A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. II. Effects of variations in ligand structure on the in situ detection of mercurated probes

In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achieved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand. In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands. The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.     

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands

Summary In the preceding paper, a method to detect specific DNA sequences with mercurated nucleic acid probes and sulfhydryl-hapten ligands has been described. Due to the instability of the bond between mercury and a negatively charged sulfhydryl-hapten ligand (trinitrophenyl-glutathione), the in situ formed hybrid could not be detected. On basis of model system experiments it was suggested that this mercury-sulfhydryl bond could be stabilized by an extra polar interaction between ligand and nucleic acid. This was achreved by reversing the net charge of the ligand. Such ligands were synthesized by reacting aliphatic diamines to the carboxyl groups of Tnp-glutathione using a water soluble carbodiimide. Gel chromatographic analysis of mercurated polynucleotide-ligand complexes showed that the stability of the mercury-sulfhydryl bond is increased by the reversal of the net charge of the ligand.In situ hybridized mercurated mouse satellite DNA to mouse liver nuclei and mercurated kinetoplast cRNA hybridized to Crithidia fasciculata were immunocytochemically detected after the introduction of these positively charged ligands.The described method is applicable for RNA and DNA probes. It has a sensitivity comparable to other non-autoradiographic methods, is relatively simple to perform and can be carried out with ordinary laboratory chemicals.This investigation was supported by the Netherlands Foundation for Medical Research Fungo (grant nr 13-54-21)     

A new hybridocytochemical method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. I. Stability of the mercury-sulfhydryl bond and influence of the ligand structure on immunochemical detection of the hapten

The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.     

Mercurated nucleic acid probes, a new principle for non-radioactive in situ hybridization

This report describes the localization of specific nucleic acid sequences in interphase nuclei and metaphase chromosomes by a new hybridocytochemical method based on the use of mercurated nucleic acid probes. After the hybridization a sulfhydryl-hapten compound is reacted with the hybrids formed. A number of such ligands were synthesized and tested. A fluorescyl ligand could be used for the direct visualization of highly repetitive sequences. For indirect immunocytochemical visualization trinitrophenyl ligands were found to be more sensitive than biotinyl analogues. These ligands were applied for the detection of target sequences in metaphase chromosomes and interphase nuclei of somatic cell hybrids, human lymphoid cell lines and blood cell cultures. The sequences were in the range of high to low copy numbers. The lower limit of sensitivity is indicated by the visualization of two human unique DNA fragments (40 and 15.6 kb) in human metaphases. The method is rapid, gives consistent results and can be used for both RNA and DNA probes. Other potentials of the new principle are discussed.     

Effect of ligand-induced conformational changes on the reactivity of specific sulfhydryl residues in rat brain hexokinase

Rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) contains 21 cysteine residues. On the basis of the amino acid sequence of the enzyme, these are predicted to be distributed among 14 peptides produced by tryptic digestion. Ten of these peptides, containing cysteine residues derivatized by reaction with the specific sulfhydryl reagent 2-bromoacetamido-4-nitrophenol have been identified in HPLC peptide maps; the four missing peptides are predicted to be relatively large and hydrophobic in character, properties that may have prevented their detection under the present conditions. The sequences encompassed by the 10 identified peptides include 12 of the 21 cysteine residues in the enzyme. The relative reactivity of these sulfhydryl groups with 2-bromoacetamido-4-nitrophenol has been assessed, and is in general accord with what might be predicted on the basis of their accessibility in the previously proposed structure for this enzyme. The effect of various ligands on reactivity of identified sulfhydryl groups has been determined; unique patterns of altered reactivity, resulting from ligand-induced conformational changes, have been observed. Biphasic effects were observed with increasing concentrations of either glucose 6-phosphate (Glc-6-P) or Pi. In both cases, decreased reactivity of sulfhydryls in the N-terminal half of the molecule was observed at low concentrations of the ligand, while further increase in ligand concentration resulted in decreased reactivity of sulfhydryl groups in the C-terminal half. In contrast, sulfhydryls in both N- and C-terminal halves were protected concomitantly by increasing concentrations of Glc. These results are consistent with previous studies that indicated (a) the existence of two sites for binding of Glc-6-P or Pi, a high affinity site in the N-terminal half and a site with lower affinity in the C-terminal half of the brain hexokinase molecule, and (b) binding of Glc to a single site located in the C-terminal half but evoking conformational effects throughout the molecule; the glucose analog, N-acetylglucosamine, previously shown to have more limited effects on conformation, affected reactivity of sulfhydryl groups only in the C-terminal half of the molecule. As reflected by effects on reactivity of sulfhydryl groups, conformational changes induced by binding of nucleotides depends markedly on the specific nature of the purine or pyrimidine base as well as the length and chelation status of the polyphosphate side chain. These results focus attention on specific regions of the molecule (the immediate environment of the sulfhydryl groups) that are affected by the binding of these ligands.     

