Cyclic Hydroxylamines as Monitors of Peroxynitrite and Superoxide-Revisited.

There is a considerable need for methods that allow quantitative determination in vitro and in vivo of transient oxidative species such as peroxynitrite (ONOOH/ONOO-) and superoxide (HO2•/O2•-). Cyclic hydroxylamines, which upon oxidation yield their respective stable nitroxide radicals, have been suggested as spin probes of peroxynitrite and superoxide. The present study investigated this approach by following the kinetics of peroxynitrite decay in the absence and presence of various 5-membered and 6-membered ring hydroxylamines, and comparing the yield of their respective nitroxides using electron paramagnetic spectroscopy. The results demonstrate that hydroxylamines do not react directly with peroxynitrite, but are oxidized to their respective nitroxides by the radicals formed during peroxynitrite self-decomposition, namely •OH and •NO2. The accumulated nitroxides are far below their expected yield, had the hydroxylamines fully scavenged all these radicals, due to multiple competing reactions of the oxidized forms of the hydroxylamines with •NO2 and ONOO-. Therefore, cyclic hydroxylamines cannot be used for quantitative assay of peroxynitrite in vitro. The situation is even more complex in vivo where •OH and •NO2 are formed also via other oxidizing reactions systems. The present study also compared the yield of accumulated nitroxides under constant flux of superoxide in the presence of various cyclic hydroxylamines. It is demonstrated that certain 5-membered ring hydroxylamines, which their respective nitroxides are poor SOD-mimics, might be considered as stoichiometric monitors of superoxide in vitro at highest possible concentrations and pH.

Bifunctional Au-templated RNA nanoparticles enable direct cell uptake detection and GRP75 knockdown in prostate cancer.

Nucleic acids templated on gold (Au) surfaces have led to a wide range of functional materials ranging from microarrays, sensors and probes in addition to drug delivery and treatment. In this application, we describe a simple and novel method for templating amino-functionalized RNA onto Au surfaces and their self-assembly into small, discrete nanoparticles. In our method, sample hybridization with a complementary RNA strand with and without a fatty acid (palmitamide) appendage produced functionalized double-stranded RNA on the Au surface. The resulting Au-functionalized RNA particles were found to be stable under reducing conditions according to UV-Vis spectroscopy. Sample characterization by DLS and TEM confirmed self-assembly into primarily small (∼10-40 nm) spherical shaped nanoparticles expected to be amenable to cell biology. However, fluorescence emission (λexc: 350 nm, λem: 650 nm) revealed radiative properties which limited cell uptake detection. Introduction of FITC within the Au-functionalized RNA particles produced a bifunctional probe, in which FITC fluorescence emission (λexc: 494 nm, λem: 522 nm) facilitated cell uptake detection, in a time-dependent manner. The dual encapsulation-release profiles of the FITC-labeled Au-functionalized RNA particles were validated by time-dependent UV-Vis spectroscopy and spectrofluorimetry. These experiments respectively indicated an increase in FITC absorption (λabs: 494 nm) and fluorescence emission (λem: 522 nm) with increased sample incubation times, under physiological conditions. The release of Au-functionalized siRNA particles in prostate cancer (PC-3) cells resulted in concomitant knockdown of GRP75, which led to detectable levels of cell death in the absence of a transfection vector. Thus, the formulation of stable, small and discrete Au-functionalized RNA nanoparticles may prove to be valuable bifunctional probes in the theranostic study of cancer cells.

Electrochemical Resistive-Pulse Sensing.

Resistive-pulse sensing with biological or solid-state nanopores and nanopipettes has been widely employed in detecting single molecules and nanoparticles. The analytical signal in such experiments is the change in ionic current caused by the molecule/particle translocation through the pipet orifice. This paper describes a new version of the resistive-pulse technique based on the use of carbon nanopipettes (CNP). The measured current is produced by electrochemical oxidation/reduction of redox molecules at the carbon surface and responds to the particle translocation. In addition to counting single entities, this technique enables qualitative and quantitative analysis of the electroactive material they contain. Using liposomes as a model system, we demonstrate the capacity of CNPs for (1) conventional resistive-pulse sensing of single liposomes, (2) electrochemical resistive-pulse sensing, and (3) electrochemical identification and quantitation of redox species (e.g., ferrocyanide, dopamine, and nitrite) contained in a single liposome. The small physical size of a CNP suggests the possibility of single-entity measurements in biological systems.

