N-Dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols

Ramachandran, Niroshan; Jacob, Sanjay; Zielinski, Barbara S.; Curatola, G; Mazzanti, Laura; Mutus, Bülent

doi:10.1016/s0167-4838(98)00286-6

Cited by 16 publications

(14 citation statements)

References 8 publications

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“…In a previous study, we have shown that the S-nitrosation of DnsHCys resulted in Ϸ90% quenching of the dansyl fluorescence (14). This effect is believed to arise from the energy transfer from the dansyl (excitation 320 nm, emission 520 nm) to the n-* transition state of the S-NO moiety ( max , 543 nm; ref.…”

Section: Resultsmentioning

confidence: 95%

Mechanism of transfer of NO from extracellular S -nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

Ramachandran

Root

Jiang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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167

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N itric oxide is a secondary messenger involved in the cGMP cascade, vasodilation, and a known inhibitor of platelet aggregation. S-nitrosothiols (RSNOs) are formed via nitrosation of thiols under aerobic conditions (1). Apart from being cellular sources of NO, they also prolong its half-life (2, 3). RSNOs are involved in signaling pathways, immune responses, and the actions of nitrovasodilating compounds (4-7). Therefore, a study involving the effects and transport of S-nitroso-BSA (BSA-NO) into live cells is of utmost physiological importance and the center of pharmacological interest (8).Many indirect, discontinuous fluorescent, electrochemical, and colorometric assays have been developed for RSNOs (8, 9). However, none of these are conducive to measuring the transport of RSNO-bound NO into live cells, in vitro. The probe for NO influx presented here is N-dansylhomocysteine (DnsHCys). The fluorescence of this compound is completely quenched on its S-nitrosation, yielding N-dansyl-S-nitrosohomocysteine (DnsHCysNO). Here we show that DnsHCysNO is a direct fluorogenic substrate for protein disulfide isomerase (PDI).PDI acts as a chaperone molecule in the endoplasmic reticulum where it catalyses protein thiol exchange reactions. Hotchkiss et al. (10) have reported that PDI is also secreted by endothelial cells as well as deposited on the cell surface. Recent studies have presented indirect evidence for the involvement of cell-surface PDI (csPDI) in the influx of 12). Stamler and coworkers (13) also have shown that the export of intracellular NO from red blood cells to be facilitated by S-nitrosation of the cysteine residues in the hemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Membranebound PDI may catalyze the formation of AE1-SNO and the subsequent export of cytosolic NO.Here, the extracellular RSNO-dependent quenching of the DnsHCys fluorescence was shown to be a csPDI-dependent process with the aid of antisense-mediated underexpression of PDI and the sense-mediated overexpression of PDI in HT1080 fibrosarcoma cells as well as with a cell-impermeant inhibitor that reacts with vicinal dithiols. In addition, N-dansylhomocystine (DnsHCys 2 ) is shown to be a sensitive intracellular probe for the kinetic characterization of csPDI in human umbilical vein endothelial cells (HUVECs), hamster lung fibroblasts, and HT1080 fibrosarcoma cells. Based on this data, a mechanism for csPDI-meditated intracellular S-nitrosation, by extracellular RSNOs, has been proposed. Materials and Methods Synthesis of S-Nitrosoglutathione (GSNO).Glutathione (GSH, Sigma) was dissolved in ice-cold 0.5 M HCl. Equimolar sodium nitrite was added and the reaction was carried out in the dark at 4°C for 40 min. The pH of the reaction mixture was adjusted to 7.0 and crystallized by the slow addition of cold acetone. BSA-NO was synthesized by using the above-mentioned protocol (3). Synthesis of DnsHCysNO.HCysNO was prepared by treating HCys (Sigma) with acidified nitrite. Dansylation was carried out in 0.1 M phosphate buffer (pH ...

