Optical Zeeman experiments were performed on Cr:forsterite with the magnetic field applied along ... more Optical Zeeman experiments were performed on Cr:forsterite with the magnetic field applied along the crystallographic a and c axes. The strong agreement between these optical results and previous EPR studies for both alignments establishes the identity of the near infrared emission center as tetrahedral Cr4+.
Proceedings of the National Academy of Sciences, 1992
Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing fac... more Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.
ABSTRACT Optical Zeeman spectra of the near-IR luminescent center in Cr:forsterite were obtained ... more ABSTRACT Optical Zeeman spectra of the near-IR luminescent center in Cr:forsterite were obtained with an external magnetic field applied along the crystallographic a and c axes. The field-dependent sublevel splittings of the triplet spin ground state were resolved through fluorescence line narrowing (FLN) techniques. The excellent correspondence between the observed splittings and those calculated for the tetrahedral Cr4+ center, previously identified and characterized in forsterite by electron paramagnetic resonance, firmly establishes the identity of the near-IR center as tetrahedral Cr4+. A simple model for the FLN excitation-emission cycle, including the influence of the external field on the optical line positions and intensities, is described.
• , iI L tO Wa , Qn ~ww cabi w" t"rv t~ OlN for lo~" 0 ina 'tww&~ l s AIoorc o 1d15 m@H1iaes t 0A... more • , iI L tO Wa , Qn ~ww cabi w" t"rv t~ OlN for lo~" 0 ina 'tww&~ l s AIoorc o 1d15 m@H1iaes t 0A11 J. REPORT TYPE AND DATES COVERED I 1994-04-25 FTNA, RFPOv 1-May 90. -31 nge-QI 4. TITLE AND SUSTITE! S. FUNOING NUMBERS Optical and Magnetic Resonance Investigations of 3d ? -" 11. SUPPLEMENTARY NOTES Cr:forsterite laer system, our results prove that the near IR lasing center is tetrahedral Cr 4 + . In S Mn:Ba3(VO4)2 EPR spectroscopy shows that tetrahedral Mn 5 + and Mn 4 + centers are both present S in the material; a novel spin-echo pulsed laser double resonance method is introduced to prove that Ithe tetrahedral Mn 5+ is the active center. Previously unpublished results of chromium-doped gehlenites and garnets are also report& .. 4.SJSET EMI KS NUMSIR OPPAI 184 N 117CUtY .IATI)N |I.E |URITY OLAS|iStCATi10 i0. ITMOAWNON OP ATtRACT Ol 01POT OF THIS PAGE OF ABSTRACT UNCLASSIFIED UNCLASSIFIED I UNCLASSIFIED ___________ N 1140-00 Standard Form 296 (Rev 2691 P. wcosd W M %csW 3 ,IS () ZIS.I
High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxi... more High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK) 2 CAAAKA-NH 2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperaturedependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy Ќ / ሻ (ϵ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.
ABSTRACT We report a 13C DNP NMR study of the organic conductor fluoranthenyl (FA) radical cation... more ABSTRACT We report a 13C DNP NMR study of the organic conductor fluoranthenyl (FA) radical cation salt (FA)2+PF6−. We have been able to suppress the Knight shift by saturating the ESR line. Overhauser enhancements achieved were large enough to detect 1016 carbons after 1 s of microwave saturation.
Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the act... more Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl ␣-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nuclei was studied in ras p21 ⅐ Mn(II) ⅐ 5guanylylimido-diphosphate (p21 ⅐ Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [ 2 H-3]Thr, the Mn(II)-[ 2 H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2 H ESEEM in solutions made with d 5 -glycerol and d 7methyl ␣-D-glucopyranoside (d 7 -MG). In p21 ⅐ Mn(II)GMPPNP, the large deuterium modulations from the d 5 -glycerol exhibit saturation behavior with increasing d 5 -glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d 7 -MG than with d 5 -glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21 ⅐ guanosine 5-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.
Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this app... more Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its in...
Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thro... more Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H202 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine. (J.