Chloroperoxidase: P-450 type absorption in the absence of sulfhydryl groups.

The oxidation state of the two half-cystine residues in the native ferric form of chloroperoxidase and in the reduced ferrous chloroperoxidase has been examined in order to evaluate the role of sulfhydryl groups as determinants of P-450 type spectra. M?ssbauer and optical spectroscopy studies indicate that the ferrous forms of P-450cam and chloroperoxidase have very similar or identical heme environments. Model studies have suggested that sulfhydryl groups may function as axial ligands for developing P-450 character. However, chemical studies involving both sulfhydryl reagents and amperometric titrations show that neither the ferric nor the chemically produced ferrous forms of chloroperoxidase contain a sulfhydryl group. These results rule out the hypothesis that sulfhydryl groups are unique components for P-450 absorption characteristics. The optical and electron paramagnetic resonance (EPR) spectra of the nitric oxide complex of chloroperoxidase have been obtained and compared to those of myoglobin, hemoglobin, and cytochrome c and horseradish peroxidase. The EPR spectrum of the NO-ferrous chloroperoxidase complex, which is similar to that of cytochrome P-450cam, does not show the extra nitrogen hyperfine structure which appears to be characteristic of those hemoproteins which have a nitrogen atom as an axial heme ligand.     

Novel trifunctional carrier molecule for the fluorescent labeling of haptens

We developed a novel trifunctional carrier molecule for the synthesis of hapten-fluorophore conjugates as reporter molecules in immunoassays. This carrier eliminates some of the disadvantages associated with currently used fluorophore-labeling procedures including high nonspecific binding. The backbone of the carrier consists of the 21 amino acid residues of the insulin A-chain molecule. This polypeptide provides a single site (terminal amino group) for covalent coupling of the hapten, three carboxyl groups for the attachment of fluorophores, and four sulfhydryl groups for derivatization with hydrophilic residues to compensate for the hydrophobic effect of the attached fluorophores. The sites for fluorophore attachment are 4, 17, and 21 amino acids away from the hapten attachment site. This spatial separation minimizes quenching of the fluorescence signal due to interaction of the fluorophores with each other and with the attached hapten. In this study, 2,4-dinitrophenol (DNP) was selected as model hapten, fluorescein as label, and S-sulfonate groups as hydrophilic residues. The properties of the DNP-insulin A-chain-fluorescein conjugate (DNP-Ins-Fl) were compared to those of a DNP derivative labeled with a single fluorescein moiety via a small lysine spacer (DNP-Lys-Fl). The DNP-Ins-Fl conjugate exhibited a 3-fold lower nonspecific adsorption to immobilized non-immune IgG contributing to an approximately 3-fold more efficient displacement from the binding sites of an immobilized monoclonal anti-DNP antibody by the antigen DNP-lysine. Furthermore, at equimolar concentrations the DNP-Ins-Fl generated a 2.6-fold higher fluorescent signal than DNP-Lys-Fl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.     

