Sol-gel Encapsulation Research Articles

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.

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.

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (alphaFeO2)2-(betaZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70-200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl-. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

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.

Sol–gel encapsulation extends diatom viability and reveals their silica dissolution capability

Several strains of diatom exhibit a long-term viability in silica gels and demonstrate the ability to dissolve the silica in their surroundings.

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.

Sol–gel encapsulation: An efficient and versatile immobilization technique for cutinase in non-aqueous media

Cutinase from Fusarium solani pisi was encapsulated in sol–gel matrices prepared with a combination of alkyl-alkoxysilane precursors of different chain-lengths. The specific activity of cutinase in a model transesterification reaction at fixed water activity in n-hexane was highest for the precursor combination tetramethoxysilane/ n-butyltrimetoxysilane (TMOS/BTMS) in a 1:5 ratio, lower and higher chain lengths of the mono-alkylated precursor or decreasing proportions of the latter relative to TMOS leading to lower enzyme activity. Results obtained using combinations of three precursors confirmed the beneficial effect of the presence of BTMS in the preparations. Scanning electron microscopy of the 1:5 TMOS/ n-alkylTMS gels showed a direct correlation between the macropore dimensions and the alkyl chain length of the alkylated precursor and revealed that TMOS/ n-octylTMS gels suffered extensive pore collapse during the drying process. The specific activity of TMOS/BTMS sol–gel entrapped cutinase was similar to that exhibited by the enzyme immobilized by adsorption on zeolite NaY. However, the incorporation of different additives (zeolites, silica, Biogel, grinded sol–gel, etc.) having in common the capability to react with residual silanol groups of the sol–gel matrix brought about remarkable enhancements of cutinase activity, despite the fact that the global porosity of the gels did not change. The behavior of the gels in supercritical CO 2 (sc-CO 2) paralleled that exhibited in n-hexane, although cutinase activity was ca. one order of magnitude lower (i.e. sol–gel encapsulation did not prevent the deleterious effect of CO 2). The impact that functionalization of some of the additives had on cutinase activity indicates that the enzyme/matrix interactions must play an important role. Some of the best additives from the standpoint of enzyme activity were also the best from the standpoint of its operational stability (ca. 80% retention of enzyme activity at the tenth reutilization cycle). None of the additives that proved effective for cutinase could improve the catalytic activity of sol–gel encapsulated Pseudomonas cepacia lipase.

Electronic nose screening of ethanol release during sol–gel encapsulation

Porous silica matrices prepared by sol-gel process yield biocompatible materials adequate for encapsulation of biomolecules or drugs. The procedure is simple and fast, but when alkoxyde precursors like tetraethoxysilane (TEOS) are used the polymerisation reaction leads to the formation of alcohol as a by-product, which can produce undesirable effects on the activity of entrapped enzymes or modify a drug release kinetic. Therefore, it is critical to determine that no remnant ethanol is left prior using or storing the obtained biomaterial. In this regard, the technique used in the alcohol determination should be non-invasive and non-destructive to preserve the encapsulation device intact and ready to use. In this work we have successfully used a portable electronic nose (e-nose) for the screening of silica polymerisation process during theophylline encapsulation. TEOS reaction was "smelt" since precursor pre-hydrolysis until the end of ethanol release, sensed directly at the headspace of matrices slabs. Measurements showed that ethanol was negligible since 10th day in polymeric slabs of 10 mm width and 2 cm diameter. This first use of e-nose following a polymerisation reaction opens a wide number of putative applications in pharmaceutical and biochemical fields.