Size Matters: Arginine-Derived Peptides Targeting the PSMA Receptor Can Efficiently Complex but Not Transfect siRNA.

Oligoarginine sequences conjugated to a short cancer-targeting peptide (CTP) selective for the prostate-specific membrane antigen (PSMA) receptor was developed for selective small interfering RNA (siRNA) delivery to a human metastatic/castration-resistant prostate cancer (PCa) cell line, which expresses PSMA on the surface. The PSMA-Rn (n = 6 and 9) peptides were synthesized by solid-phase peptide synthesis, characterized by liquid chromatography-mass spectrometry (LC-MS) and condensed with glucose-regulated protein (GRP)-silencing siRNAs. Native gels showed formation of stable CTP:siRNA ionic complexes. Furthermore, siRNA release was effected by heparin competition, supporting the peptides' capabilities to act as condensing and releasing agents. However, dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies revealed large anionic complexes that were prone to aggregation and limited cell uptake for RNAi activity. Taken together, these data support the notion that the development of efficient peptide-based siRNA delivery systems is in part contingent on the formulation of discrete nanoparticles that can effectively condense and release siRNA in cells.

Direct Transfection of Fatty Acid Conjugated siRNAs and Knockdown of the Glucose-Regulated Chaperones in Prostate Cancer Cells.

The emerging field of RNAi nanotechnology has led to rapid advances in the applications of siRNAs in chemical biology, medicinal chemistry, and biotechnology. In our RNAi approach, bioconjugation of linear, V-, and Y-shaped RNA templates were designed using a series of saturated and unsaturated fatty acids to improve cell uptake and knockdown efficacy of the oncogenic glucose regulated proteins (GRPs) in prostate (PC-3) cancer cells. An optimized HCTU-coupling procedure was developed for tagging variable saturated and unsaturated fatty acids onto the 5'-ends of linear and V-shaped RNA templates that were constructed by semiautomated solid phase RNA synthesis. Hybridization and self-assembly of complementary strands yielded linear, V-, and Y-shaped fatty acid-conjugated siRNAs which were characterized by native PAGE. CD spectroscopy confirmed their A-type helix conformations. RP IP HPLC provided trends in amphiphilic properties, whereas DLS and TEM confirmed multicomponent self-assembled structures that were prone to aggregation. Subsequently, the fatty acid conjugated siRNA bioconjugates were tested for their RNAi activity by direct transfection within PC-3 cells known to overexpress oncogenic GRP activity. The siRNA bioconjugates with sense strand modifiers provided more potent GRP knockdown relative to the antisense modified siRNAs, but to a lesser extent when compared to the unconjugated siRNA controls that were transfected with the commercial Trans-IT X2 dynamic delivery system. Flow cytometry revealed that the latter may be at least in part attributed to limited cell uptake of the fatty acid conjugated siRNAs. Nonetheless, these new constructs represent an entry point in modifying higher-order siRNA constructs that may lead to the generation of more efficient siRNA bioconjugates for screening important oncogene targets and for cancer gene therapy applications.

RNAi Screening of the Glucose-Regulated Chaperones in Cancer with Self-Assembled siRNA Nanostructures.

