A fluorescence energy transfer study of lecithin-cholesterol vesicles in the presence of phospholipase C.

We demonstrate F?rster resonance energy transfer from dehydroergosterol to dansylated lecithin in lecithin-cholesterol vesicles and characterize the vesicles in the presence of the pro-nucleating enzyme, phospholipase C (PLC). Exposure to phospholipase C causes a temporary decrease in the dehydroergosterol to dansyl fluorescence ratio followed by an increase to and above the initial value. The temporary decrease in the fluorescence ratio results from an increase in the dansylated lecithin intensity that coincides with a dansyl blue shift. The extent of the blue shift correlates with the level of diacylglycerol generated in situ by PLC, suggesting an increased association between dansylated lecithin and cholesterol as membrane fluidity increases and membrane polarity decreases. The subsequent increase in the fluorescence ratio results from both an increase in the dehydroergsterol intensity and a concomitant decrease in the dansylated lecithin intensity of equal magnitude. This signifies a reduction in energy transfer from dehydroergosterol to dansylated lecithin and indicates an increased separation between the two fluorophores. The increase in the fluorescence ratio persists beyond the time scales for vesicle aggregation and fusion, as measured by turbidity, and precedes the onset of macroscopic cholesterol crystals observed with an optical microscope. Thus, the increased separation between dehydroergosterol and dansylated lecithin is consistent with a mechanism of cholesterol nucleation from the vesicles. Moreover, the onset and rate of increase in the fluorescence ratio correlate with the cholesterol:lecithin mole ratio of the vesicles. Fluorescence energy transfer from dehydroergosterol to dansylated lecithin therefore shows potential as a methodology for measuring cholesterol nucleation in model bile.     

Simultaneous liquid chromatography determination of polyamines and arylalkyl monoamines

The diamines putrescine (PUT) and diaminopropane (DAP), the polyamines spermidine (SPD) and spermine (SPM), and the arylalkyl amines phenethylamine (PEA), tyramine (TYR), dopamine (DA), and salsolinol (SAL) were dansylated and baseline separated by LC using a Waters ODS-2 column. The dansyl derivatives were detected by fluorescence (lambda(ex): 337 nm; lambda(em): 520 nm). Besides the amine function, the phenolic OH groups of TYR, DA, and SAL were also dansylated (LC-MS, formation of N,O-didansyl [TYR] and N,O,O'-tridansyl derivatives [DA and SAL]). Calibration curves revealed response factors being appreciably lower for (N,O-didansyl) aminophenol TYR and (N,O,O'-tridansyl) DA and SAL than for N-dansylamines. However, the method is suitable as a cheap alternative to LC-MS for the simultaneous determination of polyamines and arylalkyl amines of large quantities of samples.     

Fluorescent-labeled collagen: Age-related differences and fluorescence changes during the lag phase of fibril formation

Acid-soluble collagens isolated from young and old rat tail tendon were fluorescent-labeled with dansyl hydrazine, which is capable of reacting with aldehyde groups in collagen. The dansyl fluorescence of aged collagen exhibited a weak peak at 525 nm, whereas that of young collagen had a stronger broad peak at 500 nm. Fibril formation in vitro was partially inhibited in these dansylated collagens. During the turbidity lag phase, the dansyl fluorescence was found to increase (30–50%), also shifting to 485 nm. These changes reveal the telopeptide conformation changes occurring during this period. A new fluorescence peak at 420 nm also increased during fibril formation. When the dansylated collagen was irradiated in air with uv light (340 nm), a rapid decrease of the dansyl fluorescence with a concurrent shift to 490 nm occurred. Also, the formation of fibrils was further inhibited. With increasing temperature, the dansyl fluorescence of young collagen decreased, whereas that of old collagen substantially increased, particularly at the denaturation temperature around 38°C. After denaturation, both fluorescences became similar in their intensity and position (490 nm). These findings are discussed in connection with both age-related structural changes of collagen and the mechanism of fibril formation.     

