Polyethylene Glycol Camouflaged Earthworm Hemoglobin.

Nearly 21 million components of blood and whole blood and transfused annually in the United States, while on average only 13.6 million units of blood are donated. As the demand for Red Blood Cells (RBCs) continues to increase due to the aging population, this deficit will be more significant. Despite decades of research to develop hemoglobin (Hb) based oxygen (O2) carriers (HBOCs) as RBC substitutes, there are no products approved for clinical use. Lumbricus terrestris erythrocruorin (LtEc) is the large acellular O2 carrying protein complex found in the earthworm Lumbricus terrestris. LtEc is an extremely stable protein complex, resistant to autoxidation, and capable of transporting O2 to tissue when transfused into mammals. These characteristics render LtEc a promising candidate for the development of the next generation HBOCs. LtEc has a short half-life in circulation, limiting its application as a bridge over days, until blood became available. Conjugation with polyethylene glycol (PEG-LtEc) can extend LtEc circulation time. This study explores PEG-LtEc pharmacokinetics and pharmacodynamics. To study PEG-LtEc pharmacokinetics, hamsters instrumented with the dorsal window chamber were subjected to a 40% exchange transfusion with 10 g/dL PEG-LtEc or LtEc and followed for 48 hours. To study the vascular response of PEG-LtEc, hamsters instrumented with the dorsal window chamber received multiple infusions of 10 g/dL PEG-LtEc or LtEc solution to increase plasma LtEc concentration to 0.5, then 1.0, and 1.5 g/dL, while monitoring the animals' systemic and microcirculatory parameters. Results confirm that PEGylation of LtEc increases its circulation time, extending the half-life to 70 hours, 4 times longer than that of unPEGylated LtEc. However, PEGylation increased the rate of LtEc oxidation in vivo. Vascular analysis verified that PEG-LtEc showed the absence of microvascular vasoconstriction or systemic hypertension. The molecular size of PEG-LtEc did not change the colloid osmotic pressure or blood volume expansion capacity compared to LtEc, due to LtEc's already large molecular size. Taken together, these results further encourage the development of PEG-LtEc as an O2 carrying therapeutic.

Evaluating the capacity to generate and preserve nitric oxide bioactivity in highly purified earthworm erythrocruorin: a giant polymeric hemoglobin with potential blood substitute properties.

The giant extracellular hemoglobin (erythrocruorin) from the earth worm (Lumbricus terrestris) has shown promise as a potential hemoglobin-based oxygen carrier (HBOC) in in vivo animal studies. An important beneficial characteristic of this hemoglobin (LtHb) is the large number of heme-based oxygen transport sites that helps overcome issues of osmotic stress when attempting to provide enough material for efficient oxygen delivery. A potentially important additional property is the capacity of the HBOC either to generate nitric oxide (NO) or to preserve NO bioactivity to compensate for decreased levels of NO in the circulation. The present study compares the NO-generating and NO bioactivity-preserving capability of LtHb with that of human adult hemoglobin (HbA) through several reactions including the nitrite reductase, reductive nitrosylation, and still controversial nitrite anhydrase reactions. An assignment of a heme-bound dinitrogen trioxide as the stable intermediate associated with the nitrite anhydrase reaction in both LtHb and HbA is supported based on functional and EPR spectroscopic studies. The role of the redox potential as a factor contributing to the NO-generating activity of these two proteins is evaluated. The results show that LtHb undergoes the same reactions as HbA and that the reduced efficacy for these reactions for LtHb relative to HbA is consistent with the much higher redox potential of LtHb. Evidence of functional heterogeneity in LtHb is explained in terms of the large difference in the redox potential of the isolated subunits.

Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin.

Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and ß solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

Generating S-nitrosothiols from hemoglobin: mechanisms, conformational dependence, and physiological relevance.