Proceedings of the National Academy of Sciences, 1993
Recent discoveries suggesting essential bioactivities of nitric oxide (NO') in the lung are diffi... more Recent discoveries suggesting essential bioactivities of nitric oxide (NO') in the lung are difficult to reconcile with the established pulmbnary cytotoxicity of this common air pollutant. These conflicting observations suggest that metabolic intermediaries may exist in the lung to modulate the bioactivity and toxicity of NO'. We report that S-nitrosothiols
Proceedings of the National Academy of Sciences, 1992
We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generat... more We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endotheliumderived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for -0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S-NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosodiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains =7 pIM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitrosoerum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at -1 #M; 60 min after administration of NG-monomethyl-Larginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by "40% (>95% of which were accounted for by S-nitrosoproteins, and ""80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.
Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biologic... more Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biological functions. Details of its biochemistry, however, remain poorly understood. The broader chemistry of nitrogen monoxide (NO) involves a redox array of species with distinctive properties and reactivities: NO+ (nitrosonium), NO., and NO- (nitroxyl anion). The integration of this chemistry with current perspectives of NO biology illuminates many aspects of NO biochemistry, including the enzymatic mechanism of synthesis, the mode of transport and targeting in biological systems, the means by which its toxicity is mitigated, and the function-regulating interaction with target proteins.
Proceedings of the National Academy of Sciences, 1992
Endothelium-derived relaing factor (EDRF) activity has been attributed to the highly labile nitri... more Endothelium-derived relaing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular mifleux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhy--I dryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism ofNO and raise the possibility ofan additional type of cellular regulatory mechanism.
A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with ... more A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both t...
Optical Zeeman experiments were performed on Cr:forsterite with the magnetic field applied along ... more Optical Zeeman experiments were performed on Cr:forsterite with the magnetic field applied along the crystallographic a and c axes. The strong agreement between these optical results and previous EPR studies for both alignments establishes the identity of the near infrared emission center as tetrahedral Cr4+.
Proceedings of the National Academy of Sciences, 1992
Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing fac... more Tissue-type plasminogen activator (t-PA) reacts upon exposure to endothelium-derived relaxing factor (EDRF) by way of the enzyme's single free sulfhydryl to form a stable S-nitrosothiol protein adduct. S-nitrosylation endows t-PA with potent vasodilatory and antiplatelet properties that are accompanied by elevations in intracellular cyclic GMP analogous to those induced by low molecular weight (e.g., S-nitroso amino acid) S-nitrosothiols. Moreover, this chemical modification does not adversely affect the catalytic efficiency of t-PA, the fibrin stimulation of this activity, the binding of t-PA to fibrinogen, or the interaction of the enzyme with its physiologic serine protease inhibitor, plasminogen-activator inhibitor type I. The coupling of vasodilatory, antiplatelet, and fibrinolytic properties in one molecule makes the S-nitrosylated t-PA a unique molecular species and may provide insight into the mechanisms by which the endothelium maintains vessel patency. These data also suggest a pharmacologic approach to treatment of thromboocclusive disorders.
ABSTRACT Optical Zeeman spectra of the near-IR luminescent center in Cr:forsterite were obtained ... more ABSTRACT Optical Zeeman spectra of the near-IR luminescent center in Cr:forsterite were obtained with an external magnetic field applied along the crystallographic a and c axes. The field-dependent sublevel splittings of the triplet spin ground state were resolved through fluorescence line narrowing (FLN) techniques. The excellent correspondence between the observed splittings and those calculated for the tetrahedral Cr4+ center, previously identified and characterized in forsterite by electron paramagnetic resonance, firmly establishes the identity of the near-IR center as tetrahedral Cr4+. A simple model for the FLN excitation-emission cycle, including the influence of the external field on the optical line positions and intensities, is described.