Determination of phenylalanine in serum using reversed-phase liquid chromatography and fluorescence detection

A liquid chromatography procedure is reported for determining phenylalanine in small volumes of serum. A 10-μl volume of serum was deproteinized with ethanol and an aliquot was derivatized with dansyl chloride reagent. The dansylated phenylalanine and the norleucine internal standard were separated using reversed-phase chromatography and measured with a fluorescence detector. Linearity was excellent over the range 50–800 mg/l. Within-run precision was better than 4%. Total analysis time including chromatography was approximately 40 min. As little as 300 pg of dansylated phenylalanine was detected.     

Oxidative side reactions during dansylation of SH-compounds.

Dansyl chloride can act as an oxidizing agent on compounds which are easily oxidized. During the reaction of mercaptanes, e.g. cysteine, homocysteine, cysteamine, with dansyl chloride, the corresponding disulfides are formed and dansyl chloride is reduced to 5-dimethylaminonaphthalene 1-sulfinic acid. This reaction is so rapid that the normal dansylation can take place only after complete oxidation of all SH-compounds. Therefore only the dansyl derivatives of the corresponding disulfides are formed during normal dansylation of SH-compounds. If different SH-compounds are present in the reaction mixture mixed disulfides are formed as well. These can be separated by microchromatography on 3 X 3 cm micropolyamide sheets. Dependent on the concentration of dansyl chloride, 6 or even 15 different dansylated disulfides are formed from three different SH-compounds so that interpretation of these chromatograms is difficult. The actual dansylmercaptanes (e.g., dansylcysteine, dansylhomocysteine, dansylcysteamine) can be prepared by reduction of the dansylated disulfides with suitable reducing agents.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Fluorescence analysis of denaturation and reassembly of dansylated creatine kinase

Upon exposure of rabbit muscle creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) that has been dansylated at the two reactive lysines to 8 M urea, the maximum emission of the extrinsic fluorophore shifts 4 nm towards the blue, this being accompanied by a small decrease in intensity. The fluorescence emission and excitation spectra of the reassembled and native proteins are the same. Denaturation is accompanied by a rapid decrease in fluorescence which is complete in 10 s. This suggests that denaturation is accompanied by an early disorganization at the catalytic center, where the reactive lysines are located. Reassembly is associated with a rapid increase in dansyl fluorescence followed by a slower decrease that is complete in 6 min. Since reactivation is not complete until 20 min, minor additional structural changes are needed for the reacquisition of catalytic activity. The intrinsic protein fluorescence (eight tryptophans per dimer) of dansylated creatine kinase is approximately 60% less than that of the unlabelled enzyme, which may be attributed to resonance energy transfer, indicating that the reactive lysine is located near one or more of the tryptophans. A more limited quenching of intrinsic fluorescence is observed when dansylated creatine kinase is exposed to 8 M urea. Reassembly, monitored by a decrease in intrinsic fluorescence, reveals that the dansylated protein achieves its final fluorescence after 18 min of renaturation compared with 30 min for unlabelled enzyme. The powerful quenching by the dansyl group may limit the ability to monitor changes in the tryptophan environment. Kinetics of fluorescence polarization changes during denaturation are consistent with a mechanism involving rapid dissociation, followed by a subunit disorganization and possible aggregation. Reassembly would appear to involve first a refolding of the disorganized monomers and subsequent association. These results correspond to our previous observations that subunit renaturation precedes dimerization.     

Neurophysin-neurohypophyseal hormone interactions: studies using a dansylated vasotocin analogue.

