Measurements of nitric oxide on the heme iron and β-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation

Xu, Xiuli; Cho, Man; Spencer, Netanya Y.; Patel, Neil K.; Huang, Zhi; Shields, Howard; King, S. Bruce; Gladwin, Mark T.; Hogg, Neil; Kim‐Shapiro, Daniel B.

doi:10.1073/pnas.2033883100

Cited by 81 publications

(60 citation statements)

References 45 publications

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“…The reactivity of S-nitrosylated Hb depends on the redox valency state, the ligation state of the hemes, and the number of SNO/Hb (5,12,37,(47)(48)(49). SNO-Hb standards previously used in triiodide assays (40)(41)(42)(43)(44)(45)(46) were synthesized with a large excess of low-molecular-weight SNO, which produces SNO-Hbs with Ϸ2 SNOs per tetramer; heme valency and ligation states of the standards were not characterized, and the samples were not assayed at physiological ratios of SNO/Hb. As an initial validation, we synthesized stock solutions of SNO oxyHb (50-100 M SNO/100 M Hb as assayed by photolysis; Ϸ15% Met) and verified under these conditions that amounts of SNO when measured by photolysis, triiodide, and Saville assays (39) were in good agreement (values by triiodide and photolysis were within 5%, P was not significant).…”

Section: See Also Triiodide Cautionary Notes In Si Text)mentioning

confidence: 99%

“…In this assay, biological samples are placed in acid plus triiodide to liberate NO. Other reagents [potassium ferricyanide (FeCN), potassium cyanide, N-ethylmaleimide (NEM), sulfanilamide (SAA), and mercurous chloride] are incorporated to selectively eliminate or block formation of one or another NO species (41)(42)(43)(44)(45). For example, pretreatment of biological samples with FeCN (3-200 mM followed by desalting) is reported to selectively remove NO from hemes, and SAA/HCl is reported to selectively eliminate nitrite; NEM and cyanide supposedly stabilize SNOs by blocking reactive thiols and hemes, respectively.…”

mentioning

confidence: 99%

“…Notably, the overall chemistry of triiodide that would specifically identify FeNOs has not been described (40)(41)(42)(43)(44)(45); thus, the chemistry behind the putative effects of added reagents (oxidants, reductants, electrophiles, and nucleophiles) that are used to differentiate FeNO, SNO, and nitrite remains unclear. Moreover, few NO standards have actually been tested, and no basis has been provided for asserting that response of these standards captures the general behavior.…”

mentioning

confidence: 99%

“…Moreover, few NO standards have actually been tested, and no basis has been provided for asserting that response of these standards captures the general behavior. Recovery of certain FeNO standards is reported to be as low as zero (40), and the one SNO-Hb standard that has been widely used (an R-structured Hb that contains Ϸ2 NO per tetramer) (40)(41)(42)(43)(44)(45)(46) is neither characteristic of general SNO-Hb reactivity nor of the reactivity of the micropopulation found in RBCs (a valency hybrid, estimated 1 NO per tetramer) (5,47). Furthermore, the claimed effects of added reagents in triiodide assays, including FeCN and NEM (alone and in combination), are not supported, and they, along with the acidic and denaturing conditions of the triiodide assay, can alter the reactivities of SNO and FeNO as well as disrupt the partitioning of NO species within hydrophobic compartments and thus lead to their misidentification.…”

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confidence: 99%

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Assessment of nitric oxide signals by triiodide chemiluminescence

Hausladen

Rafikov

Angelo

et al. 2007

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

101

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Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe-and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures. red blood cell vasodilation ͉ S-nitrosohemoglobin ͉ S-nitrosylationT he biological effects of nitric oxide (NO) are mediated in large part through binding to transition metals and cysteine thiols at active or allosteric sites within regulatory proteins (1), which elicits changes in protein activity, protein-protein interactions, and protein location (1). Within tissues, many dozens of S-nitrosylated proteins have been identified, and signatures of NO bound to nonheme and heme iron have been detected (1, 2). Additionally, NO can be transported in endocrine or paracrine fashion by reacting with heme iron and cysteine thiols in proteins [hemoglobin (Hb) and albumin] and peptides (glutathione and cysteinlyglycine) to form NO adducts with longer biological lifetimes (3-5); release of NO bioactivity from stable adducts is effected by allosteric and redox-based mechanisms that alter FeNO or S-nitrosothiol (SNO) reactivity (5, 6). An updated discussion of the factors influencing reactivity of S-nitrosohemoglobin, S-nitrosoalbumin, and low-molecular-weight SNO in the context of vasoregulation (5-15) can be found in supporting information (SI) Text.The dynamic distribution of protein and low-molecular-weight NO compounds that subserve NO transport and signaling instantiate the variation in both FeNO and SNO reactivities (4,6,13...

