Synthesis and biological applications of two novel fluorescent proteins-labeling probes

Two novel chlorinated fluoresceins 2′,4′,5′,7′-tetrachloro-6-(5-carboxypentyl)-4,7-dichloro fluorescein succinimidyl ester (1G) and 2′,4′,5′,7′-tetrachloro-6-(3-carboxypropyl)-4,7-dichlorofluorescein succinimidyl ester (2G) were synthesized as fluorescent probes for labeling proteins. Structures of target compounds and intermediates were determined via IR, MS, ¹H NMR and element analysis. The investigation in immunofluorescence histochemistry showed them had strong fluorescence, high photostability and good biocompatibility.     

Dependence of properties of polyclonal antibodies to (2'-5')-oligoadenylates on the method of hapten binding to an immunogen

To elucidate the antibody-(2'-5')oligoadenylate relation to the mode of the hapten-immunogen conjugation, a new (2'-5')oligoadenylic acid trimer derivative containing a 2'-terminal N6-(5-carboxypentyl)adenosine and its 125I-labeled immunogenic conjugate were synthesized. The immunization with this conjugate and with a conjugate based on the 2',3'-O-[1-(2-carboxyethyl)]ethylidene derivative of the (2'-5')triadenylic acid gave antisera with different affinities toward modified (2'-5')oligonucleotides. Epitopes involved in the (2'-5')oligomer-binding to different antisera were found.     

Lysine-derived fluorophores formed by autoxidation of linoleic acid

To obtain an insight into fluorophores formed in proteins during lipid peroxidation, a lysine residue analogue (N(alpha)-hippuryllysine) was exposed to autoxidation of linoleic acid catalyzed by iron(III)-EDTA and L-ascorbic acid. The reaction predominantly produced two fluorescent products, N,N'-bis[5-(N-benzoylglycylamino)-5-carboxypentyl]-2-hydroxy-2-pentyl-3-imino-l,2-dihydropyrrole (II) and N,N'-bis[5-(N-benzoylglycylamino)-5-carboxypentyl]-2-hydroxy-2-(7-carboxyheptyl)-3-imino-1,2-dihydropyrolle (I).     

Convenient synthesis of GOLD and MOLD and identification of their oxidation products in vitro and in vivo

Summary. Two Lys–Lys crosslinks, 1,3-bis-(5-amino-5-carboxypentyl)-1H-imidazolium (GOLD) and 1,3-bis(5-amino-5-carboxypentyl)-4-methyl-1H-imidazolium (MOLD) salts, have been synthesized by the reaction of imidazole or 4(5)-methyl imidazole with 5-(4-bromobutyl)-hydantoin followed by the hydrolysis of 1,3-substituted imidazolium derivatives by 6.0 N HCL at 110 °C. Treatment of GOLD and MOLD with hydrogen peroxide in acetic acid leads to MOLD oxidation only. The oxidation product of MOLD was detected in cataractous lens proteins.     

Reaction of N(alpha)-hippuryllysine with 2-hydroxyheptanal: a model for lysine-directed protein modifications by lipid peroxidation

2-Hydroxyheptanal (2-HH) is one of the major aldehydes derived from peroxidation of polyunsaturated fatty acids. In the present study, to obtain an insight into the contributions of 2-HH to protein modifications during lipid peroxidation, a lysine-containing dipeptide, N(alpha)-hippuryllysine (N-benzoylglycyl-L-lysine, BGL), was reacted with 2-HH at neutral pH. The products were characterized on the basis of LC/MS and NMR spectroscopy. The reaction afforded a 2:1 2-HH-lysine adduct, 1-[5-(N-benzoylglycylamino)-5-carboxypentyl]-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one (I). In addition, we obtained a 1:1 2-HH-lysine adduct, N-[5-(N-benzoylglycylamino)-5-carboxypentyl]-1-amino-2-heptanone (III). The treatment of the purified III with 2-HH produced I. On the other hand, when the reaction mixture was allowed prolonged standing, I was slowly oxidized to 1-[5-(N-benzoylglycylamino)-5-carboxypentyl]-4-butyl-5-pentyl-3-hydroxypyridinium (V). This conversion was strongly accelerated by the addition of copper(II) ion and 2,2'-bipyridyl. We propose here that the above series of conversions is the main pathway for the modification of lysine residues of proteins by 2-HH.     

