to nonspecific DNA. The operator binding activity, in contrast, was abolished at excesses of reagent 20.75 mercurials/monomer. Reversal of the effects on operator and inducer binding activity could be obtained by addition of dithiothreitol to the modified repressor protein. Mapping studies to determine the extent of reaction at each of the cysteines indicated that with excesses of fluorescein mercuric acetate below 0.5 molecules/monomer, cysteine 268 was less reactive than either cysteine 107 or cysteine 140; at the point of maximum reaction, each of the cysteines was approximately 50% reacted. Using 2-bromoacetamido-4-nitrophenol as a selective blocking agent for cysteines 107 and 140 prior to reaction with fluorescein mercuric acetate, the loss of operator binding activity was decreased, while no effect was observed on the increased affinity for inducer noted for mercurial modified protein. The loss of operator DNA binding activity with simultaneous maintenance of nonspecific DNA binding activity suggests that, while the determinants for binding may overlap, there are also independent determinants. It is of interest to note that loss of operator binding occurred with modification of residues in the core region of the molecule rather than in the NH2 terminus. Perturbations of the protein structure by inducer, anti-inducer, and nonspecific DNA were reflected in the fluorescein absorbance spectrum. Inducer difference spectra and anti-inducer difference spectra exhibited opposite characteristics; this is consistent with their different effects on the function of the protein molecule. Nonspecific DNA also perturbed the fluorescein spectrum; part of this difference spectrum involves direct interaction of the fluorescein mercuric acetate with the nucleic acid, but the spectrum also contained components attributable to perturbation of the fluorescein spectrum indirectly through the protein. This constitutes the first direct spectral evidence for the effect of nonspecific DNA on the protein structure. It is interesting to note that the spectral changes arise from residues which are in the core region of the molecule

The mechanism of the neocarzinostatin-induced cleavage of DNA

The single strand scission of DNA by neocarzinostatin (NCS) requires oxygen and is inhibited by free radical and thiol group trapping agents. Photoreduction of the two disulfide links in NCS in the presence of sodium hypophosphite affords the sulfhydryl group, the oxidation of which is responsible for nicking the DNA. The latter process and consequently the nicking of DNA by NCS is catalyzed by Cu(II) ion. Cleavage of DNA by NCS is prevented by selective oxidation with performic acid of the two disulfide links, the removal of which is monitored by a fluorescein mercuric acetate assay. In contrast to bleomycin, NCS is not activated by traces of Fe(II) ion and there is no evidence for strong binding of NCS to DNA.

Mercury and Organomercurial Resistances Determined by Plasmids in Pseudomonas

Mercury and organomercurial resistance determined by genes on ten Pseudomonas aeruginosa plasmids and one Pseudomonas putida plasmid have been studied with regard to the range of substrates and the range of inducers. The plasmidless strains were sensitive to growth inhibition by Hg(2+) and did not volatilize Hg(0) from Hg(2+). A strain with plasmid RP1 (which does not confer resistance to Hg(2+)) similarly did not volatilize mercury. All 10 plasmids determine mercury resistance by way of an inducible enzyme system. Hg(2+) was reduced to Hg(0), which is insoluble in water and rapidly volatilizes from the growth medium. Plasmids pMG1, pMG2, R26, R933, R93-1, and pVS1 in P. aeruginosa and MER in P. putida conferred resistance to and the ability to volatilize mercury from Hg(2+), but strains with these plasmids were sensitive to and could not volatilize mercury from the organomercurials methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids, in addition, conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate. The other plasmids, FP2, R38, R3108, and pVS2, determined resistance to and decomposition of a range of organomercurials, including methylmercury, ethylmercury, phenylmercury, and thimerosal. These plasmids also conferred resistance to the organomercurials merbromin, p-hydroxymercuribenzoate, and fluorescein mercuric acetate by a mechanism not involving degradation. In all cases, organomercurial decomposition and mercury volatilization were induced by exposure to Hg(2+) or organomercurials. The plasmids differed in the relative efficacy of inducers. Hg(2+) resistance with strains that are organomercurial sensitive appeared to be induced preferentially by Hg(2+) and only poorly by organomercurials to which the cells are sensitive. However, the organomercurials p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate were strong gratuitous inducers but not substrates for the Hg(2+) volatilization system. With strains resistant to phenylmercury and thimerosal, these organomercurials were both inducers and substrates.

Kinetics of complex formation between fluorescein mercuric acetate and DNA.

