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PubMed1983

Dansylarginine N-(3-ethyl-1.5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase.

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 x 10(-7) M) but not of acetylcholinesterase (IC50 = 4 x 10(-4) M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a Kd of 4.5 x 10(-7) M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.

Animals↗
PubMed1984

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors.

A dansyl-L-lysine analogue of methotrexate, N alpha-(4-amino-4-deoxy-10- methylpteroyl )-N epsilon-(5-[N,N-dimethylamino]-1-naphthalenesulfonyl)-L-lysine, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [3H]methotrexate transport with a Ki of 7.02 microM, a value virtually identical to the Kt for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-L-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.

Animals↗
PubMed1989

A pitfall in the use of double-reciprocal plots to estimate the intrinsic molar fluorescence of ligands bound to albumin.

Double-reciprocal plots of fluorescence intensity versus protein concentration are often used to obtain the intrinsic molar fluorescence (Fb) of ligands bound to acceptor molecules such as albumin. In this paper we show that these plots develop upward concave curvature as the concentration of albumin increases. Thus linear extrapolation of such plots cannot be employed to provide accurate values of Fb. It is suggested that a direct plot of fluorescence intensity versus log protein concentration should be employed to obtain Fb.

Albumins↗
PubMed1991

Analysis of the binding of fluorescent ligands to soluble proteins. Use of simultaneous non-linear least squares regression to obtain estimates of binding parameters.

The binding of three fluorescent ligands (warfarin, dansylsarcosine and 1-anilino-8-naphthalene sulphonate) to human albumin was analysed using simultaneous non-linear least squares regression analysis. Both mock and actual fluorescence data were examined and the results indicated that reliable estimates of the binding parameters as well as the molar fluorescence of bound ligand could be obtained. The advantage of this method of analysis is that it makes full use of all the experimental data and it eliminates the need for the graphical procedures usually employed to estimate the molar fluorescence of bound ligand and its binding constants. This type of analysis can be extended to other systems where some physical property of the bound ligand varies with increasing protein concentration.

Albumins↗
PubMed1991

High prostaglandin-E1 binding to serum protein in allergic subjects.

Protein binding to both salicylate and 3H-labelled prostaglandin-E1 ([3H]PGE1) was examined in the sera of 22 allergic and 16 normal individuals. Protein binding to salicylate (P less than 0.001) and to [3H]PGE1 (P less than 0.01) was significantly greater in the allergic than in the normal group. The nature of the binding sites of salicylate and PGE1 was investigated with two fluorescent probes, dansylamide and dansylsarcosine as specific marker ligands for established Sites I and II, found to be specific for anionic drugs. The serum protein of 11 allergic subjects showed a higher binding at Site I (P less than 0.05) and a lower binding at Site II (P less than 0.05) than that of seven normal subjects. Salicylate and [3H]PGE1 bound competitively at the two sites. It was concluded, when comparing allergic to normal subjects, that the high protein binding of allergic individuals to salicylate and PGE1 could be attributed to qualitative and/or quantitative differences in the lipophilic substances which are tightly bound to the albumin of normal sera, causing a reduction in binding ability at Site I.

Alprostadil↗
PubMed1991

Effect of valproic acid, its unsaturated metabolites and some structurally related fatty acids on the binding of warfarin and dansylsarcosine to human albumin.

The sites to which valproic acid and its main unsaturated metabolites (2-en-2-propyl pentanoic acid and 4-en-2-propyl pentanoic acid) bind to on human albumin were investigated by (1) measuring their ability to displace the fluorescent probes warfarin and dansylsarcosine and (2) by assessing the extent to which they inhibited the hydrolysis of 4-nitrophenyl acetate. Valproate and its metabolites displaced both warfarin and dansylsarcosine, and they also inhibited the hydrolysis of 4-nitrophenyl acetate. The order of potency for inhibition of both binding and hydrolysis was: 2-en-2-propyl pentanoic acid greater than 4-en-2-propyl pentanoic acid greater than or equal to valproate. It is concluded that valproic acid and its unsaturated metabolites can displace ligands from the warfarin binding site (site I) and the benzodiazepine/indole binding site (site II), but the primary interaction is with site II. Furthermore, the introduction of a double bond into the carbon backbone of valproate increases affinity for albumin at both sites.