In vitro, ferrous deoxy-hemes in hemoglobin (Hb) react with nitrite to generate nitric oxide (NO) through a nitrite reductase reaction. In vivo studies indicate Hb with nitrite can be a source of NO bioactivity. The nitrite reductase reaction does not appear to account fully for this activity because free NO is short lived especially within the red blood cell. Thus, the exporting of NO bioactivity both out of the RBC and over a large distance requires an additional mechanism. A nitrite anhydrase (NA) reaction in which N2O3, a potent S-nitrosating agent, is produced through the reaction of NO with ferric heme-bound nitrite has been proposed (Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N., Ghosh, A., Gladwin, M. T., and Kim-Shapiro, D. B. (2007) Nat. Chem. Biol. 3, 785-794) as a possible mechanism. Legitimate concerns, including physiological relevance and the nature of the mechanism, have been raised concerning the NA reaction. This study addresses these concerns demonstrating NO and nitrite with ferric hemes under near physiological conditions yield an intermediate having the properties of the purported NA heme-bound N2O3 intermediate. The results indicate that ferric heme sites, traditionally viewed as a source of potential toxicity, can be functionally significant, especially for partially oxygenated/partially met-R state Hb that arises from the NO dioxygenation reaction. In the presence of low levels of nitrite and either NO or a suitable reductant such as L-cysteine, these ferric heme sites can function as a generator for the formation of S-nitrosothiols such as S-nitrosoglutathione and, as such, should be considered as a source of RBC-derived and exportable bioactive NO.

Enhanced nitrite reductase activity associated with the haptoglobin complexed hemoglobin dimer: functional and antioxidative implications.

The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αß dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb-Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αß Hb dimer. The presented results show that the Hb dimer in the Hb-Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb-Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb-Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.

Structural and functional studies indicating altered redox properties of hemoglobin E: implications for production of bioactive nitric oxide.

Hemoglobin (Hb) E (ß-Glu26Lys) remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms; for example, EE individuals exhibit a mild chronic anemia, and HbE/ß-thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac dysfunction. The purpose of this study was to determine and evaluate structural and functional consequences of the HbE mutation that might account for the pathophysiology. Functional studies indicate minimal allosteric consequence to both oxygen and carbon monoxide binding properties of the ferrous derivatives of HbE. In contrast, redox-sensitive reactions are clearly impacted as seen in the following: 1) the ∼2.5 times decrease in the rate at which HbE catalyzes nitrite reduction to nitric oxide (NO) relative to HbA, and 2) the accelerated rate of reduction of aquometHbE by L-cysteine (L-Cys). Sol-gel encapsulation studies imply a shift toward a higher redox potential for both the T and R HbE structures that can explain the origin of the reduced nitrite reductase activity of deoxyHbE and the accelerated rate of reduction of aquometHbE by cysteine. Deoxy- and CO HbE crystal structures (derived from crystals grown at or near physiological pH) show loss of hydrogen bonds in the microenvironment of ßLys-26 and no significant tertiary conformational perturbations at the allosteric transition sites in the R and T states. Together, these data suggest a model in which the HbE mutation, as a consequence of a relative change in redox properties, decreases the overall rate of Hb-mediated production of bioactive NO.

Sustained release nitric oxide from long-lived circulating nanoparticles.

The current limitations of nitric oxide (NO) delivery systems have stimulated an extraordinary interest in the development of compounds that generate NO in a controlled and sustained manner with a heavy emphasis on the treatment of cardiovascular disease states. This work describes the positive physiological response to the infusion of NO-releasing nanoparticles prepared using a new platform based on hydrogel/glass hybrid nanoparticles. When exposed to moisture, these nanoparticles slowly release therapeutic levels of NO, previously generated through thermal reduction of nitrite to NO trapped within the dry particles. The controlled and sustained release of NO observed from these nanoparticles (NO-np) is regulated by its hydration over extended periods of time. In a dose-dependent manner, circulating NO-np both decreased mean arterial blood pressure and increased exhaled concentrations of NO over a period of several hours. Circulating NO-np induced vasodilatation and increased microvascular perfusion during their several hour circulation lifetime. Control nanoparticles (control-np; without nitrite) did not induce changes in arterial pressure, although a decrease in the number of capillaries perfused and an increase in leukocyte rolling and immobilization in the microcirculation were observed. The NO released by the NO-np prevents the inflammatory response observed after infusion of control-np. These data suggest that NO release from NO-np is advantageous relative to other NO-releasing compounds, because it does not depend on chemical decomposition or enzymatic catalysis; it is only determined by the rate of hydration. Based on the observed physiological properties, NO-np has clear potential as a therapeutic agent and as a research tool to increase our understanding of NO signaling mechanisms within the vasculature.

NO reactions with sol-gel and solution phase samples of the ferric nitrite derivative of HbA.