The emerging field of RNA nanotechnology has been used to design well-programmed, self-assembled nanostructures for applications in chemistry, biology, and medicine. At the forefront of its utility in cancer is the unrestricted ability to self-assemble multiple siRNAs within a single nanostructure formulation for the RNAi screening of a wide range of oncogenes while potentiating the gene therapy of malignant tumors. In our RNAi nanotechnology approach, V- and Y-shape RNA templates were designed and constructed for the self-assembly of discrete, higher-ordered siRNA nanostructures targeting the oncogenic glucose regulated chaperones. The GRP78-targeting siRNAs self-assembled into genetically encoded spheres, triangles, squares, pentagons and hexagons of discrete sizes and shapes according to TEM imaging. Furthermore, gel electrophoresis, thermal denaturation, and CD spectroscopy validated the prerequisite siRNA hybrids for their RNAi application. In a 24 sample siRNA screen conducted within the AN3CA endometrial cancer cells known to overexpress oncogenic GRP78 activity, the self-assembled siRNAs targeting multiple sites of GRP78 expression demonstrated more potent and long-lasting anticancer activity relative to their linear controls. Extending the scope of our RNAi screening approach, the self-assembled siRNA hybrids (5 nM) targeting of GRP-75, 78, and 94 resulted in significant (50-95%) knockdown of the glucose regulated chaperones, which led to synergistic effects in tumor cell cycle arrest (50-80%) and death (50-60%) within endometrial (AN3CA), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines. Interestingly, a nontumorigenic lung (MRC5) cell line displaying normal glucose regulated chaperone levels was found to tolerate siRNA treatment and demonstrated less toxicity (5-20%) relative to the cancer cells that were found to be addicted to glucose regulated chaperones. These remarkable self-assembled siRNA nanostructures may thus encompass a new class of potent siRNAs that may be useful in screening important oncogene targets while improving siRNA therapeutic efficacy and specificity in cancer.

Synthesis, characterization and anti-cancer activity of a peptide nucleolipid bioconjugate.

The synthesis, characterization and anti-cancer activity of a novel peptide nucleolipid bioconjugate is reported in this study. The prerequisite 5'-carboxy derived nucleolipid was synthesized following a five-step solution-phase approach and then coupled to the cytotoxic D-(KLAKLAK)2 sequence by solid-phase bioconjugation. The biophysical and structural properties of the peptide-nucleolipid bioconjugate were evaluated and compared to the peptide controls. These characterization studies revealed that the amphiphilic peptides favored helical-type secondary structures and well-defined nanoparticle formulations that were found to be contributive towards their biological activity. The peptide-nucleolipid bioconjugate displayed greater lethality in comparison to the native D-(KLAKLAK)2AK sequence when treated within the human A549 non-small cell lung carcinoma cell line. Thus, the amphiphilic peptide-nucleolipid forms a new class of anti-cancer peptides that may be developed into promising leads in the fight against cancer.

Nitroxide radicals as research tools: Elucidating the kinetics and mechanisms of catalase-like and "suicide inactivation" of metmyoglobin.

BACKGROUND: Metmyoglobin (MbFe(III)) reaction with H(2)O(2) has been a subject of study over many years. H(2)O(2) alone promotes heme destruction frequently denoted "suicide inactivation," yet the mechanism underlying H(2)O(2) dismutation associated with MbFe(III) inactivation remains obscure. METHODS: MbFe(III) reaction with excess H(2)O(2) in the absence and presence of the nitroxide was studied at pH 5.3-8.1 and 25°C by direct determination of reaction rate constants using rapid-mixing stopped-flow technique, by following H(2)O(2) depletion, O(2) evolution, spectral changes of the heme protein, and the fate of the nitroxide by EPR spectroscopy. RESULTS: The rates of both H(2)O(2) dismutation and heme inactivation processes depend on [MbFe(III)], [H(2)O(2)] and pH. Yet the inactivation stoichiometry is independent of these variables and each MbFe(III) molecule catalyzes the dismutation of 50±10 H(2)O(2) molecules until it is inactivated. The nitroxide catalytically enhances the catalase-like activity of MbFe(III) while protecting the heme against inactivation. The rate-determining step in the absence and presence of the nitroxide is the reduction of MbFe(IV)O by H(2)O(2) and by nitroxide, respectively. CONCLUSIONS: The nitroxide effects on H(2)O(2) dismutation catalyzed by MbFe(III) demonstrate that MbFe(IV)O reduction by H(2)O(2) is the rate-determining step of this process. The proposed mechanism, which adequately fits the pro-catalytic and protective effects of the nitroxide, implies the intermediacy of a compound I-H(2)O(2) adduct, which decomposes to a MbFe(IV)O and an inactivated heme at a ratio of 25:1. GENERAL SIGNIFICANCE: The effects of nitroxides are instrumental in elucidating the mechanism underlying the catalysis and inactivation routes of heme proteins.