High sensitive protein detection by hollow fiber membrane interface based protein enrichment and in situ fluorescence derivatization

A novel device, composed of a syringe pump for sample loading, a hydrophilic hollow fiber membrane interface for protein concentration and small molecules removal, and a centrifugation tube for buffer exchange, was designed for protein preconcentration and in situ fluorescence derivatization. With the outlet of the interface blocked, denatured proteins were continually introduced. Restricted by the membrane with the molecular weight cutoff (MWCO) of 3000 Da, proteins were concentrated within the membrane. However, denaturant and other small molecules, which might affect the further fluorescence derivatization, were driven out of the membrane. Then, the membrane with proteins restricted inside was directly put into the fluorescence derivatization buffer. Here, the water-soluble sulfo-3H-indocyanine dye, the active N-hydroxysuccinimide of 3H-indolium,1-(5-carboxypentyl)-2-[3-[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-5-sulfo-,monopotassium salt (sw-cy3-NHS), synthesized in our lab, was used for protein labeling. By such a method, the detection sensitivity of bovine serum albumin (BSA) was improved by nearly 200 folds, compared to that obtained by direct in-solution derivatization. Through the derivatization of a fraction of E. coli protein separated by reversed phase HPLC, proteins with low concentration were efficiently labeled, which indicated the potential merit of the developed method for the high sensitive detection of low abundance proteins.     

Ultrabright fluorescein-labeled antibodies near silver metallic surfaces

Fluorescein-labeled antibodies are widely used in clinical assays and fluorescence microscopy. The fluorescent signal per labeled antibody is limited by fluorescein self-quenching, which occurs when the antibody is heavily labeled with multiple fluoresceins. We examined immunoglobulin G (IgG) when labeled with 0.7 to about 30 fluoresceins per antibody molecule. The extent of self-quenching was decreased, and the signal increased, when the labeled antibody was in close proximity to metallic silver particles. Time-resolved measurements showed that the intensity increase was due in part to a silver-induced increase in the radiative decay rate. These results suggest the use of labeled antibodies conjugated to silver particles as ultrabright probes for imaging or analytical applications.     

Beta-oxidation of 2-ethyl-5-carboxypentyl phthalate in rodent liver

[7-14C]-2-Ethyl-5-carboxypentyl phthalate was isolated and purified from urine of rats given [7-14C]-di-(2-ethylhexyl) phthalate. This metabolite was shown to serve as a precursor for 2-ethyl-3-carboxypropyl phthalate in vivo. 2-Ethyl-5-carboxypentyl phthalate was oxidized to 2-ethyl-3-carboxypropyl phthalate in liver slices from control or, much more rapidly, from clofibrate-pretreated rats. Inhibition by KCN in liver slices from untreated rats, and strong inhibition by acrylate, suggested that formation of 2-ethyl-3-carboxypropyl phthalate involved mitochondrial beta-oxidation. The strong enhancement of the production of this compound by clofibrate (a very weak inducer for mitochondrial dehydrogenases), and strong inhibition by chlorpromazine suggested that peroxisomes may also be able to oxidize 2-ethyl-5-carboxypentyl phthalate. We were able to detect beta-oxidation of 2-ethyl-5-carboxypentyl phthalate to 2-ethyl-3-carboxypropyl phthalate using purified mitochondria, but strong phthalate monoester hydrolase activity observed during incubation of the former compound with purified peroxisomes made it impossible to determine whether 2-ethyl-3-carboxypropyl phthalate could be produced in the latter organelle or not. 2-Ethyl-5-carboxypentyl phthalate was such an inefficient substrate for beta-oxidation compared to palmitic acid that it is unlikely that it contributes significantly to the production of H2O2 in rats chronically exposed to di-(2-ethylhexyl) phthalate. Normal fatty acids are most likely to serve as the dominant substrates for peroxisomal beta-oxidase.     