We have synthesized a neurohypophyseal hormone analogue containing an extrinsic fluorescence probe by linking a dansyl (DNS) group to the epsilon-amino group of the lysine at residue 8 of vasotocin. The fluorescence properties of this analogue have been characterized by steady-state and time-resolved spectroscopic methods and compared with those of epsilon-DNS-lysine and the dansylated carboxyl terminal tripeptide Pro-Lys(DNS)-GlyNH2. The binding of this hormone analogue to purified isoforms of bovine neurophysins, the natural carrier proteins of the neurohypophyseal hormones, results in changes in several fluorescence parameters of the dansyl probe. These changes include an increase in intensity and average lifetime, a shift of the emission band to higher energies, and an increase in the emission anisotropy. Anisotropy changes have been used to determine dissociation constants for binding to these neurophysin isoforms. Based on the changes in the fluorescence properties of the dansyl probe, the dansyl group itself interacts with the protein. The degree of the dansyl-neurophysin interaction, however, appears to be different for the full sequence isoform of neurophysin I and the Val89 isoform of neurophysin II.     

Dansylated analogues of the opioid peptide [Dmt1]DALDA: in vitro activity profiles and fluorescence parameters

Dansylated analogues of the potent and selective micro opioid peptide agonist [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2); Dmt = 2',6'-dimethyltyrosine) were prepared either by substitution of N(beta)-dansyl-alpha,beta-diaminopropionic acid or N(epsilon)-dansyllysine for Lys(4), or by attachment of a dansyl group to the C-terminal carboxamide function via a linker. All three analogues displayed high micro agonist potency in vitro and the C-terminally dansylated one retained significant micro receptor selectivity. The three analogues showed interesting differences in their fluorescence emission maxima and quantum yields, indicating that the dansyl group in two of them was engaged in intramolecular hydrophobic interactions. These dansylated [Dmt(1)]DALDA analogues represent valuable tools for binding studies, cellular uptake and intracellular distribution studies, and tissue distribution studies.     

Estrogen determination using liquid chromatography with precolumn fluorescence labeling

A liquid chromatography procedure is described for the determination of some estrogens using fluorescence detection. The estrogens are labeled by precolumn derivatization with 5-dimethylaminonaphthalene-1-sulfonylchloride (dansyl chloride) and chromatographed on a reversed-phase, C-18 column with a mobile phase consisting of methanol, water, and acetic acid. The eluted analytes are measured with a fluorescence detector using excitation and emission wavelengths of 350 and 540 nm, respectively. The chief advantage of this new procedure is its sensitivity, requiring smaller amounts of sample to detect and quantitate estrogens in biological materials. We could detect less than 400 pg of estriol. With our procedure, this corresponded to about 25 ng in the final reaction mixture, before derivatization. The use of smaller sample volumes could improve this limit. Linearity for dansylated estriol, estrone, and estradiol was excellent over the estrogen range below 100 μg in the sample. This corresponds to approximately 1.7 μg on the column. Within-run precision was better than 5% for the full extraction and derivatization procedure for estriol from pregnancy urine samples. Chromatography is complete within 10 min for dansylated estriol, estrone, and estradiol.     

Separation and quantitation of polyamines in plant tissue by high performance liquid chromatography of their dansyl derivatives

High performance liquid chromatography in combination with fluorescence spectrophotometry can be used to separate and quantitate polyamines (putrescine, cadaverine, spermidine, spermine), prepared as their dansyl derivatives, from plant tissue. The procedure gives sensitive and consistent results for polyamine determinations in plant tissue. In a standard mixture, the minimal detection level was less than 1 picomole of polyamines.     

Lectin binding to complex carbohydrate at the interface: a study by resonance energy transfer.

The binding affinity of the oligosaccharide moiety of a neutral glycosphingolipid, asialoGM1, towards Ricinus communis agglutinin (RCAI) was determined for the first time by fluorescence resonance energy transfer (RET). The asialoGM1 was incorporated into a phospholipid (DMPC) vesicle doped with dansylated DPPE and then titrated with an increasing amount of the galactose specific RCAI. The efficiency of RET was determined by a saturable increase in the quenching of 'donor' fluorescence, i.e. the 'trp' residue of RCAI, due to the energy transfer from the 'acceptor' dansyl group on the surface of the vesicle. The apparent binding constant was found to be in the range of 10(5)-10(6) M-1 at 27 degrees C.     