Synergistic activity of acetohydroxamic acid on prokaryotes under oxidative stress: the role of reactive nitrogen species.

One-electron oxidation of acetohydroxamic acid (aceto-HX) initially gives rise to nitroxyl (HNO), which can be further oxidized to nitric oxide (NO) or react with potential biological targets such as thiols and metallo-proteins. The distinction between the effects of NO and HNO in vivo is masked by the reversible redox exchange between the two congeners and by the Janus-faced behavior of NO and HNO. The present study examines the ability of aceto-HX to serve as an HNO donor or an NO donor when added to Escherichia coli and Bacillus subtilis subjected to oxidative stress by comparing its effects to those of NO and commonly used NO and HNO donors. The results demonstrate that: (i) the effects of NO and HNO on the viability of prokaryotes exposed to H2O2 depend on the type of the bacterial cell; (ii) NO synergistically enhances H2O2-induced killing of E. coli, but protects B. subtilis depending on the extent of cell killing by H2O2; (iii) the HNO donor Angeli׳s salt alone has no effect on the viability of the cells; (iv) Angeli׳s salt synergistically enhances H2O2-induced killing of B. subtilis, but not of E. coli; (v) aceto-HX alone (1-4 mM) has no effect on the viability of the cells; (vi) aceto-HX enhances the killing of both cells induced by H2O2 and metmyoglobin, which may be attributed in the case of B. subtilis to the formation of HNO and to further oxidation of HNO to NO in the case of E. coli; (vii) the synergistic activity of aceto-HX on the killing of both cells induced by H2O2 alone does not involve reactive nitrogen species. The effect of aceto-HX on prokaryotes under oxidative stress is opposite to that of other hydroxamic acids on mammalian cells.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides.

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-µM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.

Synthesis, DNA binding and anti-leukemic activity of an aminoacyl nucleolipid.

The synthesis and characterization of a new class of DNA binding molecule exhibiting potent and selective anti-leukemic activity is described. The synthesis of an aminoacyl nucleolipid was developed from an efficient EEDQ coupling strategy, in which a series of seven bioconjugates were synthesized in yields of 53-78%. Guanosine bioconjugate 7, was used as building block for the synthesis of a target aminoacyl nucleolipid 14. Its GRP78 DNA binding affinity was confirmed by gel shift assay, CD spectroscopy, Tm measurements and dynamic light scattering experiments. Moreover, in a single dose (10 µM) screen against a panel of 60 cancer cell lines, aminoacyl nucleolipid 14 was found to selectively trigger greater than 90% cell death in a SR human leukemia cancer cell line. The reported aminoacyl nucleolipid represents a useful model for a new class of DNA binding molecules for the development of potent and selective anti-cancer agents.

The use of cyclic nitroxide radicals as HNO scavengers.

Reduction of cyclic stable nitroxides (RNO) by HNO to the respective hydroxylamines (RNO-H) has been demonstrated using EPR spectrometry. HNO shows low reactivity toward piperidine, pyrrolidine and nitronyl nitroxides with rate constants below 1.4 × 10(5)M(-1)s(-1) at pH 7.0, despite the high driving force for these reactions. The rate constants can be predicted assuming that the reactions take place via a concerted proton-electron transfer pathway and significantly low self-exchange rate constants for HNO/NO and RNO-H/RNO. NO does not react with piperidine and pyrrolidine nitroxides, but does add to HNO forming the highly oxidizing and moderately reducing hyponitrite radicals. In this work, the radicals are produced by pulse radiolysis and the rate constants of their reactions with 2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and 3-carbamoyl-PROXYL have been determined at pH 6.8 to be (2.4 ± 0.2)× 10(6), (9.8 ± 0.2)× 10(5), (5.9 ± 0.5)× 10(5)M(-1)s(-1), respectively. This low reactivity implies that NO competes efficiently with these nitroxides for the hyponitrite radical. The ability of TEMPOL and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO) to oxidize HNO and their different reactivity toward NO are used to quantify HNO formed via acetohydroxamic acid oxidation. The extent of TEMPOL or C-PTIO reduction was similar to the yield of HNO formed upon oxidation by ()OH under anoxia, but not by the metmyoglobin and H(2)O(2) reaction system where both nitroxides catalytically facilitate H(2)O(2) depletion and nitrite accumulation. In this system the conversion of C-PTIO into 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (C-PTI) is a minor reaction, which does not provide any mechanistic insight.