General strategy for the preparation of membrane permeable fluorogenic peptide ester conjugates for in vivo studies of ester prodrug stability

To study ester prodrug stability properties in living cells we have conjugated fluorogenic esters to the cell membrane permeable peptide Arg9. The desired conjugates are prepared by coupling N-maleoyl amino acid esters of monoalkylated fluoresceins or fluorescein to TyrArg9Cys. The photophysical properties of the monoalkylated fluorescein derivatives are described.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.     

Continuous measurement of mitochondrial pH gradients in isolated hepatocytes by difference ratio spectroscopy

The pH gradient, delta pH, present across the inner mitochondrial membrane in isolated rat hepatocytes was continuously monitored with a novel spectroscopic technique that utilizes the weak acid fluorescein. Unlike most cytosolic pH indicators, such as 2',7'-bis(carboxyethyl)-5,(6)-carboxyfluorescein (BCECF), fluorescein freely distributes between the cytosolic and mitochondrial compartments. As is typical for weak acids, the distribution between these two compartments is governed by the magnitude of the pH gradient. Since fluorescein has two ionizable groups, the fluorescein dianion is concentrated in the mitochondrial compartment 100-fold per delta pH unit. In this compartment, fluorescein absorbance (or excitation) spectra are red-shifted about 6-8 nm in the matrix environment, as compared to the cytosolic dye at equivalent pH values. The combination of favorable mitochondrial accumulation and red-shifted spectra enables mitochondrial pH to be continuously monitored qualitatively in whole cells by dual wavelength spectroscopy (510 minus 540 nm). When the cytosolic pH is determined by independent means, the mitochondrial pH can be quantitated, based on the theoretical dependence of the fluorescein distribution ratio on delta pH, the ratio of cytosolic to mitochondrial volumes, and the known extinction coefficients for the dye in the cytosolic and mitochondrial compartments. The sensitivity of the method for following kinetic responses in mitochondrial pH is especially noteworthy; a 0.1-unit change in delta pH is easily distinguished, with a time resolution of less than a second.     

Monoclonal antibodies to queuine

Monoclonal antibodies specific for queuine have been prepared. Synthetic 9-(5-carboxypentyl)queuine (cp9Q) was conjugated with bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH), and the conjugate was used to immunize BALB/c mice by intraperitoneal and subcutaneous injection. Monoclonal antibodies were subsequently obtained by fusion of spleen cells and the mouse myeloma cell line X63Ag8U1. An enzyme-linked immunoabsorbent assay (ELISA) using o-phenylenediamine as peroxidase substrate was used for screening of clones and characterization of antibodies. Inhibition experiments with various homologous nucleosides revealed that the monoclonal antibody designated as 2D8E6 has no cross-reactivity with guanosine, adenosine or 7-methylguanosine.     

Probing of the spliceosome with site-specifically derivatized 5' splice site RNA oligonucleotides.

We have developed a site-specific chemical modification technique to incorporate a photoreactive azidophenacyl (APA) group at designated internal positions along the RNA phosphodiester backbone. Using this technique, we have analyzed interactions of the 5' splice site (5'SS) RNA within the spliceosome. Several crosslinked products can be detected within complex B using the derivatized 5'SS RNAs, including U6 snRNA, hPrp8p, and 114-, 90-, 70-, 54-, and 27-kDa proteins. The 5'SS RNAs derivatized at intron positions +4 to +8 crosslink to U6 snRNA, confirming the previously reported pairing interaction between these sequences. hPrp8p and p70 are crosslinked to the 5'SS RNA when the APA is placed within the 5' exon. Finally, a set of unidentified proteins, including p114, p54, and p27, is detected with the 5'SS RNA derivatized at intron positions +4 to +8. Introduction of the bulky APA group near the 5'SS junction (positions -2 to +3) strongly interferes with complex B formation and thus no APA crosslinks are observed at these positions. Together with our earlier observation that hPrp8p crosslinks to the GU dinucleotide at the 5' end of the intron, these results suggest that the inhibitory effect of APA results from steric hindrance of the hPrp8p:5'SS interaction. Unexpectedly, thio-modifications within the region of the 5'SS RNA that is involved in base pairing to U6 snRNA strongly stimulate complex B formation.     

Carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, from yellow sweet potato "Benimasari", Ipomoea batatas Lam

A series of carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, named ipomoeaxanthins A (1), B (2), C1 (3) and C2 (4) were isolated from the flesh of yellow sweet potato "Benimasari", Ipomoea batatas Lam. Their structures were determined to be (5R,6S,3'R)-5,6-dihydro-beta,beta-carotene-5,6,3'-triol (1), (5R,6S,5'R,6'S)-5,6,5',6'-tetrahydro-beta,beta-carotene-5,6,5'6'-tetrol (2), (5R,6S,5'R,8'R)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (3), and (5R,6S,5'R,8'S)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (4) by UV-Vis, NMR, MS and CD data.     

Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human α-thrombin

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein aminelabeled thrombin. Two fluorescein amines, 4-(N-6-aminoethyl thioureal)-fluorescein and 4-(N-6-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human α-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogin to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4°C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37°C following binding at 4° C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.     

Steps in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid in methanogenic bacteria

The biosynthetic steps involved in the conversion of alpha-ketosuberate to 7-mercaptoheptanoic acid were studied in cell-free extracts of methanogenic bacteria. The pathway was established by measuring the incorporation of stable isotopically labeled precursors into the S-methyl ether methyl ester derivative of the enzymatically generated 7-mercaptoheptanoic acid by using gas chromatography-mass spectrometry (GC-MS). Quantitation of the 7-mercaptoheptanoic acid produced in the incubations with the substrates was accomplished by using an internal standard of 6-mercaptohexanoic acid. [4,4,6,6-2H4]-2-Oxosuberic acid, [7-2H]-7-oxoheptanoic acid, [2-2H]-2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid, and S-(6-carboxyhexyl)cysteine were each shown to be converted to 7-mercaptoheptanoic acid. Incubation of cell extracts with a mixture of 2(RS)-(5-carboxypentyl)thiazolidine-4(R)-carboxylic acid and [2-2H]-2-(RS)-(5-carboxypentyl)-[34S]thiazolidine-4(R)-carboxylic acid showed that both 34S and 2H are incorporated into the 7-mercaptoheptanoic acid but only after separation of the cysteine from the [7-2H]-7-oxyheptanoic acid portion of the molecule. Furthermore, the sulfur from the cysteine was incorporated into the thiol only after its elimination from the cysteine and subsequent mixing with an unlabeled sulfur source which had a molecular weight of sufficient size that it was excluded from Sephadex G-25. Hydrogen sulfide was found to supply the sulfur for the production of the 7-mercaptoheptanoic acid in a reaction that was shown to obtain its reducing equivalents from hydrogen via an F420-dependent hydrogenase.     

Hypersensitive substrate for ribonucleases.

A substrate for a hypersensitive assay of ribonucleolytic activity was developed in a systematic manner. This substrate is based on the fluorescence quenching of fluorescein held in proximity to rhodamine by a single ribonucleotide embedded within a series of deoxynucleotides. When the substrate is cleaved, the fluorescence of fluorescein is manifested. The optimal substrate is a tetranucleotide with a 5',6-carboxyfluorescein label (6-FAM) and a 3',6-carboxy-tetramethylrhodamine (6-TAMRA) label: 6-FAM-dArUdAdA-6-TAMRA. The fluorescence of this substrate increases 180-fold upon cleavage. Bovine pancreatic ribonuclease A (RNase A) cleaves this substrate with a k (cat)/ K (m)of 3.6 x 10(7)M(-1)s(-1). Human angiogenin, which is a homolog of RNase A that promotes neovascularization, cleaves this substrate with a k (cat)/ K (m)of 3. 3 x 10(2)M(-1)s(-1). This value is >10-fold larger than that for other known substrates of angio-genin. With these attributes, 6-FAM-dArUdAdA-6-TAMRA is the most sensitive known substrate for detecting ribo-nucleolytic activity. This high sensitivity enables a simple protocol for the rapid determination of the inhibition constant ( K (i)) for competitive inhibitors such as uridine 3'-phosphate and adenosine 5'-diphos-phate.     