Photo-induced proton gradients and ATP biosynthesis produced by vesicles encapsulated in a silica matrix

Sol-gel immobilization of soluble proteins has proven to be a viable method for stabilizing a wide variety of proteins in transparent inorganic matrices. The encapsulation of membrane-bound proteins has received much less attention, although work in this area suggests potential opportunities in microarray technology and high-throughput drug screening. The present paper describes a liposome/sol-gel architecture in which the liposome provides membrane structure and protein orientation to two transmembrane proteins, bacteriorhodopsin (bR) and F(0)F(1)-ATP synthase; the sol-gel encapsulation converts the liposomal solution into a robust material without compromising the intrinsic activity of the incorporated proteins. Here we report on two different proteoliposome-doped gels (proteogels) whose properties are determined by the transmembrane proteins. Proteogels containing bR proteoliposomes exhibit a stable proton gradient when irradiated with visible light, whereas proteogels containing proteoliposomes with both bR and F(0)F(1)-ATP synthase couple the photo-induced proton gradient to the production of ATP. These results demonstrate that materials based on the liposome/sol-gel architecture are able to harness the properties of transmembrane proteins and enable a variety of applications, from power generation and energy storage to the powering of molecular motors, and represent a new technology for performing complex chemical synthesis in a solid-state matrix.

Engineering hybrid nano-devices powered by the F<SUB align=right>1-ATPase biomolecular motor

Hybrid nano-devices that encompass organic and inorganic components offer truly unique and flexible functionalities. With integrated biomolecules and active proteins, it is plausible to envision seamless interfacing with nastive biological environments. The ability to incorporate inorganic features not only enables the synthesis of complex systems, it provides a myriad of options in custom tailoring devices to pertinent biomedical applications as well. This is especially true with the advent of more advanced nano-fabrication technologies in recent years. F1-ATPase is a motor protein whose rotary motion has been well characterised [1,2]. The biomolecular motor hydrolyses ATP to generate forces compatible with currently fabricated nano-mechanical structures. We have fabricated and operated a hybrid organic-inorganic nano-device powered by the F1-ATPase biomolecular motor [3–5]. The scope of the hybrid device developments was manifold including the local movement of surrounding suspended particles by functional organic-inorganic devices and in other efforts, the capability of implementing control mechanisms [6]. In addition, a renewable biological fuel (ATP) source has recently been developed through sol–gel encapsulation of proteoliposomes in an effort towards creating a self-sustaining device. The following paper is a review of the knowledge that has transpired upon developing hybrid organic-inorganic nano-devices to hopefully elicit future advancements of bio-nano-systems.

Effect of Sol?Gel Encapsulation on Lipase Structure and Function: A Small Angle Neutron Scattering Study

The application of small angle neutron scattering (SANS) to the characterisation of sol–gel hosts containing biomolecules offers the opportunity to explore the relationship between gel structure and catalyst. A model system involving the immobilisation of Candida antarctica lipase B (CALB) was investigated. Gels were produced by fluoride-catalysed hydrolysis of fixed ratios of tetramethylorthosilicate (TMOS) and methyltrimethoxysilane (MTMS). Phase separation between the enzyme and the evolving sol–gel matrix was minimised by incorporating glycerol into the sol–gel precursor solution. The potential stabilising effect of the NaF catalyst upon the enzyme was also investigated. Scattering studies were conducted on both immobilised lipase, and lipase in free solution. Scattering studies on free enzyme provided evidence of multiple populations of enzyme aggregates and showed that choice of solvent affected the degree of aggregation. Both NaF and glycerol affected neutron scattering, indicating changes in lipase conformation. Increasing glycerol concentration increased the degree of aggregation and produced differences in solvent packing on the surface of protein molecules. Initial evidence from SANS data indicated that the presence of the enzyme during gel formation conferred structural changes on the gel matrix. Modelling the effect of sol–gel encapsulation on lipase requires comparison of data from free enzyme to the immobilised form. Removal of the enzyme from the sol–gel structure, post gelation, is necessary to better characterise the modified matrix. This methodological problem will be the subject of future investigations.

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol–gel glasses

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase–peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase–peroxidase system was immobilized in a single sol–gel matrix. The enzymatic activity of the encapsulated superoxide dismutase–peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol–gel encapsulation preserved the hierarchy of the enzyme’s activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.

