PubMed Health⌕ Search

Biomedical subjects

David J Singel

Publications and source records attributed to David J Singel.

13 recordsLinked to original sources
PubMed2006

An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate.

Red blood cells (RBCs) act as O(2)-responsive transducers of vasodilator and vasoconstrictor activity in lungs and tissues by regulating the availability of nitric oxide (NO). Vasodilation by RBCs is impaired in diseases characterized by hypoxemia. We have proposed that the extent to which RBCs constrict vs. dilate vessels is, at least partly, controlled by a partitioning between NO bound to heme iron and to Cysbeta93 thiol of hemoglobin (Hb). Hemes sequester NO, whereas thiols deploy NO bioactivity. In recent work, we have suggested that specific micropopulations of NO-liganded Hb could support the chemistry of S-nitrosohemoglobin (SNO-Hb) formation. Here, by using nitrite as the source of NO, we demonstrate that a (T state) micropopulation of a heme-NO species, with spectral and chemical properties of Fe(III)NO, acts as a precursor to SNO-Hb formation, accompanying the allosteric transition of Hb to the R state. We also show that at physiological concentrations of nitrite and deoxyHb, a S-nitrosothiol precursor is formed within seconds and produces SNO-Hb in high yield upon its prompt exposure to O(2) or CO. Deoxygenation/reoxygenation cycling of oxyHb in the presence of physiological amounts of nitrite also efficiently produces SNO-Hb. In contrast, high amounts of nitrite or delays in reoxygenation inhibit the production of SNO-Hb. Collectively, our data provide evidence for a physiological S-nitrosothiol synthase activity of tetrameric Hb that depends on NO-Hb micropopulations and suggest that dysfunction of this activity may contribute to the pathophysiology of cardiopulmonary and blood disorders.

Hemoglobins↗
PubMed2005

Characterization of heterogeneously functionalized dendrimers by mass spectrometry and EPR spectroscopy.

Starburst dendrimers are receiving considerable attention as templates for the assembly of structured arrays of molecular components. This research motivates the development of improved methods for dendrimer characterization-specifically, for determining the numbers, distributions of numbers, and spatial distribution of molecular species synthetically attached to macromolecular templates. Such information provides the basis for advancing strategies aimed at controlling dendrimer functionalization, and thus represents enabling technology for tailoring the composition and structure of molecular arrays fashioned on dendrimer templates. Moreover, this information is vital to the proper interpretation of ongoing experiments in which dendrimers sparsely functionalized with reporter groups are used as probes. In this article, we report MALDI-TOF mass spectrometry and EPR spectroscopy of heterogeneously functionalized G(4)-PAMAM dendrimers bearing nitroxide spin-labels.

Dendrimers↗
PubMed2005

A nitric oxide processing defect of red blood cells created by hypoxia: deficiency of S-nitrosohemoglobin in pulmonary hypertension.

The mechanism by which hypoxia [low partial pressure of O(2) (pO(2))] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO(2-)governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). S-nitrosothiol (SNO)-deficient RBCs produce impaired vasodilator responses in vitro and exaggerated pulmonary vasoconstrictor responses in vivo and are defective in oxygenating the blood. RBCs from hypoxemic patients with elevated pulmonary arterial pressure (PAP) exhibit a similar FeNO/SNO imbalance and are thus deficient in pO(2)-coupled vasoregulation. Chemical restoration of SNO-Hb levels in both animals and patients restores the vasodilator activity of RBCs, and this activity is associated with improved oxygenation and lower PAPs.

Animals↗
PubMed2005

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions.

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the beta-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb--under conditions that suppress SNO-Hb formation--exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.

Biosensing Techniques↗
PubMed2005

Chemical physiology of blood flow regulation by red blood cells: the role of nitric oxide and S-nitrosohemoglobin.

Blood flow in the microcirculation is regulated by physiological oxygen (O2) gradients that are coupled to vasoconstriction or vasodilation, the domain of nitric oxide (NO) bioactivity. The mechanism by which the O2 content of blood elicits NO signaling to regulate blood flow, however, is a major unanswered question in vascular biology. While the hemoglobin in red blood cells (RBCs) would appear to be an ideal sensor, conventional wisdom about its chemistry with NO poses a problem for understanding how it could elicit vasodilation. Experiments from several laboratories have, nevertheless, very recently established that RBCs provide a novel NO vasodilator activity in which hemoglobin acts as an O2 sensor and O2-responsive NO signal transducer, thereby regulating both peripheral and pulmonary vascular tone. This article reviews these studies, together with biochemical studies, that illuminate the complexity and adaptive responsiveness of NO reactions with hemoglobin. Evidence for the pivotal role of S-nitroso (SNO) hemoglobin in mediating this response is discussed. Collectively, the reviewed work sets the stage for a new understanding of RBC-derived relaxing activity in auto-regulation of blood flow and O2 delivery and of RBC dysfunction in disorders characterized by tissue O2 deficits, such as sickle cell disease, sepsis, diabetes, and heart failure.

Blood Circulation↗
PubMed2004

EPR and affinity studies of mannose-TEMPO functionalized PAMAM dendrimers.

Mannose-TEMPO functionalized G4-PAMAM dendrimers with increasing mannose loadings have been synthesized and characterized by MALDI-TOF MS and EPR spectroscopy. Analysis of linebroadening effects in the EPR spectra of these dendrimers allowed us to determine the relative presentation of mannose and TEMPO on the dendrimer surface. Hemagglutination assays and affinity chromatography/EPR experiments to assess the activity of the mannose-TEMPO dendrimers with Concanavalin A are presented.

Chromatography, Affinity↗
PubMed2003

Altering the strength of lectin binding interactions and controlling the amount of lectin clustering using mannose/hydroxyl-functionalized dendrimers.

Protein-carbohydrate interactions play a critical role in many biological recognition events. Multivalent therapeutic agents that utilize protein-carbohydrate interactions have proven difficult to design, primarily because the fundamental requirements of protein-carbohydrate interactions are not well understood. Here, we report a systematic study of the effect on lectin binding of varying the loading of mannose surface residues on generations three through six PAMAM dendrimers. The degree of mannose functionalization was controlled by stoichiometric addition, and dendrimers were characterized using NMR and MALDI-TOF MS. Hemagglutination assays and quantitative precipitation assays were performed to determine the relative activity of the dendrimers. Using the mannose/hydroxyl-functionalized dendrimers reported here, we could systematically control both the degree of lectin clustering and the overall activity of the lectin with the dendrimer.

Concanavalin A↗
PubMed2003

Routes to S-nitroso-hemoglobin formation with heme redox and preferential reactivity in the beta subunits.

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the alpha subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the beta subunit (cysbeta-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of S-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the beta subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of beta subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme-iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.

Heme↗
PubMed2002

Nitric oxide in the human respiratory cycle.

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O(2)) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO(2) saturation (FeO(2)). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO(2) over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO(2). Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O(2) uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O(2) and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.

Erythrocytes↗