Antonini and Brunori's 1971 book "Hemoglobin and Myoglobin in Their Reactions with Ligands" was a truly remarkable publication that summarized almost 100 years of research on O2 binding to these globins. Over the ensuing 50 years, ultra-fast laser photolysis techniques, high-resolution and time resolved X-ray crystallography, molecular dynamics simulations, and libraries of recombinant myoglobin (Mb) and hemoglobin (Hb) variants have provided structural interpretations of O2 binding to these proteins. The resultant mechanisms provide quantitative descriptions of the stereochemical factors that govern overall affinity, including proximal and distal steric restrictions that affect iron reactivity and favorable positive electrostatic interactions that preferentially stabilize bound O2. The pathway for O2 uptake and release by Mb and subunits of Hb has been mapped by screening libraries of site-directed mutants in laser photolysis experiments. O2 enters mammalian Mb and the α and β subunits of human HbA through a channel created by upward and outward rotation of the distal His at the E7 helical position, is non-covalently captured in the interior of the distal cavity, and then internally forms a bond with the heme Fe(II) atom. O2 dissociation is governed by disruption of hydrogen bonding interactions with His (E7), breakage of the Fe(II)-O2 bond, and then competition between rebinding and escape through the E7-gate. The structural features that govern the rates of both the individual steps and overall reactions have been determined and provide the framework for: (1) defining the physiological functions of specific globins and their evolution; (2) understanding the clinical features of hemoglobinopathies; and (3) designing safer and more efficient acellular hemoglobin-based oxygen carriers (HBOCs) for transfusion therapy, organ preservation, and other commercially relevant O2 transport and storage processes.

Globins play a key role in regulating nitric oxide (NO) levels in all forms of life. Five key reactions of NO with mammalian muscle myoglobin (Mb) and red blood cell hemoglobin (Hb) have been examined: (1) reversible NO binding to Fe(II) forms; (2) reversible NO binding to Fe(III) forms; (3) NO dioxygenation by Fe(II)O-2 complexes; (4) autoxidation of Fe(II)NO complexes in the presence of O-2; and (5) autoreduction of Fe(III)NO complexes. NO reacts rapidly and almost irreversibly with deoxyMb(FeII) in the absence of O-2, whereas it reacts much more slowly and weakly with metMb(FeIII). The reaction of NO with Mb(FeII)O-2 is very rapid and results in oxidation of the iron atom and dioxygenation of NO to nitrate. Autoxidation of Mb(FeII)NO in air is determined by the slow rate of NO dissociation from the Fe(II)NO complex, which is followed by rapid O-2 binding to the newly formed deoxyMb(FeII) and dioxygenation of the displaced NO to generate NO3- and metMb(FeIII). MetMb(FeIII)NO autoreduces slowly by addition of a hydroxide ion to bound NO to generate nitrous acid and reduced deoxyMb (FeII), which immediately binds another NO to generate Mb(FeII)NO as the final product. The reverse of this process involves nitrite reduction to NO by deoxyMb(FeII), which can occur on physiological time scales when the globin concentration is in the millimolar range. The relevance of these processes to the regulation of NO metabolism by hemoglobins and myoglobins in humans and other organisms is discussed.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It actively acquires the essential nutrient iron from human hemoglobin (Hb) using the iron-regulated surface-determinant (Isd) system. This process is initiated when the closely related bacterial IsdB and IsdH receptors bind to Hb and extract its hemin through a conserved tri-domain unit that contains two NEAr iron Transporter (NEAT) domains that are connected by a helical linker domain. Previously, we demonstrated that the tri-domain unit within IsdH (IsdHN2N3) triggers hemin release by distorting Hb's F-helix. Here, we report that IsdHN2N3 promotes hemin release from both the α- and β-subunits. Using a receptor mutant that only binds to the α-subunit of Hb and a stopped-flow transfer assay, we determined the energetics and micro-rate constants of hemin extraction from tetrameric Hb. We found that at 37 °C, the receptor accelerates hemin release from Hb up to 13,400-fold, with an activation enthalpy of 19.5 ± 1.1 kcal/mol. We propose that hemin removal requires the rate-limiting hydrolytic cleavage of the axial HisF8 Nϵ–Fe3+ bond, which, based on molecular dynamics simulations, may be facilitated by receptor-induced bond hydration. Isothermal titration calorimetry experiments revealed that two distinct IsdHN2N3·Hb protein·protein interfaces promote hemin release. A high-affinity receptor·Hb(A-helix) interface contributed ∼95% of the total binding standard free energy, enabling much weaker receptor interactions with Hb's F-helix that distort its hemin pocket and cause unfavorable changes in the binding enthalpy. We present a model indicating that receptor-introduced structural distortions and increased solvation underlie the IsdH-mediated hemin extraction mechanism.