Sol–Gel Encapsulation of Molecules to Generate Functional Optical Materials: A Molecular Programming Approach

The extraordinary opportunities offered by integrating solution chemistry of molecular entities with the solid-state nature of the gel provide the basis for designing a number of novel molecular materials. Herein, we present a strategy based on encapsulation of suitable response active species to impart useful optical properties to sol–gel glasses. The basic concept of this molecular programming approach is based on deliberate incorporation of response-active species in the silica gel framework to elicit specific optical responses. Design of molecular materials for device applications depends on selection of molecules which exhibit well-defined electronic or optical response, and assembly of these molecular components into a geometric structure that retains the rigidity, addressability, and stability necessary for practical applications. The approach is based on using molecules as active species and sol–gel glass as structural matrix in which the molecules are selectively integrated. A designer approach that employs specific molecules for generating optical signals is described. As such the properties of these silica-based glasses can be tuned by varying the composition of encapsulated species. These modified glasses exhibit substantially altered optical properties as compared to pristine silica sol–gels. The optical response of these materials provide initial examples toward designing novel materials whose optical and/or photonic responses can be modulated by structural integration of specific dopant entities.

Aqueous sol–gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates

An all-aqueous sol–gel method for encapsulation of bacterial cells in porous silicate matrices towards the development of a biosensor is described. The sol–gel encapsulation of cells is achieved at room temperature and neutral pH. Furthermore, use of sodium silicate as precursor avoids generation of alcohol that can be detrimental to cells in contrast to the traditional alkoxide sol–gel encapsulation process. Moraxella spp. cells engineered to express recombinant organophosphorus hydrolase (OPH) on the cell surface were encapsulated and OPH enzymatic activity was measured for paraoxon hydrolysis. Kinetic parameters ( K m and V max) as well as pH behavior of surface-expressed OPH were determined to evaluate the effect of encapsulation. Cells encapsulated by the sodium silicate method displayed higher activity retention compared to those by the traditional alkoxide process. Time-course studies over a 2-month period indicate that immobilization through the sodium silicate process led to a reduction in activity of ∼5% as compared to ∼30% activity reduction in case of free cells in buffer indicating that immobilization leads to stabilization, a key parameter in biosensor development.

Utilization of a sol–gel method for encapsulation of doxorubicin

The sol-gel pre-doping method was used to encapsulate doxorubicin in silica gels and optimum conditions of preparation of drug-loaded gel were established, ensuring both reproducible and effective results of quantitative encapsulation of doxorubicin and its gradual but complete release. Doxorubicin was encapsulated in polysiloxane polymers using the method based on sol-gel encapsulation without a catalyst, with an acid catalyst (HCl) and a base catalyst (NH3). The time of gelation of the gel loaded with doxorubicin, encapsulation efficiency of the drug and the degree of release of the drug from the gel are all affected by the kind of catalyst (acidic or basic) or its absence at the gel preparation stage, and the temperature of the gelation process. The time of sol gelation when using the NH3 or HCl catalyst was 9 days at 21°C, 2 days at 30°C and 1.5 days at 37°C, while for the gel prepared without a catalyst it was 90 days at 21°C, 75 days at 30°C and 70 days at 37°C. The efficiency of doxorubicin encapsulation was 99.5 ± 0.5% (w/w) for acid-catalyzed gel, 98.9 ± 1.01% (w/w) for base-catalyzed gel and 86.4 ± 11.6% (w/w) for non-catalyzed gel. A 100% (w/w) release of doxorubicin by diffusion through pores was found only in the case of base-catalyzed gel after a 140-h incubation time. For acid-catalyzed gel and non-catalyzed gel, the total amounts of released doxorubicin after 140 h of incubation were 3-5% (w/w) and 9-11% (w/w), respectively. The stability of doxorubicin encapsulated in the three kinds of gel matrices was found to be improved compared to the stability of a free form of the drug in solution.

Distal heme pocket conformers of carbonmonoxy derivatives of Ascaris hemoglobin: evidence of conformational trapping in porous sol-gel matrices.

We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.

A reagent less fluorescent sol–gel biosensor for uric acid detection in biological fluids

The simultaneous encapsulation of a coupled uricase–peroxidase system and amplex red in a sol–gel matrix allows one to obtain a reagent-less and ready-to-use fluorescent biosensor for the accurate detection of uric acid in highly diluted biological fluids. The detection limit of the prepared biosensor was found to be 20 nM and was linear up to 1 μM. The high sensitivity found for the biosensor permitted a reliable determination of uric acid concentrations in the presence of interfering species (e.g., ascorbic acid) just by sample dilution (up to 50,000 for urine and 10,000 for serum and blood). The sol–gel encapsulation preserved the hierarchy of the enzyme activity as demonstrated by the performance of the fluorescent biosensor.