AbstractThe kinetics of complex formation between fluorescein mercuric acetate and heat‐denatured DNA were studied by measuring the fluorescence quenching of this reagent. This quenching process involved no immeasurably rapid phase and it was shown that this reaction follows simple second‐order kinetics. The rate constant at 25°C was estimated to be 2.9 × 104M−1 sec−1 for calf‐thymus DNA (42% G + C) and 1.1 × 104M−1 sec−1 for Micrococcus lysodeikticus DNA (72% G + C). Activation parameters for this reaction were calculated from the temperature dependence of the reaction rate, and the activation entropy was found to be highly negative (−27.5 cal/mol deg for calf‐thymus DNA and −25.5 cal/mol deg for M. lysodeikticus DNA). The binding of fluorescein mercuric acetate to native DNA, which requires the opening of the double‐helical structure, was also followed by measuring the absorbance change of this reagent. There was a lag phase in this binding process, and the enthalpy change for the opening step corresponded roughly to that for the opening of one base pair. These findings are discussed in relation to the results of a similar study with formaldehyde.

Mercury and organomercurial resistances determined by plasmids in Staphylococcus aureus.

Penicillinase plasmids of Staphylococcus aureus often contain genes conferring resistance to inorganic mercury (Hg(2+)) and the organomercurial phenylmercury acetate. The mechanism of resistance was found to be the enzymatic hydrolysis of the organomercurial phenylmercury to benzene plus inorganic ionic mercury, which was then enzymatically reduced to metallic mercury (Hg(0)). The Hg(0) was rapidly volatilized from the medium into the atmosphere. After the mercurial was degraded and the mercury was volatilized, the resistant cells were able to grow. These plasmids also conferred the ability to volatilize mercury from thimerosal, although the plasmid-bearing strains were equally as thimerosal sensitive as the S. aureus without plasmids. None of the plasmids conferred the ability to volatilize mercury from several other organomercurials, however: methylmercury, ethylmercury, p-hydroxymercuribenzoate, merbromin, and fluorescein mercuric acetate. (Organomercurial resistance-conferring plasmids of Escherichia coli and Pseudomonas aeruginosa that we have been studying confer the ability to degrade two or three of these organomercurials.) Although mercury was not volatilized from p-hydroxymercuribenzoate or fluorescein mercuric acetate, the plasmid-bearing strains were resistant to these organomercurials. The ability to volatilize mercury from Hg(2+) and phenylmercury was inducible. The range of inducers included Hg(2+), phenylmercury, and several organomercurials that were not substrates for the degradation system. Mercury-sensitive mutants have been isolated from the parental plasmids pI258 and pII147. Thirty-one such mercury-sensitive strains fall into three classes: (i) mercury-sensitive strains totally devoid of the phenylmercury hydrolase and Hg(2+) reductase activities; (ii) mutants with normal hydrolase levels and no detectable reductase; and (iii) mutants with essentially normal hydrolase levels and low and variable (5 to 25%) levels of reductase activities. The mercury-sensitive strains were also sensitive to phenylmercury, including those with the potential for hydrolase activity.

A partial characterization of human fibroblast plasma membranes isolated by the fluorescein mercuric acetate method of Barland and Schroeder

Plasma membranes were isolated from cultured human fibroblasts by the method of Barland and Schroeder [Barland, P., and Schroeder, L.A. (1970) J. Cell Biol. 45, 662–668.]. The proteins of the membrane preparations were separated on a specially adapted polyacrylamide gel system employing acetic acid and urea. In this nondetergent system, approximately 40 protein bands can be resolved with a relatively high degree of clarity. Analyses of phosphorus content and of the retention of 32P revealed a selective loss of phospholipids from the membranes during their isolation. The level of total sialic acids in the isolated surface membranes of growing cells was found to be greater than that for confluent cultures of fibroblasts (15.1 ± 2.8 vs 6.4 ± 0.79 SE nmol/mg of protein). Electron microscopic and other studies indicate a need for caution concerning the size of yields obtained by this method of plasma membrane isolation.

Use of fluorescein mercuric acetate as a probe in studies of thiol-containing proteins.

The reaction of fluorescein mercuric acetate (FMA) with thiol groups is inhibited by barbital (Veronal). However, barbital has no effect on the reaction of 5,5'-dithiobis(2-nitrobenzoic acid) with thiol groups. Complex formation between FMA and barbital was shown by an increase in the fluorescence polarization of FMA in the presence of barbital. These findings indicate that the inhibitory effect of barbital is due to its interaction with FMA. The extent of this inhibition, which depends on the nature of the thiol-containing substance, presumably reflects the structure surrounding the thiol groups. Furthermore, the absorption and fluorescence properties of FMA conjugated with thiol groups are sensitive to the environment. Because of these properties, FMA can be used to investigate the environment of thiol groups.

Fluorometric method using fluorescein mercuric acetate reagent for determination of hydrogen sulfide in the atmosphere

Fluorometric method using fluorescein mercuric acetate reagent for determination of hydrogen sulfide in the atmosphere