The reaction of nitric oxide (NO) with the ferric (met) nitrite derivative of human adult hemoglobin Hb is probed for both solution phase and sol-gel encapsulated populations. The evolution of both the Q band absorption spectrum and fitted populations of Hb derivatives are used to show the sequence of events occurring when NO interacts with nitrite bound to a ferric heme in Hb. The sol-gel is used to compare the evolving populations as a function of quaternary state for the starting met-nitrite populations. The redox status of intermediates is probed using the CN(-) anion to trap ferric heme species. The emergent presence of reactive NO species such as N(2)O(3) during the course of the reaction is probed using the fluorescent probe DAF-2 whereas the fluorophore Chemifluor is used as an indirect measure of the ability of the reaction to create S-nitrosothiols on glutathione. The results are consistent with the formation of a stable reactive intermediate capable of generating bioactive forms of NO. The patterns observed are consistent with a proposed mechanism whereby NO reacts with the ferric nitrite derivative to generate N(2)O(3).

Conformational dependence of hemoglobin reactivity under high viscosity conditions: the role of solvent slaved dynamics.

The concept of protein dynamic states is introduced. This concept is based on (i) protein dynamics being organized hierarchically with respect to solvent slaving and (ii) which tier of dynamics is operative over the time window of a given measurement. The protein dynamic state concept is used to analyze the kinetic phases derived from the recombination of carbon monoxide to sol-gel-encapsulated human adult hemoglobin (HbA) and select recombinant mutants. The temperature-dependent measurements are made under very high viscosity conditions obtained by bathing the samples in an excess of glycerol. The results are consistent with a given tier of solvent slaved dynamics becoming operative at a time delay (with respect to the onset of the measurement) that is primarily solvent- and temperature-dependent. However, the functional consequences of the dynamics are protein- and conformation-specific. The kinetic traces from both equilibrium populations and trapped allosteric intermediates show a consistent progression that exposes the role of both conformation and hydration in the control of reactivity. Iron-zinc symmetric hybrid forms of HbA are used to show the dramatic difference between the kinetic patterns for T state alpha and beta subunits. The overall results support a model for allostery in HbA in which the ligand-binding-induced transition from the deoxy T state to the high -affinity R state proceeds through a progression of T state intermediates.

Ligand recombination and a hierarchy of solvent slaved dynamics: the origin of kinetic phases in hemeproteins.

Ligand recombination studies play a central role both for characterizing different hemeproteins and their conformational states but also for probing fundamental biophysical processes. Consequently, there is great importance to providing a foundation from which one can understand the physical processes that give rise to and modulate the large range of kinetic patterns associated with ligand recombination in myoglobins and hemoglobins. In this work, an overview of cryogenic and solution phase recombination phenomena for COMb is first reviewed and then a new paradigm is presented for analyzing the temperature and viscosity dependent features of kinetic traces in terms of multiple phases that reflect which tier(s) of solvent slaved protein dynamics is (are) operative on the photoproduct population during the time course of the measurement. This approach allows for facile inclusion of both ligand diffusion among accessible cavities and conformational relaxation effects. The concepts are illustrated using kinetic traces and MEM populations derived from the CO recombination process for wild type and mutant myoglobins either in sol-gel matrices bathed in glycerol or in trehalose-derived glassy matrices.

Molecular level probing of preferential hydration and its modulation by osmolytes through the use of pyranine complexed to hemoglobin.

Two spectroscopic probes are used to expose molecular level changes in hydration shell water interactions that directly relate to such issues as preferential hydration and protein stability. The major focus of the present study is on the use of pyranine (HPT) fluorescence to probe as a function of added osmolytes (PEG, urea, trehalose, and magnesium), the extent to which glycerol is preferentially excluded from the hydration shell of free HPT and HPT localized in the diphosphoglycerate (DPG) binding site of hemoglobin in both solution and in Sol-Gel matrices. The pyranine study is complemented by the use of vibronic side band luminescence from the gadolinium cation that directly exposes the changes in hydrogen bonding between first and second shell waters as a function of added osmolytes. Together the results form the basis for a water partitioning model that can account for both preferential hydration and water/osmolyte-mediated conformational changes in protein structure.

Nitrite reductase activity of sol-gel-encapsulated deoxyhemoglobin. Influence of quaternary and tertiary structure.

Nitrite reductase activity of deoxyhemoglobin (HbA) in the red blood cell has been proposed as a non-nitric-oxide synthase source of deliverable nitric oxide (NO) within the vasculature. An essential element in this scheme is the dependence of this reaction on the quaternary/tertiary structure of HbA. In the present work sol-gel encapsulation is used to trap and stabilize deoxy-HbA in either the T or R quaternary state, thus allowing for the clear-cut monitoring of nitrite reductase activity as a function of quaternary state with and without effectors. The results indicate that reaction is not only R-T-dependent but also heterotropic effector-dependent within a given quaternary state. The use of the maximum entropy method to analyze carbon monoxide (CO) recombination kinetics from fully and partially liganded sol-gel-encapsulated T-state species provides a framework for understanding effector modulation of T-state reactivity by influencing the distribution of high and low reactivity T-state conformations.