Mediator-free phenol sensor based on titania sol–gel encapsulation matrix for immobilization of tyrosinase by a vapor deposition method

A novel amperometric phenol sensor was constructed by immobilizing tyrosinase in a titania sol–gel matrix. The tyrosinase entrapped sol–gel film was obtained with a vapor deposition method, which simplified the traditional sol–gel process and avoided the shrinkage and cracking of conventional sol–gel-derived glasses. This matrix provided a microenvironment for retaining the native structure and activity of the entrapped enzyme and a very low mass transport barrier to the enzyme substrates. Phenol could be oxidized by dissolving oxygen in presence of immobilized tyrosinase to form a detectable product, which was determined at −150 mV without any mediator. The phenol sensor exhibited a fast response (less than 5 s) and sensitivity as high as 103 μA/mM, which resulted from the porous structure and high enzyme loading of the sol–gel matrix. The linear range for phenol determination was from 1.2×10 −7 to 2.6×10 −4 M with a detection limit of 1.0×10 −7 M. The apparent Michaelis–Menten constant of the encapsulated tyrosinase was calculated to be (0.29±0.02) mM. The stability of the biosensor was also evaluated.

Functional and spectroscopic characterization of half-liganded iron-zinc hybrid hemoglobin: evidence for conformational plasticity within the T state.

Functionally distinct conformations of HbA (human adult hemoglobin) were probed using deoxy and diliganded derivatives of symmetric Fe-Zn hybrids of HbA. To expand the range of accessible structures, different environments were utilized including solution, sol-gel encapsulation, and crystals. Further structural and functional modulation was achieved by the addition of allosteric effectors. Functional characterization included oxygen affinity measurements, CO combination rates, and geminate and bimolecular CO recombination, after photodissociation. The conformational properties were studied using visible resonance Raman spectroscopy as a probe of local tertiary structure at the iron-containing hemes and UV resonance Raman spectroscopy as a probe of elements of the globin known to be sensitive to quaternary structure. The combined results show a pattern in which there is a progression of conformational and functional properties that are consistent with a picture in which the T quaternary structure can accommodate a range of tertiary conformations (plasticity). At one end of the distribution is the equilibrium deoxy T state conformation that has the lowest ligand reactivity. At the other end of the distribution are T state conformations with higher ligand reactivity that exhibit "loosened" T state constraints within the globin including the alpha(1)beta(2) interface and reduced proximal strain at the heme.

Quaternary structure dependence of kinetic hole burning and conformational substates interconversion in hemoglobin.

Using a sol-gel encapsulation technique, we have prepared samples of CO saturated human adult hemoglobin locked in the R or T quaternary conformation. We report time-resolved spectra of these samples in the Soret region following flash photolysis, in the time interval ranging from 250 ns to 200 ms and in the temperature interval of 100-170 K. A suitable analysis of the measured difference spectra enables us to obtain the spectral contribution of deoxyHb and HbCO molecules as a function of time and/or of the fraction N(t) of deoxyHb molecules. In our experimental time window geminate CO rebinding to hemoglobin in the T quaternary conformation is about 2 orders of magnitude slower than to hemoglobin in the R conformation: this suggests that the barrier distribution for the CO rebinding, g(H), depends strongly on the protein quaternary structure. In our temperature interval, spectral shifts due to kinetic hole burning (KHB) are present: for HbCO the KHB effect is large in the R conformation and small in the T conformation. For deoxyHb the opposite is true. We attribute the observed behavior to the effect of interconversion between the relevant substates. This effect is stronger for HbCO molecules in the T conformation and for deoxyHb molecules in the R conformation; it confirms the quaternary structure dependence of the hemoglobin energy landscape and suggests enhanced dynamics of ligation intermediate species such as T-state HbCO or R-state deoxyHb.