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Section: Resultsmentioning

confidence: 95%

Mechanism of transfer of NO from extracellular S -nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

Ramachandran

Root

Jiang

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

167

137

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“…This is evidenced by the intrinsic fibrinogen fluorescence quenching by GSNO, which is attributed to energy transfer between Trp residues (excitation 290 nm; emission 330 nm) of the protein and the S-NO moiety of GSNO (acceptorabsorbance maxima 343 nm, 543 nm). We have previously observed that the fluorescence of N-dansylhomocysteine was quenched on its S-nitrosation (26). The quenching was attributed to energy transfer between the dansyl moiety donor (excitation 340 nm; emission 540 nm) and RS-NO (acceptor, absorbance 543 nm), and, in order for this process to occur, the RS-NO must ''lay over'' the dansyl ring.…”

Section: Discussionmentioning

confidence: 99%

Evidence for S -nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation

Akhter

Vignini

Zhang

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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S-nitrosoglutathione (GSNO, 50 M) inhibited the initial rate of thrombin-catalyzed human and bovine fibrinogen polymerization by Ϸ50% to 68% respectively. Inhibition was also observed with other structurally varied S-nitrosothiols (RSNOs) including sugar derivatives of S-nitroso-N-acetylpenicillamine (SNAP). The fact that the same concentration of GSNO had no effect on thrombindependent hydrolysis of tosylglycylprolylarginine-4-nitroanilide acetate suggested that this inhibition was due to GSNO-induced changes in fibrinogen structure. This result was confirmed by CD spectroscopy where GSNO or S-nitrosohomocysteine increased the ␣-helical content of fibrinogen by Ϸ15% and 11%, respectively. S-carboxymethylamido derivatives of glutathione or homocysteine had no effect on the fibrinogen secondary structure. The GSNO-dependent secondary structural effects were reversed on gel filtration chromatography, suggesting that the effects were allosteric. Further evidence for fibrinogen-GSNO interactions was obtained from GSNO-dependent quenching of the intrinsic fibrinogen Trp fluorescence and the perturbation of the GSNO circular dichroic absorbance as a function of [fibrinogen]. The K ds of 3 to 10 M for fibrinogen-GSNO interactions with a stoichiometry of 2:1 (GSNO:fibrinogen) were estimated from isothermal titration calorimetry and fluorescence quenching, respectively. These results suggest that RSNOs induce changes to fibrinogen structure by interacting at specific aromatic rich domains. Three such putative RSNO-binding domains have been identified in the unordered, aromatic residue-rich C-termini of the ␣-chains of fibrinogen.

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“…This new type of S-nitrosothiol, which coupled the functional S-NO group with the fluorescent molecule, was recently prepared for detecting intracellular thiols and S-nitrosothiols (58). Thus, it could be used as a fluorescent probe to identify the modified form of Cys 25 in papain.…”

Section: Inhibition Of Papain By S-nitrosothiolsmentioning

confidence: 99%

Inhibition of Papain by S-Nitrosothiols

Xian

Chen

Liu

et al. 2000

Journal of Biological Chemistry

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S-Nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by NO. The cysteine protease papain has a critical thiol residue (Cys 25 ). It has been demonstrated that NO or NO donors such as sodium nitroprusside and N-nitrosoaniline derivatives can reversibly inhibit this enzyme by S-NO bond formation in its active site. In this study, a different regulated mechanism of inactivation was reported using S-nitrosothiols as the NO donor. Five S-nitroso compounds, Snitroso-N-acetyl-DL-penicillamine, S-nitrosoglutathione, S-nitrosocaptopril, glucose-S-nitroso-N-acetyl-DL-penicillamine-2, and the S-nitroso tripeptide acetyl-Phe-Gly-S-nitrosopenicillamine, exhibited different inhibitory activities toward the enzyme in a time-and concentrationdependent manner with second-order rate constants (k i / K I ) ranging from 8.9 to 17.2 M ؊1 s ؊1 . The inhibition of papain by S-nitrosothiol was rapidly reversed by dithiothreitol, but not by ascorbate, which could reverse the inhibition of papain by NOBF 4 . Incubation of the enzyme with a fluorescent S-nitroso probe (S-nitroso-5-dimethylaminonaphthalene-1-sulfonyl) resulted in the appearance of fluorescence of the protein, indicating the formation of a thiol adduct. Moreover, S-transnitrosylation in the incubation of S-nitroso inactivators with papain was excluded. These results suggest that inactivation of papain by S-nitrosothiols is due to a direct attack of the highly reactive thiolate (Cys 25 ) in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between the inactivator and papain.

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N-Dansyl-S-nitrosohomocysteine a fluorescent probe for intracellular thiols and S-nitrosothiols

Cited by 16 publications

References 8 publications

Mechanism of transfer of NO from extracellular S -nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

Mechanism of transfer of NO from extracellular S -nitrosothiols into the cytosol by cell-surface protein disulfide isomerase

Evidence for S -nitrosothiol-dependent changes in fibrinogen that do not involve transnitrosation or thiolation

Inhibition of Papain by S-Nitrosothiols

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