Preparation of anomeric pairs of 1-thioglycosides: use of anomerization catalyzed by boron trifluoride

Anomeric pairs of some alkyl 1-thioaldopyranosides of d-galactose, d-glucose, d-mannose, 2-acetamido-2-deoxy-d-glucose, 2-acetamido-2-deoxy-d-galactose, and l-fucose were prepared. The per-O-acetylated, 1,2-trans anomers of 6-(trifluoroacetamido)hexyl 1-thioaldopyranosides and 5-(methoxycarbonyl)pentyl 1-thioaldopyranosides were anomerized with boron trifluoride in dichloromethane. The anomeric mixtures were then separated by chromatography, using columns of either silica gel or an ion-exchange resin. De-blocking of the separated compounds provided pure anomers of 6-aminobexyl 1-thioaldopyranosides or 5-carboxypentyl 1-thioaldopyranosides. The aglycons of the latter glycosides were further extended by reaction with aminoacetaldehyde diethyl acetal, which, after deacetalization of the products, provided an ω-aldehydo group. These series of glycosides could be readily coupled to proteins or solid matrices.     

Interaction of muscle glycogen phosphorylase b with riboflavin, its tetraacetyl derivative and their analogs

The interaction of rabbit skeletal muscle glycogen phosphorylase b with riboflavin, 2',3',4',5'-tetraacetylriboflavin and their analogues, containing different substituents in the positions 6, 8 and 8 alpha, has been studied. Dissociation constant for the complex of the enzyme and riboflavin was determined to be 12.5 microM (pH 6.8; 20 degrees C) by sedimentation velocity method. Riboflavin and its analogues have been found to inhibit glycogen phosphorylase b. The inhibitor half-saturation concentration values increase in the following order: riboflavin (18 microM), 8-methoxy(nor)rifoblavin (23 microM), 8 alpha-bromo-2',3',4',5'-tetraacetylriboflavin (40 microM), 6-bromoriboflavin (40 microM), 8 alpha-hydroxyriboflavin (60 microM), 8-hydroxy(nor)riboflavin (90 microM), 8 alpha-(gamma-carboxypropylamino-2',3',4',5'-tetraacetylriboflav in (90 microM), 8 alpha-[p-(5-ethyl-1,3,4-thiodiazol-2-ylsulfamido)phenylamino ]- 2',3',4',5'-tetraacetylriboflavin (100 microM), 8 alpha-(L-methionyno)-2',3',4',5'-tetraacetylriboflavin (120 microM), 8 alpha-[p-(thiazol-2-ylsulfamido)phenylamino]- 2',3',4',5'-tetraacetylriboflavin (140 microM), 8 alpha-(p-sulfamidophenylamino)-2',3',4',5'-tetraacetylriboflavi n (180 microM), 8 alpha-(p-carboxyphenylamino)-2',3',4',5'-tetraacetylriboflavin+ ++ (210 microM), 2',3',4',5'-tetraacetylriboflavin (250 microM), 8 alpha-(L-homoserino)-2',3',4',5'-tetraacetylriboflavin (340 microM), 8 alpha-(L-glutamo)-2',3',4',5'-tetraacetylriboflavin (360 microM). The existence of glycogen phosphorylase b complexes with riboflavin and its analogues has been proved by methods of absolute and difference spectrophotometry.