• , iI L tO Wa , Qn ~ww cabi w" t"rv t~ OlN for lo~" 0 ina 'tww&~ l s AIoorc o 1d15 m@H1iaes t 0A... more • , iI L tO Wa , Qn ~ww cabi w" t"rv t~ OlN for lo~" 0 ina 'tww&~ l s AIoorc o 1d15 m@H1iaes t 0A11 J. REPORT TYPE AND DATES COVERED I 1994-04-25 FTNA, RFPOv 1-May 90. -31 nge-QI 4. TITLE AND SUSTITE! S. FUNOING NUMBERS Optical and Magnetic Resonance Investigations of 3d ? -" 11. SUPPLEMENTARY NOTES Cr:forsterite laer system, our results prove that the near IR lasing center is tetrahedral Cr 4 + . In S Mn:Ba3(VO4)2 EPR spectroscopy shows that tetrahedral Mn 5 + and Mn 4 + centers are both present S in the material; a novel spin-echo pulsed laser double resonance method is introduced to prove that Ithe tetrahedral Mn 5+ is the active center. Previously unpublished results of chromium-doped gehlenites and garnets are also report& .. 4.SJSET EMI KS NUMSIR OPPAI 184 N 117CUtY .IATI)N |I.E |URITY OLAS|iStCATi10 i0. ITMOAWNON OP ATtRACT Ol 01POT OF THIS PAGE OF ABSTRACT UNCLASSIFIED UNCLASSIFIED I UNCLASSIFIED ___________ N 1140-00 Standard Form 296 (Rev 2691 P. wcosd W M %csW 3 ,IS () ZIS.I
High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxi... more High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence Ac-(AAAAK) 2 CAAAKA-NH 2 3K-11, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperaturedependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy Ќ / ሻ (ϵ N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.
ABSTRACT We report a 13C DNP NMR study of the organic conductor fluoranthenyl (FA) radical cation... more ABSTRACT We report a 13C DNP NMR study of the organic conductor fluoranthenyl (FA) radical cation salt (FA)2+PF6−. We have been able to suppress the Knight shift by saturating the ESR line. Overhauser enhancements achieved were large enough to detect 1016 carbons after 1 s of microwave saturation.
Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the act... more Electron spin-echo envelope modulation (ESEEM) spectroscopy is widely used to investigate the active sites of biological molecules in frozen solutions. Various cryoprotection techniques, particularly the addition of co-solvents, are commonly employed in the preparation of such samples. In conjunction with ESEEM studies of Mn(II) guanosine nucleotide complexes of p21 ras, we have investigated the effects of cryoprotection on the spectroscopy, the structure, and the activity of this protein. Echo decay times, which typically govern ESEEM spectral resolution, were found to vary linearly with the concentration of glycerol or methyl ␣-D-glucopyranoside (MG), with both additives equally effective on a per-mole basis. The effect of glycerol and MG on the ESEEM amplitudes of various protein nuclei was studied in ras p21 ⅐ Mn(II) ⅐ 5guanylylimido-diphosphate (p21 ⅐ Mn(II)-GMPPNP) complexes: these additives did not alter the distances of these nuclei from the Mn(II) ion. In particular, in p21 incorporating [ 2 H-3]Thr, the Mn(II)-[ 2 H-3]Thr35 distance was found to be unaffected by the concentration of cryoprotectant or the rate of freezing. The proximity of the cryoprotectants to the Mn(II) ion was probed by 2 H ESEEM in solutions made with d 5 -glycerol and d 7methyl ␣-D-glucopyranoside (d 7 -MG). In p21 ⅐ Mn(II)GMPPNP, the large deuterium modulations from the d 5 -glycerol exhibit saturation behavior with increasing d 5 -glycerol concentration, implying that glycerol, a widely used cryoprotectant, replaces the aquo ligands of the Mn(II) ion. The interaction between the Mn(II) ion of p21 and MG, however, is less intimate: the deuterium ESEEM amplitudes are much smaller for samples prepared with d 7 -MG than with d 5 -glycerol. Several polyhydroxylic compounds were found to have essentially no effect on the ability of the guanosine 5-triphosphate (GTP) hydrolysis activating protein, GAP334, to catalyze hydrolysis of p21 ⅐ guanosine 5-triphosphate. This observation implies that the introduction of cryoprotectant does not significantly perturb the structure of p21 and gives insight into the mechanism of the GTPase reaction.
Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this app... more Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its in...
Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thro... more Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H202 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine. (J.
Proceedings of the National Academy of Sciences, 1993
Recent discoveries suggesting essential bioactivities of nitric oxide (NO') in the lung are diffi... more Recent discoveries suggesting essential bioactivities of nitric oxide (NO') in the lung are difficult to reconcile with the established pulmbnary cytotoxicity of this common air pollutant. These conflicting observations suggest that metabolic intermediaries may exist in the lung to modulate the bioactivity and toxicity of NO'. We report that S-nitrosothiols
Proceedings of the National Academy of Sciences, 1992
We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generat... more We have recently shown that nitric oxide or authentic endothelium-derived relaxing factor generated in a biologic system reacts in the presence of specific protein thiols to form S-nitrosoprotein derivatives that have endotheliumderived relaxing factor-like properties. The single free cysteine of serum albumin, Cys-34, is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK; given its abundance in plasma, where it accounts for -0.5 mM thiol, we hypothesized that this plasma protein serves as a reservoir for nitric oxide produced by the endothelial cell. To test this hypothesis, we developed a methodology, which involves UV photolytic cleavage of the S-NO bond before reaction with ozone for chemiluminescence detection, with which to measure free nitric oxide, S-nitrosodiols, and S-nitrosoproteins in biologic systems. We found that human plasma contains =7 pIM S-nitrosothiols, of which 96% are S-nitrosoproteins, 82% of which is accounted for by S-nitrosoerum albumin. By contrast, plasma levels of free nitric oxide are only in the 3-nM range. In rabbits, plasma S-nitrosothiols are present at -1 #M; 60 min after administration of NG-monomethyl-Larginine at 50 mg/ml, a selective and potent inhibitor of nitric oxide synthetases, S-nitrosothiols decreased by "40% (>95% of which were accounted for by S-nitrosoproteins, and ""80% of which was S-nitroso-serum albumin); this decrease was accompanied by a concomitant increase in mean arterial blood pressure of 22%. These data suggest that naturally produced nitric oxide circulates in plasma primarily complexed in S-nitrosothiol species, principal among which is S-nitroso-serum albumin. This abundant, relatively long-lived adduct likely serves as a reservoir with which plasma levels of highly reactive, short-lived free nitric oxide can be regulated for the maintenance of vascular tone.
Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biologic... more Nitric oxide (NO.), a potentially toxic molecule, has been implicated in a wide range of biological functions. Details of its biochemistry, however, remain poorly understood. The broader chemistry of nitrogen monoxide (NO) involves a redox array of species with distinctive properties and reactivities: NO+ (nitrosonium), NO., and NO- (nitroxyl anion). The integration of this chemistry with current perspectives of NO biology illuminates many aspects of NO biochemistry, including the enzymatic mechanism of synthesis, the mode of transport and targeting in biological systems, the means by which its toxicity is mitigated, and the function-regulating interaction with target proteins.
Proceedings of the National Academy of Sciences, 1992
Endothelium-derived relaing factor (EDRF) activity has been attributed to the highly labile nitri... more Endothelium-derived relaing factor (EDRF) activity has been attributed to the highly labile nitric oxide radical (NO). In view of the fact that the plasma and cellular mifleux contain reactive species that can rapidly inactivate NO, it has been postulated that NO is stabilized by a carrier molecule that preserves its biological activity. Reduced thiol species are candidates for this role, reacting readily in the presence of NO to yield biologically active S-nitrosothiols that are more stable than NO itself. Because sulfhydryl groups in proteins represent an abundant source of reduced thiol in biologic systems, we examined the reaction of several sulfhy--I dryl-containing proteins of diverse nature and function upon exposure to authentic NO and EDRF. We demonstrate that S-nitroso proteins form readily under physiologic conditions and possess EDRF-like effects of vasodilation and platelet inhibition. These observations suggest that S-nitrosothiol groups in proteins may serve as intermediates in the cellular metabolism ofNO and raise the possibility ofan additional type of cellular regulatory mechanism.
A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with ... more A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both t...
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Papers by David Singel