Synthesis of a stable and specific surface plasmon resonance biosensor surface employing covalently immobilized peptide nucleic acids

Biosensors allow the real-time and label-free observation of biochemical reactions between various ligands including antigen-antibody reactions and nucleic acids hybridizations. In our studies, we used a surface plasmon resonance biosensor to elucidate the hybridization characteristics of a peptide nucleic acid (PNA) ligand immobilized on sensor surfaces either through covalent or streptavidin-biotin coupling. A biotin-labeled PNA was employed in the latter approach whereas the covalent immobilization included the following steps: A maleimide group was attached to the N-terminal of the PNA using N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). To generate free thiol groups for coupling, a carboxylated dextran matrix of the sensor surface was activated with N-hydroxysuccinimide (NHS) and N-ethyl-N'-(dimethylaminopropyl)-carbodiimide (EDC) and thiolated by addition of cystamine dihydrochloride followed by reduction with 1, 4-dithioerythrite (DTE). Finally, the modified PNA was coupled to the sulfhydryl groups of the activated dextran matrix. Repetitive hybridizations of a single-stranded synthetic DNA oligomer to the PNAs demonstrated the superior stability of covalent immobilization compared to noncovalent immobilization. Differentiation of point mutations in the analyte molecule was accomplished at 40 degrees C using guanidine thiocyanate concentrations of 1.5-1.7 M. In further experiments, we showed that a perfectly matched PNA allows the detection of a single-stranded DNA at a sensitivity of less than 1% in a background of single-stranded DNA having a single C to T point mutation in the region complementary to the PNA. Consequently, covalently bound PNAs provide a stable and reproducible environment for the development of mutation-specific DNA analysis assays.     

Selective isolation of mercurated DNA by affinity chromatography on thiol matrices

A method for isolating picomole quantities of nascent mercurated DNA from a mixture of cellular nucleic acids using affinity chromatography on thiol-agarose is described. Analysis of mercurated DNA (HgDNA) isolated in the presence of in vivo-labeled cellular RNA or in vitro-synthesized RNA showed a low level of RNA contamination, about 0.04-0.16%, in the HgDNA. Comparative binding studies on different thiol matrices showed that the efficiency of binding of HgDNA was related to the nature but not to the SH content of the matrix used. Another important parameter for binding was the structure of HgDNA. The recovery was 98% with large nascent HgDNA sedimenting at about 30 S, whereas for short pulse-labeled single-stranded HgDNA (20-50 nucleotides long), the maximum recovery was 60%. The effect of the structure of HgDNA on the binding to the thiol matrix was probed using a variety of well-defined mercurated structures obtained from phage DNA and their restriction fragments. For DNA containing one 5-mercuricytidine 5'-triphosphate (HgdCMP) residue at each 3'-end, short fragments (size range, 230-510 bp) were bound quantitatively. With larger fragments (size range, 490-1100 bp), the binding decreased progressively with increasing size. DNA fragments larger than 1060 bp did not bind to the matrix. Single-stranded DNA containing only one HgdCMP at one end did not bind to the matrix even in the size range 200-1100 nucleotides. In contrast, continuous stretches of HgdCMP residues in one strand or short stretches of HgdCMP residues at random in both strands permit quantitative binding irrespective of size.     

Introduction of sulfhydryl groups into proteins at carboxyl sites

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.     

Affinity electrophoresis and its applications to studies of immune response

Affinity electrophoresis (AEP) is a useful technique for separation of biomolecules such as plasma proteins, enzymes, nucleic acids, lectins, receptors, and extracellular matrix proteins by specific interactions with their ligands in electric fields and for the determination of dissociation constants for those interactions. Two-dimensional affinity electrophoresis (2-D AEP), which was newly developed by a combination of isoelectric focusing with AEP, has been used for studies on immune response to haptens. Antihapten antibodies, which were induced by immunization of a mouse with the hapten-conjugated bovine serum albumin, were separated by 2-D AEP into a large number of groups of IgG spots with a few microliters of antiserum. Each group of spots showed an identical affinity for the hapten but different isoelectric points as in the case of monoclonal antibodies specific to the hapten. This enabled us to study the diversification and affinity maturation of antihapten antibodies in the course of immunization of a single mouse. Furthermore, effects of a carrier and a hapten array on the production of antihapten antibodies and the cause of charge heterogeneity of monoclonal antibodies were also examined to understand the molecular basis of the immune response in vivo.     