Binding Sites↗
PubMed1992

A comparison of drug binding sites on mammalian albumins.

The fluorescent probes warfarin and dansylsarcosine are known to selectively interact with binding sites I and II, respectively, on human albumin. This paper investigates whether similar binding sites exist on bovine, dog, horse, sheep and rat albumins. Binding sites on albumins were studied by: (1) displacement of warfarin and dansylsarcosine by site I (phenylbutazone) and site II (diazepam) selective ligands; (2) the effects of non-esterified fatty acids (carbon chain lengths: C5-C20) and changes in pH (6-9) on the fluorescence of warfarin and dansylsarcosine; and (3) the ability of site selective ligands to inhibit hydrolysis of 4-nitrophenyl acetate. For bovine, dog, horse, human and sheep albumins the fluorescence of bound warfarin and dansylsarcosine was selectively decreased by phenylbutazone and diazepam, respectively. For these albumins medium chain fatty acids (C1-C12) reduced the fluorescence of dansylsarcosine (maximum inhibition with C9) whereas long chain acids (C12-C20) enhanced the fluorescence of warfarin (maximum increases with C12). In addition, changes in pH from 6 to 9 increased the fluorescence of warfarin and although site I ligands (warfarin/phenylbutazone) had no pronounced effects on 4-nitrophenyl acetate hydrolysis, site II ligands (dansylsarcosine/diazepam) significantly inhibited this reaction. Rat albumin behaved differently from the other albumins studied in that the C12-C20 fatty acids and changes in pH did not enhance the fluorescence of warfarin. Moreover, the differential effects of site I and site II ligands on the fluorescence of warfarin/dansylsarcosine and hydrolysis of 4-nitrophenyl acetate were less apparent with rat albumin. The results suggest bovine, dog, horse and sheep albumins have binding sites for warfarin and dansylsarcosine with similar properties to sites I and II on human albumin. By contrast, the warfarin binding site and to a lesser degree the dansylsarcosine site, of rat albumin have different characteristics from these sites on the other albumins studied.

Albumins↗
PubMed1992

Mixed-type inhibition of bovine lung angiotensin converting enzyme by lisinopril and its dansyl derivative.

The steady-state inhibition of bovine lung angiotensin converting enzyme (ACE; EC 3.4.15.1) by the slow-binding inhibitor lisinopril and its dansyl derivative conformed to a linear mixed inhibition model with inhibitor binding to ES as well as to E. Studied at pH8, 35 degrees, and using N-(3-[2-furyl]-acryloyl)phe-gly-gly as substrate, the approach to steady-state activity at different substrate concentrations pointed to slow isomerizations in both EI and EIS. While an inhibitory scheme involving a single I-binding site adequately accounts for the data presented, information relating to the primary structure of ACE brings up a two-site alternative which remains to be tested.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1993

Study of interaction of carprofen and its enantiomers with human serum albumin--I. Mechanism of binding studied by dialysis and spectroscopic methods.

The binding of carprofen, a non-steroidal anti-inflammatory drug of the aryl propionic acid class [2-(6-chlorocarbazole)propionic acid], and its enantiomers to human serum albumin (HSA) has been studied by dialysis and spectroscopic methods. Binding parameters obtained by different methods were in close agreement. The binding of carprofen to HSA by both fluorescence and equilibrium dialysis (ED) methods is characterized by two sets of association constants [K1 = 5.1 x 10(6) M-1 (fluorescence) and 3.7 x 10(6) M-1 (ED), K2 = 3.7 x 10(5) M-1 (fluorescence) and 1.3 x 10(5) M-1 (ED)]. The S(+)-enantiomer of carprofen showed slightly higher affinity for HSA than its corresponding antipode by both methods. Different analyses of the binding to HSA suggested the presence of one high affinity site and five to seven low affinity sites for carprofen and its enantiomers on HSA. Fluorescence displacement data implied that carprofen primarily binds to site II, the benzodiazepine site, while the low affinity site of carprofen is site I, the warfarin site. Circular dichroism data suggested different mechanisms for the high affinity and the low affinity binding of carprofen to HSA. The data are consistent with the major part of the binding energy at site II being electrostatic and hydrophobic interactions, whereas for the low affinity binding, hydrophobic interactions. Binding was exothermic, entropy driven and spontaneous, as indicated by the thermodynamic analyses. From binding data with chemically modified HSA derivatives, it is likely that tyrosine, lysine and histidine residues are especially involved in carprofen binding to HSA, and it is most likely that the high affinity binding of carprofen is located in the N-terminal part of domain III or that section of protein plus the C-terminal part of domain II of the HSA molecule. When the binding of carprofen to HSA was compared to the binding of carprofen methyl ester to HSA (K = 0.1 x 10(6) M-1), the carboxyl group of carprofen was found to play an important role especially in the high affinity binding of carprofen to HSA. The high affinity of carprofen to HSA was independent of the conformational changes on HSA caused by N-B transition.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1978