Rapid kinetic studies on calcium interactions with native and fluorescently labeled calmodulin

Stopped-flow studies on calcium binding to calmodulin showed that under pseudo first order conditions the reaction was complete within 2.5 milliseconds. The time course for calcium dissociation from the native protein showed a single kinetic phase (τ1^?1 = 10S^?1) while that from the dansylated derivative revealed a second slower kinetic phase (τ1^?1 = 10S^?1, τ2^?1 = 0.31S^?1) that accounted for about one-half of the total fluorescence decrease. Therefore the dansyl derivative of calmodulin may provide a useful tool for studying conformational changes in the protein not reflected by the active site tyrosines.     

Detection and quantitation of 8-hydroxydeoxyguanosine 5'-monophosphate in X-irradiated calf-thymus DNA by fluorescence postlabeling

8-Hydroxydeoxyguanosine 5'-monophosphate (8-OH dGmp) was synthesized from deoxyguanosine 5'-monophosphate (dGmp) by ascorbic acid in the presence of hydrogen peroxide and labeled with dansyl chloride through a phosphoramidate linkage with ethylenediamine (EDA). A DNA model 8-OHd(TACG), isolated intact by high pressure liquid chromatography (HPLC) from x-irradiated d(TACG) and characterized by nmr, was digested enzymatically to 5'-mononucleotides. The modified nucleotide was enriched by HPLC and dansylated. Analysis of the dansylated product by HPLC, using a fluorescent detector, detected a peak with retention time corresponding to that of the dansyl labeled authentic marker. The same overall procedure was used to detect 8-OHdGmp from x-irradiated calf-thymus DNA. The content of 8-OHdGmp in the irradiated DNA increased linearly with increasing levels of x-irradiation in the dose range of 6-60 Gy.     

Polyamine content of the Plodia interpunctella granulosis virus and its larval host

The presence of polyamines in the granulosis virus and its larval host, Plodia interpunctella, was examined using thin-layer chromatography of the dansylated compounds. The uninfected and infected larvae contain the three polyamines, putrescine, spermidine, and spermine. Although purified granulosis virus infecting this host does not contain any typical polyamines, an acidsoluble dansylated compound with properties indicative of a polyamine was observed.     

Thermal tolerance role of novel polyamine,caldopentamine, identified in fifth instar Bombyx mori

Silkworms have limited ability to regulate their body temperature; therefore, environmental changes, such as global warming, can adversely affect their viability. Polyamines have shown protection to various organisms against heat stress. This study evaluated the qualitative and quantitative changes in heat-stressed Bombyx mori larvae polyamines. Fifth instar Bombyx mori larvae were divided into two groups; control group, reared at room temperature, i.e., 28?±?2 °C, and the heat shock group, exposed to 40 °C. Dansylation of the whole worm polyamines and subsequent thin-layer chromatography revealed the presence of components with the same Rf value as dansyl–putrescine, spermidine, and spermine. The dansyl–putrescine, spermidine, and spermine polyamines were identified by mass spectrometric analyses. After heat shock, the thin-layer chromatography of the whole-larvae tissue extracts showed qualitative and quantitative changes in dansylated polyamines. A new polyamine, caldopentamine, was identified, which showed elevated levels in heat-stressed larvae. This polyamine could play a role in helping the larvae tolerate various stress, including thermal stress. No significant changes in silk fiber’s economic and mechanical properties were observed in our study. This study indicated that PA, caldopentamine, supplementation could improve heat-stress tolerance in Bombyx mori.

     

A novel technique to assay adducts of DNA induced by anticancer agent cis-diamminedichloroplatinum(II).