A simple linear transform for the Folin-Lowry protein calibration curve to 1.0 mg/ml

The stability of covalently mercurated DNAs during DNA:DNA reassociation, heteroduplex recovery on sulfhydryl-Sepharose, and S₁ nuclease digestion under a variety of solvent and temperature conditions is described. The nonspecific loss of²⁰³Hg from mercurated DNA can be minimized by use of aqueous formamide solvents in reassociation experiments and by minimizing exposure to sulfhydryl reagents and temperatures above 35°C. Single-stranded DNA is shown to be more sensitive to demercuration than is native, duplex DNA.     

The reaction of sulfhydryl groups of sodium and potassium ion-activated adenosine triphosphatase with N-ethylmaleimide. The relationship between ligand-dependent alterations of nucleophilicity and enzymatic conformational states

The reaction between N-ethylmaleimide and (Na+ + K+)-ATPase, performed under ligand conditions which produce each of the kinetic states of the enzyme and their associated conformational forms, was examined through an analysis of the inhibition of enzymatic activity and the incorporation of radiolabeled reagent into the enzyme. The inactivation reactions displayed pseudo-first order kinetics with respect to the concentration of active enzyme, indicating that the loss of activity is associated with the alkylation of a unique sulfhydryl group. In the absence of enzyme phosphorylation, the nucleophilicity of this sulfhydryl group is affected primarily by the nature of the monovalent cation present and does not correlate with the conformational state. A method for determining the actual concentration and specific radioactivity of radiolabeled N-ethylmaleimide during the reaction with (Na+ + K+)-ATPase was developed, allowing the measurement of the total reactive sulfhydryl groups of native (Na+ + K+)-ATPase under conditions identical with those of the inactivation studies. The labeling of the enzyme complex is associated almost exclusively with the large polypeptide, which contains four sulfhydryl groups which react with this reagent. One of these residues is presumably the sulfhydryl responsible for inactivation of the enzyme. Two react stoichiometrically and rapidly with N-ethylmaleimide under all conditions. The nucleophilicity of the fourth sulfhydryl group is governed by the conformational state of the enzyme, but the alkylation of this residue does not result in loss of enzymatic activity.     

Chemical Modifications of Melatonin Receptors in Chicken Brain

Abstract: The membrane-bound or solubilized melatonin receptors were treated with protein-modifying agents under specific conditions and then assayed for ¹²⁵I-melatonin binding in order to obtain information on amino acids present in the ligand binding domain. The reagents specific for sulfhydryl ( N -ethylmaleimide and p -chloromercuribenzoate), guanidyl (phenylglyoxal), and amino groups (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1-fluoro-2,4-dinitrobenzene) inhibited ¹²⁵I-melatonin binding in a dose-dependent manner, and their effects were prevented by pretreatment with cold melatonin. These results suggest the presence of cysteine, arginine, and lysine residues in the melatonin binding domain. Decreased sensitivity of ¹²⁵I-melatonin binding to guanine nucleotides after N -ethylmaleimide pretreatment suggests the presence of another sulfhydryl group within the coupling domain between the receptor and G protein. Tyrosine reagents tetranitromethane, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, N -acetylimidazole, and p -nitrobenzenesulfonyl fluoride also inhibited ¹²⁵I-melatonin binding, and their effects were prevented by cold melatonin pretreatment; however, they were effective only at concentrations when cross-reaction with a sulfhydryl group may occur. Histidine reagent diethyl pyrocarbonate inhibited ¹²⁵I-melatonin binding in a dose-dependent manner, and its action was reversed by cold melatonin. However, diethyl pyrocarbonate had a smaller effect in a solubilized receptor preparation and, therefore, it could have modified a site remote from the ligand binding site. Our data do not suggest the presence of tryptophanyl, aspartic, or glutamic residues at the ligand binding domain.