The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids.

BACKGROUND: The pharmacological effects of hydroxamic acids (RC(O)NHOH, HX) are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Given the development and use of HXs as therapeutic agents, elucidation of the oxidation mechanism is needed for more educated selection of HX-based drugs. METHODS: Acetohydroxamic and glycine-hydroxamic acids were oxidized at pH 7.0 by a continuous flux of radiolytically generated (·)OH or by metmyoglobin and H(2)O(2) reactions system. Gas chromatography and spectroscopic methods were used to monitor the accumulation of N(2)O, N(2), nitrite and hydroxylamine. RESULTS: Oxidation of HXs by (·)OH under anoxia yields N(2)O, but not nitrite, N(2) or hydroxylamine. Upon the addition of H(2)O(2) to solutions containing HX and metmyoglobin, which is instantaneously and continuously converted into compound II, nitrite and, to a lesser extent, N(2)O are accumulated under both anoxia and normoxia. CONCLUSIONS: Oxidation of HXs under anoxia by a continuous flux of (·)OH, which solely oxidizes the hydroxamate moiety to RC(O)NHO(·), forms HNO. This observation implies that bimolecular decomposition of RC(O)NHO(·) competes efficiently with unimolecular decomposition processes such as internal disproportionation, hydrolysis or homolysis. Oxidation by metmyoglobin/H(2)O(2) involves relatively mild oxidants (compounds I and II). Compound I reacts with HX forming RC(O)NHO(·) and compound II, which oxidizes HX, RC(O)NHO(·), HNO and NO. The latter reaction is the main source of nitrite. GENERAL SIGNIFICANCE: HXs under oxidative stress release HNO, but can be considered as NO-donors provided that HNO oxidation is more efficient than its reaction with other biological targets.

On the distinction between nitroxyl and nitric oxide using nitronyl nitroxides.

A better understanding of the origins of NO and HNO and their activities and biological functions requires accurate methods for their detection and quantification. The unique reaction of NO with nitronyl nitroxides such as 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (C-PTIO), which yields the corresponding imino nitroxides, is widely used for NO detection (mainly by electron paramagnetic resonance spectroscopy) and for modulation of NO-induced physiological functions. The present study demonstrates that HNO readily reacts with nitronyl nitroxides, leading to the formation of the respective imino nitroxides and hydroxylamines via a complex mechanism. Through the use of the HNO donor Angeli's salt (AS) with metmyoglobin as a competing agent, the rate constant for C-PTIO reduction by HNO has been determined to be (1.4 +/- 0.2) x 10(5) M(-1) s(-1) at pH 7.0. This reaction yields the corresponding nitronyl hydroxylamine C-PTIO-H and NO, which is trapped by C-PTIO to form (*)NO(2) and the corresponding imino nitroxide, C-PTI. (*)NO(2) oxidizes the nitronyl and imino nitroxides to their respective oxoammonium cations, which decay mainly via comproportionation with the nitronyl and imino hydroxylamines. When [AS] > [C-PTIO], the reduction of C-PTI by HNO proceeds, eventually converting C-PTIO to the corresponding imino hydroxylamine, C-PTI-H. Similar results were obtained for 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). It is concluded that nitronyl nitroxide is readily reduced by HNO to nitronyl hydroxylamine and is eventually converted into imino nitroxide and imino hydroxylamine. The yield of the imino hydroxylamine increases at the expense of the imino nitroxide as the ratio [AS](0)/[nitronyl nitroxide](0) is increased. Since the reaction of NO with nitronyl nitroxide yields only the corresponding imino nitroxide, nitronyl nitroxide can discriminate NO from HNO only when present at a concentration much lower than the total production of HNO.