Direct mapping of beta-adrenergic receptors in the rat central nervous system by a novel fluorescent beta-blocker.

DL-N-(2-Hydroxy-3-napthyloxypropyl)-N'-dansylethylenediamine, dansyl analogue of propranolol (DAPN) is a novel fluorescent beta-adrenergic antagonist with high affinity to beta-receptors. The distribution pattern of DAPN fluorescence was studied in the rat central nervous system subsequent to its intravenous administration to living rats. DAPN distinctly labels specific regions and cells in the central nervous system (CNS). Highly dense DAPN fluorescence was observed in the pyramidal cell layer of the hippocampus, the granule cell layer of the dentate gyrus, the basal layers of the piriform cortex and the neocortex, the cerebellar Purkinje cell layer, and the spinal a-motoneurons. Pretreatment of control rats with DL-and L-propranolol markedly decreased the intensity and density of DAPN fluorescence in the tissue sections, whereas prior administration of D-propranolol had almost no effect. Pretreatment with large doses of reserpine did not alter the pattern of DAPN fluorescence. These findings were identical to those observed with another fluorescent beta-blocker, 9-aminoacridino-propranolol (9-AAP). Our data suggest that fluorescent beta-adrenergic antagonists may be used in vivo for the direct probing of the beta-receptors within the mammalian CNS.

Animals↗
PubMed1981

The release of endogenous amino acids into the vitreous of the intact eye of the albino rat: effect of light, potassium, and ouabain.

The vitreal space of the intact eye of albino rats was perfused in vivo. The concentration of several endogenous amino acids in the vitreal effluent was measured by the [3H]microdansylation procedure. GABA was never detected despite a sensitivity of the method of 0.5 pmol. In contrast to previous results obtained in pigmented rats, photic stimulation with flashing white light did not alter the release of glycine or any of the other amino acids. Potassium (60 mM) and ouabain (0.1 mM) evoked a specific release of glycine. The potassium-evoked release was blocked by magnesium suggesting a neuronal site of origin of glycine. Ouabain-evoked release was not blocked by magnesium. The results were contrasted with experiments on radiolabeled amino acid release from retinas preloaded and superfused in vitro, a condition in which glial localization of exogenous amino acids predominates.

Amino Acids↗
PubMed1988

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.

Chemical Phenomena↗
PubMed1977

Interactions between fluorescent labeled phosphatidyl serine and cations.

Phosphatidyl serine (PS) was reacted with dimethylaminoanphthalenesulfonyl (DNS) chloride to produce a fluorescent phospholipid derivative (DNS--PS). By comparison of the emission maximum of the fluorochrome in solvents of varying dielectric constants and in aqueous suspension it was concluded that the DNS moiety of sonicated DNS--PS micells was within the glycerol region of the lipid. Addition of CaC12 to aqueous suspensions of DNS--PS micells caused a shift in the emission maximum from 524 nm to 490 nm, a two-fold increase in the relative fluorescence and an increase in the polarization of fluorescence and finally aggregation. MgC12 produced similar but smaller changes in the fluorescence parameters. In the presence of calcium the thermal transition of the fatty acid tail region could be monitored as an increase in the fluorescence intensity of the DNS group. Without calcium an almost linear decrease in relative fluorescence was observed. These data suggested that calcium caused the fluorochrome to be shifted from the glycerol region of the lipid structure into the fatty acid tail region. These observations indicate how the conformation of a biological membrane might be altered by the interaction between acidic phospholipids and calcium.

Binding Sites↗