Ammonium 4-chloro-7-sulfobenzofurazan: a new fluorigenic thiol-specific reagent

This paper describes the synthesis and study of a new fluorigenic, thiol-specific reagent, ammonium 4-chloro-7-sulfobenzofurazan, which is readily prepared by sulfonation of 4-chlorobenzofurazan. Evaluation of ammonium 4-chloro-7-sulfobenzofurazan was undertaken using glutathione as a model thiol peptide and bovine serum albumin and jackbean urease as thiol-containing proteins. Thiol specificity of this reagent was established using various amino acids and peptides including l-cysteine, l-lysine, l-histidine, l-tyrosine, glutathione, and oxidized glutathione. Nitrogen and sulfur derivatives of ethanediol and acetic acid were also investigated. Optimum conditions for the thiol labeling reaction have been investigated using the parameters of pH, buffer type, time, temperature, and relative concentrations. Under appropriate conditions the fluorescence produced by the reaction of ammonium 4-chloro-7-sulfobenzofurazan is linearly related to thiol concentration. The fluorescence intensity of 7-sulfobenzofurazan thiol derivatives are considerably greater than the corresponding 7-nitrobenzofurazan derivatives in both aqueous and aprotic solvents, rendering this reagent highly sensitive. Our preliminary experiments with proteins labeled with ammonium 4-chloro-7-sulfobenzofurazan have shown the probe to be removable by thiolysis with excess 2-mercaptoethanol.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

A versatile, highly reactive, cross-linking reagent: 2,2'-sulfonylbis[3-methoxy-(E,E)-2-propenenitrile]

Adequate aqueous stability and cross-linking ability of the novel title reagent, recently discovered in this laboratory, have been demonstrated by comparison of its rate of hydrolysis with the rate of reaction with an amine nucleophile and by cross-linking deoxy- and oxyhemoglobins, as an example.     

A chemical-modification approach to the olfactory code. Studies with a thiol-specific reagent

The effects of thiol-specific reagents on the amplitude of the electro-olfactogram (E.O.G.) responses elicited from frog olfactory mucosa by pulses of odorant vapours was studied. The impermeant thiol-specific reagent mersalyl [(3-{[2-(carboxymethoxy)-benzoyl]amino}-2-methoxypropyl)hydroxymercury monosodium salt] brings about a rapid decrease in the E.O.G. signal obtained with the odorant pentyl acetate. The extent of the decrease is proportional to the concentration of the mersalyl applied and the effect of the reagent is partially but incompletely reversed by treatment of the labelled mucosa with dithiothreitol. The sites labelled by mersalyl can be protected by pretreating the mucosa with a dilute solution of the odorant pentyl acetate and leaving the solution in contact with the tissue after the addition of mersalyl. When the protecting odorant is washed out of the tissue, the original E.O.G. amplitude is regained. Pentyl acetate applied to the mucosa protected the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: n-butyric acid, n-butyl acetate, phenylacetaldehyde and cineole (1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane). The pentyl acetate applied to the mucosa failed to protect the E.O.G. response to vapour pulses of the following odorants from the effects of mersalyl: butan-1-ol, benzyl acetate, nitrobenzene, beta-ionone and linalyl acetate. The significance of the differential protection effects for the odour-quality-coding mechanism in the olfactory primary neurons is discussed. It is suggested that the olfactory code at this level of the olfactory system may be elucidated by chemical-modification methods.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC): a heterobifunctional reagent for immobilization of biomolecules on glass surface

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)cyclohexane-1-carboxamide (TPICC) has been developed by a simple 'two step reaction' for the preparation of bioconjugates and immobilization of biomolecules such as oligonucleotides, peptides and proteins on the glass surface. The isothiocyanate functionality at one end of the reagent, TPICC was found specific for the ligands having either aminoalkyl (RNH(2)) or mercaptoalkyl (R-SH) functionality. The synthesis of bioconjugates with the reagent was achieved through its isothiocyanate functionality at one end via the formation of stable thiourea linkage with aminoalkyl and dithiocarbamate linkage with mercaptoalkyl derivatives. The triethoxysilyl functionality of the reagent has been utilized for specific coupling with the virgin glass surface by a very simple methodology.     

3-(p-hydroxyphenyl)propionic acid as a new fluorogenic reagent for amine oxidase assays

A detailed procedure of a new and extremely sensitive fluorometric assay for amine oxidases is presented. Hydrogen peroxide, produced by the oxidase reaction, reacted with 3-(p-hydroxyphenyl)propionic acid in the presence of peroxidase to yield a fluorescent compound by which enzyme activity could be determined. The enzyme reaction was terminated by NaOH solution, which increased the fluorescence intensity three- to fivefold. The detection limit thus obtained was as little as 0.02 nmol. The alkalinization also contributed to stopping the enzyme reaction and to the clarification of assay mixtures containing turbid enzyme preparations.     