Modulation of reactivity and conformation within the T-quaternary state of human hemoglobin: the combined use of mutagenesis and sol-gel encapsulation.

A range of conformationally distinct functional states within the T quaternary state of hemoglobin are accessed and probed using a combination of mutagenesis and sol-gel encapsulation that greatly slow or eliminate the T --> R transition. Visible and UV resonance Raman spectroscopy are used to probe the proximal strain at the heme and the status of the alpha(1)beta(2) interface, respectively, whereas CO geminate and bimolecular recombination traces in conjunction with MEM (maximum entropy method) analysis of kinetic populations are used to identify functionally distinct T-state populations. The mutants used in this study are Hb(Nbeta102A) and the alpha99-alpha99 cross-linked derivative of Hb(Wbeta37E). The former mutant, which binds oxygen noncooperatively with very low affinity, is used to access low-affinity ligated T-state conformations, whereas the latter mutant is used to access the high-affinity end of the distribution of T-state conformations. A pattern emerges within the T state in which ligand reactivity increases as both the proximal strain and the alpha(1)beta(2) interface interactions are progressively lessened after ligand binding to the deoxy T-state species. The ligation and effector-dependent interplay between the heme environment and the stability of the Trp beta37 cluster in the hinge region of the alpha(1)beta(2) interface appears to determine the distribution of the ligated T-state species generated upon ligand binding. A qualitative model is presented, suggesting that different T quaternary structures modulate the stability of different alphabeta dimer conformations within the tetramer.

The position 68(E11) side chain in myoglobin regulates ligand capture, bond formation with heme iron, and internal movement into the xenon cavities.

After photodissociation, ligand rebinding to myoglobin exhibits complex kinetic patterns associated with multiple first-order geminate recombination processes occurring within the protein and a simpler bimolecular phase representing second-order ligand rebinding from the solvent. A smooth transition from cryogenic-like to solution phase properties can be obtained by using a combination of sol-gel encapsulation, addition of glycerol as a bathing medium, and temperature tuning (-15 --> 65 degrees C). This approach was applied to a series of double mutants, myoglobin CO (H64L/V68X, where X = Ala, Val, Leu, Asn, and Phe), which were designed to examine the contributions of the position 68(E11) side chain to the appearance and disappearance of internal rebinding phases in the absence of steric and polar interactions with the distal histidine. Based on the effects of viscosity, temperature, and the stereochemistry of the E11 side chain, the three major phases, B --> A, C --> A, and D --> A, can be assigned, respectively, to ligand rebinding from the following: (i) the distal heme pocket, (ii) the xenon cavities prior to large amplitude side chain conformational relaxation, and (iii) the xenon cavities after significant conformational relaxation of the position 68(E11) side chain. The relative amplitudes of the B --> A and C --> A phases depend markedly on the size and shape of the E11 side chain, which regulates sterically both ligand return to the heme iron atom and ligand migration to the xenon cavities. The internal xenon cavities provide a transient docking site that allows side chain relaxations and the entry of water into the vacated distal pocket, which in turn slows ligand recombination markedly.

Cysteine biosynthetic enzymes are the pieces of a metabolic energy pump.

Understanding the mechanisms of free energy transfer in metabolism is fundamental to understanding how the chemical forces that sustain the molecular organization of the cell are distributed. Recent studies of molecular motors (1-3) and ATP-driven proton transport (4-6) describe how chemical potential is transferred at the molecular level. These systems catalyze energy transfer through structural change and appear to be dedicated exclusively to their coupling tasks (7, 8). Here we report the discovery of a new class of energy-transfer system. It is a biosynthetic pump composed of cysteine biosynthesis enzymes, ATP sulfurylase and O-acetylserine sulfhydrylase, each with its own catalytic function and from whose interactions emerge new function: the hydrolysis of ATP. The hydrolysis is kinetically and energetically linked to the chemistry catalyzed by ATP sulfurylase, the first enzyme in the cysteine biosynthetic pathway, in such a way that each molecule of ATP hydrolyzed, each stroke of the pump, produces 1 equivalent of that enzyme's product. These findings integrate cysteine metabolism and broaden our understanding of the ways in which higher order allostery is used to effect free energy transfer.