Reactive oxygen species mediate hepatotoxicity induced by the Hsp90 inhibitor geldanamycin and its analogs.

Geldanamycin (GM), a benzoquinone ansamycin antibiotic, is a natural product inhibitor of Hsp90 with potent and broad anti-cancer properties. Because of its adverse effects on liver, its less toxic derivatives 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are currently being evaluated for the treatment of cancer. Previously, it has been demonstrated that the redox cycling of GM by NADPH-cytochrome P450 reductase leads to the formation of the GM semiquinone and superoxide radicals, the latter being identified using spin-trapping. We hypothesized that the different hepatotoxicity induced by GM, 17-AAG and 17-DMAG reflects the redox active properties of the quinone moiety and possibly the extent of superoxide formation, which may stimulate cellular oxidative injury. Our data demonstrate that superoxide can be efficiently trapped during the reduction of GM, 17-AAG and 17-DMAG by NADPH-cytochrome P450 reductase, and that superoxide formation rate followed the order 17-DMAG > 17-AAG > GM. In the absence of superoxide scavengers, the rate of NADPH oxidation followed the order 17-DMAG > GM > 17-AAG. The half-wave one-electron reduction potentials (E(1/2)) of GM, 17-AAG and 17-DMAG in DMSO have been determined to be -0.37, -0.13 and -0.015V (vs. Ag/AgCl), respectively. If the same order of E(1/2) follows in neutral aqueous media, thermodynamic considerations imply that 17-DMAG is more readily reduced by the P450 reductase as well as by superoxide. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their effect on cell viability and on intracellular oxidant level, was opposite to the order of E(1/2) of the respective quinone/semiquinone couples. These results suggest that hepatotoxicity exhibited by the Hsp90 inhibitors belonging to benzoquinone ansamycins could be attributed to superoxide. The apparent discrepancy between the order of toxicity and the orders of superoxide formation rate, which is correlated with E(1/2), is discussed.

The hemoglobins of the sub-Antarctic fish Cottoperca gobio, a phyletically basal species--oxygen-binding equilibria, kinetics and molecular dynamics.

The dominant perciform suborder Notothenioidei is an excellent study group for assessing the evolution and functional importance of biochemical adaptations to temperature. The availability of notothenioid taxa in a wide range of latitudes (Antarctic and non-Antarctic) provides a tool to enable identification of physiological and biochemical characteristics gained and lost during evolutionary history. Non-Antarctic notothenioids belonging to the most basal families are a crucial source for understanding the evolution of hemoglobin in high-Antarctic cold-adapted fish. This paper focuses on the structure, function and evolution of the oxygen-transport system of Cottoperca gobio, a sub-Antarctic notothenioid fish of the family Bovichtidae, probably derived from ancestral species that evolved in the Antarctic region and later migrated to lower latitudes. Unlike most high-Antarctic notothenioids, but similar to many other acanthomorph teleosts, C. gobio has two major hemoglobins having the beta chain in common. The oxygen-binding equilibria and kinetics of the two hemoglobins have been measured. Hb1 and Hb2 show strong modulation of oxygen-binding equilibria and kinetics by heterotropic effectors, with marked Bohr and Root effects. In Hb1 and Hb2, oxygen affinity and subunit cooperativity are slightly higher than in most high-Antarctic notothenioid hemoglobins. Hb1 and Hb2 show similar rebinding rates, but also show significant dynamic differences that are likely to have functional consequences. Molecular dynamic simulations of C. gobio Hb1 were performed on the dimeric protein in order to obtain a better understanding of the molecular basis of structure/function relationships.

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.

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.