Pyrenyldiazomethane, a versatile reagent for nucleotide phosphate alkylation

Pyrenyldiazomethane was shown to react quantitatively and selectively at phosphate with 2'-, 3'-, and 5'-nucleotide phosphates incorporating the different nucleic bases.     

Synthesis, spectral properties, and use of Thiodan, a new thiol-specific fluorescent reagent

A new fluorescent reagent, Thiodan, containing the -SO₂-S- function, is described. It can be utilized in protein chemistry as a reversible fluorescent labeling agent of cysteine residues. Thiodan is also shown to be useful for titration of sulfhydryl groups, due to the intensely fluorescent sulfinate anion released during the reaction.     

8-N(3)-3'-biotinyl-ATP, a novel monofunctional reagent: differences in the F(1)- and V(1)-ATPases by means of the ATP analogue

A novel photoaffinity label, 8-N(3)-3'-biotinyl-ATP, has been synthesized. The introduction of an additional biotin residue is advantageous for easy detection of labeled proteins. This could be first tested by reaction with the F(1)-ATPase from the thermophilic bacterium PS3 (TF(1)). UV irradiation of TF(1) in the presence of 8-N(3)-3'-biotinyl-ATP results in a nucleotide-dependent binding of the analogue in the noncatalytic alpha and the catalytic beta subunits of TF(1), demonstrating the suitability of this analogue as a potential photoaffinity label. Reaction with the V(1)-ATPase, however, led to labeling of subunit E, which has been suggested as a structural and functional homologue of the gamma subunit of the F-ATPases. MALDI-TOF mass spectrometry has been used to map the regions of subunit E involved in the binding of 8-N(3)-3'-biotinyl-ATP.     

New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent.

A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.     

Heterogeneity of protein labeling with a fluorogenic reagent, 3-(2-furoyl)quinoline-2-carboxaldehyde

Fluorogenic reagents are used for protein labeling when high-sensitivity fluorescence detection is required. Similar to traditional labeling with activated fluorescent dyes, such as fluorescein isothiocyanate, a fluorogenic reaction is expected to change the physical-chemical properties of proteins. Knowledge of these changes may be essential for efficient separation and identification of labeled proteins. Here we studied the effect of labeling of myoglobin with a fluorogenic reagent on the acid-base properties of the protein. The fluorogenic reagent used was 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ). In slab-gel isoelectric focusing, we found that the labeling reaction generated at least six species with pI values lower than that of non-labeled myoglobin. These species can be identified as products of progressive labeling of myoglobin with one to six FQ molecules. The same series of FQ-labeled species were observed when the reaction products were analyzed by capillary zone electrophoresis. The comparison of experimental and theoretical pI values allowed us to elucidate the labeling pattern--the number of FQ molecules corresponding to each labeled product detected by isoelectric focusing.     

P-Azidophenacyl bromide, a versatile photolabile bifunctional reagent. Reaction with glyceraldehyde-3-phosphate dehydrogenase.

The synthesis of the photochemically labile bifunctional reagent p-azidophenacyl bromide (1) is described. This compound may be covalently attached to a known site of an enzyme or other macromolecule by nucleophilic displacement at the alpha-bromo ketone moiety. Subsequent irradiation of the bound reagent gives a nitrene which may insert into a second portion of the enzyme forming a cross-link. Regeant 1 proved to be an excellent inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase.     

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)- alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make some comparisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, overall, comparable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non- reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta- glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl- hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.      

Photolabeling of myelin basic protein in lipid vesicles with the hydrophobic reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine

The hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine([125I]TID) was used to label myelin basic protein or polylysine in aqueous solution and bound to lipid vesicles of different composition. Although myelin basic protein is a water soluble protein which binds electrostatically only to acidic lipids, unlike polylysine it has several short hydrophobic regions. Myelin basic protein was labeled to a significant extent by TID when in aqueous solution indicating that it has a hydrophobic site which can bind the reagent. However, myelin basic protein was labeled 2-4-times more when bound to the acidic lipids phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and cerebroside sulfate than when bound to phosphatidylethanolamine, or when in solution in the presence of phosphatidylcholine vesicles. It was labeled 5-7-times more than polylysine bound to acidic lipids. These results suggest that when myelin basic protein is bound to acidic lipids, it is labeled from the lipid bilayer rather than from the aqueous phase. However, this conclusion is not unequivocal because of the possibility of changes in the protein conformation or degree of aggregation upon binding to lipid. Within this limitation the results are consistent with, but do not prove, the concept that some of its hydrophobic residues penetrate partway into the lipid bilayer. However, it is likely that most of the protein is on the surface of the bilayer with its basic residues bound electrostatically to the lipid head groups.