The dideoxynucleotides d(pGpG) and d(pApG) and the tetradeoxynucleotide d(CpTpApG) were synthesized in solution phase by a modified phosphotriester technique and reacted with the anticancer agent cis-diamminedichloroplatinum(II) (cisplatin). The major products were isolated by HPLC and characterized by NMR and mass spectrometry as cross-link adducts of cisplatin with the neighboring purine bases. The cross-link adducts of d(pGpG) and d(pApG) were dansylated through a 5'-phosphoramidate linkage with ethylenediammine. The labeling efficiency of the adducts was quantitative as in the case of the normal dinucleotides. The modified tetramer was digested with nuclease P1. The excised adduct was enriched by HPLC and labeled with dansyl chloride. The analysis of the postlabeled adduct by HPCL, using a fluorescence detector, detected a peak with retention time corresponding to that of the dansylated cis-Pt(NH3)2d(pApG). Cochromatography with the authentic marker confirmed the identification. The same overall procedure was used to assay calf thymus DNA exposed to cisplatin. The major adducts were identified as cis-Pt(NH3)2d(pGpG) and cis-Pt(NH3)2d(pApG). The quantitative labeling efficiency of platinum adducts combined with highly sensitive fluorescence detection technique (subfemtomol) suggests that fluorescence postlabeling assay could be a novel approach for real-time analysis of DNA modification induced by platinated drugs in biological system.     

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]     

Identification of dansyl dipeptides in the dansyl-Edman peptide degradation

The manual dansyl-Edman¹ degradation procedure is one of the most convenient and widely used techniques for the sequencing of peptides up to about 15 residues in length (1,2). A frequently encountered complication in this procedure is the resistance of certain peptide bonds to acid hydrolysis. If the amino terminal peptide bond of the dansylated peptide is especially resistant, the dansyl dipeptide is frequently in higher yield than the corresponding dansyl amino acid. The resistant dansyl dipeptide is often composed of two hydrophobic amino acid residues. The resistance of such peptide bonds to acid hydrolysis is well understood (3). Other resistant bonds have, however, been noted in practice, e.g., those involving a hydrophobic and a prolyl residue. This phenomenon can lead to difficulty in interpretation of the thin-layer chromatogram and to subsequent incorrect identification of amino acid residues. Extending the hydrolysis time to 24 hr still leaves especially resistant dipeptides as the major product while significantly reducing the yield of other dansylated residues, notably dansyl proline, serine, and threonine. We report here the chromatographic behavior of 18 dansyl dipeptides on polyamide thin-layers using the solvent systems commonly employed in the dansyl-Edman procedure (2). All of these dipeptides have been encountered in practice, and the extent of hydrolysis in 6 n HCl at 110°C is usually less than 20%.     

Synthesis and application of fluorescent labeled nucleotides to assay DNA damage

A facile method was developed to covalently attach a fluorophore to the 5'-phosphate of a nucleic acid. The procedure, illustrated by coupling 5'-dNmp (N = A,C,G,T) with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride, involves 5'-phosphoramidation with ethylenediamine (EDA) followed by conjugation of the free aliphatic amino group of the phosphoramidate with Dansyl chloride. This method is also applicable to multi-incorporation of fluorescent labels in the nucleic acids. The reaction of 5'-Amp with a polyamine such as poly L-lysine (PLL, mol. wt., 4000) resulted in a phosphoramidate with multiple amino groups, which after isolation and conjugation with fluorescamine gave dAmp with multilabeled fluorophores. A condition was devised to separate the four dansylated mononucleotides of DNA, conjugated via ethylenediamine linker, by reverse phase HPLC. The elution profile could be monitored with a variable wavelength detector at 254 nm and 340 nm corresponding to the absorption of the nucleotides and the dansyl moiety, respectively. The detection limit was 2 nmol at 254 nm. The use of a fluorescence detector enhanced the detection sensitivity to a sub-picomole level (200 fmol). Samples of a DNA model, d(pCpGpTpA) and calf-thymus DNA were digested enzymatically to 5'-mononucleotides and labeled with Dansyl chloride. HPLC analysis of the dansylated digests from these samples, both before and after irradiation, suggests that the combination of enzymatic digestion and fluorescence postlabeling could be a novel approach to assay DNA damage.