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Section: See Also Triiodide Cautionary Notes In Si Text)mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Assessment of nitric oxide signals by triiodide chemiluminescence

Hausladen

Rafikov

Angelo

et al. 2007

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

101

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“…The work of Gladwin and co-workers did not find evidence for the allosteric cycling of NO from the heme iron to the β93 Cys residues and they determined that generation of Cys-modified Hb did not influence the reaction kinetics [20][21][22]. These experiments were not supportive of the allosteric cycling model, and suggested that β93 Cys plays a minor role in the regulation of NO activity by Hb.…”

Section: Introductionmentioning

confidence: 97%

The role of β93 Cys in the inhibition of Hb S fiber formation

Knee

Roden

Flory

et al. 2007

Biophysical Chemistry

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Recent studies have suggested that nitric oxide (NO) binding to hemoglobin (Hb) may lead to the inhibition of sickle cell fiber formation and the dissolution of sickle cell fibers. NO can react with Hb in at least 3 ways: 1) formation of Hb(II)NO, 2) formation of methemoglobin, and 3) formation of S-nitrosohemoglobin, through nitrosylation of the β93 Cys residue. In this study, the role of β93 Cys in the mechanism of sickle cell fiber inhibition is investigated through chemical modification with N-ethylmaleimide. UV resonance Raman, FT-IR and electrospray ionization mass spectroscopic methods in conjunction with equilibrium solubility and kinetic studies are used to characterize the effect of β93 Cys modification on Hb S fiber formation. Both FT-IR spectroscopy and electrospray mass spectrometry results demonstrate that modification can occur at both the β93 and α104 Cys residues under relatively mild reaction conditions. Equilibrium solubility measurements reveal that singlymodified Hb at the β93 position leads to increased amounts of fiber formation relative to unmodified or doubly-modified Hb S. Kinetic studies confirm that modification of only the β93 residue leads to a faster onset of polymerization. UV resonance Raman results indicate that modification of the α104 residue in addition to the β93 residue significantly perturbs the α 1 β 2 interface, while modification of only β93 does not. These results in conjunction with the equilibrium solubility and kinetic measurements are suggestive that modification of the α104 Cys residue and not the β93 Cys residue leads to T-state destabilization and inhibition of fiber formation. These findings have implications for understanding the mechanism of NO binding to Hb and NO inhibition of Hb S fiber formation.

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Iron: Heme Proteins & Dioxygen Transport & StorageBased in part on the article Iron: Heme Proteins & Dioxygen Transport by Harold M. Goff which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

Hayashi

2005

Encyclopedia of Inorganic Chemistry

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Most organisms require dioxygen (O 2 ) to survive; however, simple O 2 diffusion in blood does not supply a sufficient amount of oxygen for respiration. Thus, in all mammals, O 2 is carried and stored by O 2 ‐binding heme proteins, myoglobin, and hemoglobin. Myoglobin is a single‐chain globular protein with heme as a prosthetic group, while hemoglobin consists of four heme‐containing subunits. O 2 binds to ferrous pentacoordinated heme‐iron and is stabilized by distal and proximal amino acid residues in the heme pocket. The O 2 pathway in the protein matrix and the binding mechanisms are discussed in detail by O 2 ‐binding kinetic data for a series of myoglobin and hemoglobin mutants. Particularly, several residues, B10, E7, E10, E11, F7, and F8, mainly regulate the O 2 binding in the vertebrate heme proteins. In contrast, invertebrate and plant hemoglobins with unusually high O 2 affinities have been found in nature. In Ascaris hemoglobin, O 2 dissociation is very slow, whereas leghemoglobin shows a large O 2 association rate constant. In the case of trematode hemoglobin from Paramphistonum epiclitum , both kinetic parameters are improved. Artificial molecular design to modify O 2 affinity is another way to understand the O 2 binding mechanisms of the heme proteins. The strategy of heme protein modification can be divided into at least two approaches: One is a point mutation at distal or proximal amino acid residues, and the other is reconstitution by replacement of the native heme with an artificial heme. The physiological function of vertebrate hemoglobins is O 2 transport from the lungs or gills to the tissues; thus, they are required to exhibit cooperative interactions in binding O 2 to heme‐iron in the protein matrix. Although it is well known that a Hill coefficient of approximately 3.0 has been observed for tetrameric human hemoglobin, the mechanism of allosteric interaction is still discussed based on theoretical and experimental data. At present, an a two‐state (MWC) model proposed by Monod, Wyman, and Changeux for cooperative ligand binding is preferred to a sequential model. In addition, a study on the allosteric effectors for hemoglobins seems to be attractive from physiological aspects. The conventional effectors such as organic polyphosphate for human hemoglobin reduce the O 2 affinity of the T‐state, while it has been found that bezafibrate (BZF) or inositol hexaphosphate (IHP) can regulate O 2 binding not only for the T‐state but also for the R‐state. Finally, the interaction of NO with the heme proteins and modification of myoglobin for conversion into a new oxidase are briefly described.

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Measurements of nitric oxide on the heme iron and β-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation

Cited by 81 publications

References 45 publications

Assessment of nitric oxide signals by triiodide chemiluminescence

Assessment of nitric oxide signals by triiodide chemiluminescence

The role of β93 Cys in the inhibition of Hb S fiber formation

Iron: Heme Proteins & Dioxygen Transport & StorageBased in part on the article Iron: Heme Proteins & Dioxygen Transport by Harold M. Goff which appeared in theEncyclopedia of Inorganic Chemistry, First Edition.

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