Hemoglobin functions as an oxygen transport protein, with each subunit containing a heme cofactor. We have developed a global disassembly model for human hemoglobin, linking hemin (ferric heme) disassociation and apo(heme-free)-protein unfolding pathways. The model was based on the evaluation of circular dichroism and visible absorbance measurements of guanidine hydrochloride-induced disassembly of holo (heme-bound)-hemoglobin and previous measurements of apohemoglobin unfolding. The populations of holo-intermediates and equilibrium disassembly parameters were determined quantitatively for adult and fetal hemoglobins. The key stages for disassembly into unfolded monomers are characterized by hemichrome intermediates with molten globule characteristics. Hemichromes, which occur when both hemin iron axial sites coordinate amino acids, are not energetically favored in native human hemoglobins. However, these hexacoordinate iron complexes are important for preventing hemin disassociation from partially unfolded species during early disassembly and late stage assembly events. Both our model evaluation and independent small angle X-ray scattering measurements demonstrate that heme disassociation during early disassembly leads to loss of tetrameric structural integrity. Dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells where the hemoglobin concentration is very high. This prediction explains why in the red cells of patients with unstable hemoglobinopathies, misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway is dominated by early hemin disassociation events, which leads to the generation of higher fractions of apo-subunits and free hemin known to damage to the integrity of blood vessel walls. Thus, our model illuminates the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis, and provides insights into the factors governing hemoglobin assembly during erythropoiesis. Significance Our deconvolution and global analysis of spectral data led to both the characterization of “hidden” hemichrome intermediates and the development of a quantitative model for human hemoglobin disassembly/assembly. The importance of this mechanism is several-fold. First, the hemoglobin system serves as a general biological model for understanding the role of oligomerization and cofactor binding in facilitating protein folding and assembly. Second, the fitted parameters provide: (a) estimates of hemin affinity for apoprotein states; (b) quantitative interpretations of the pathophysiology of hemoglobinopathies and other diseases associated with unstable globins and red cell lysis; (c) insights into the factors governing hemoglobin assembly during erythropoiesis; and (d) a framework for designing targeted hemoglobinopathy therapeutics.

Recent Advances: In this review, I have described four key conclusions from work done by my group and our close colleagues. (i) O-2 uptake by mammalian red cells is limited by diffusion through unstirred water layers adjacent to the cell surface and across cell-free layers adjacent to vessel walls. (ii) In most vertebrates, hemoglobins (Hbs) and myoglobins (Mbs), the distal histidine at the E7 helical position donates a strong hydrogen bond to bound O-2, which selectively enhances O-2 affinity, prevents carbon monoxide poisoning, and markedly slows autoxidation. (iii) O-2 binding to mammalian Hbs and Mbs occurs by migration of the ligand through a channel created by upward rotation of the His(E7) side chain, capture in the empty space of the distal pocket, and then coordination with the ferroprotoporphyrin IX (heme) iron atom. (iv) The assembly of Mbs and Hbs occurs by formation of molten globule intermediates, in which the N- and C-terminal helices have almost fully formed secondary structures, but the heme pockets are disordered and followed by high-affinity binding of heme. Critical Issues: These conclusions indicate that there are often compromises between O-2 transport function, holoprotein stability, and the efficiency of assembly. Future Directions: However, the biochemical mechanisms underlying these conclusions provide the framework for understanding globin evolution in greater detail and for engineering more efficient and stable globins.

We depend on the protein hemoglobin to transport oxygen from our lungs to our cells. Functional hemoglobin is a heterotetramer with each subunit encapsulating a metallocofactor heme within a cavity. Hemoglobinopathies resulting in cardiovascular complications could manifest via hemoglobin misassembly or instability events. These events can be triggered by accumulation of unstable disassembly intermediates or heme disassociation, facilitated following ferrous heme auto-oxidation into ferric heme (hemin). We used a combination of spectroscopy and molecular dynamics (MD) simulations approaches to resolve structural mechanisms of human hemoglobin disassembly, uncover hidden disassembly intermediates, and identify potential hemoglobinopathy therapeutic targets. Measurements of guanidine hydrochloride triggered reversible disassembly of hemoglobin were done of protein secondary structure loss through circular dichroism; and hemin environment changes through visible absorbance. Using these spectral measurements, we then developed a quantitative hemoglobin disassembly model that predicts the disassembly pathway would differ markedly between erythrocytes and acellular blood plasma environments. In hemoglobin packed erythrocytes, molten globule intermediates with hemichrome characteristics will dominate the pathway. Hemichromes have been implicated in hemoglobinopathies and are characterized by amino acid coordination at each of the hemin iron axial site. With cell lysis, hemoglobin dilution into plasma will promote hemin disassociation. Atomic level MD simulations were then performed to probe hemin disassociation pathways in folded hemoglobins. The simulations revealed that local protein backbone arrangements around the heme cavities sometimes lead to a bis-histidine hemichrome as an on-pathway intermediate. This intermediate has not been experimentally resolved and was seen to dampen hemin disassociation. We also discovered new electrostatics and pi-stacking interactions between hemin and certain residues lining the heme cavity. Thermodynamics integration method of MD simulations is independently being used to evaluate inter-subunit interactions and energetics related to destabilization of hemoglobin tetramer interfaces resulting from clinically relevant mutations.

After reacting with hydrogen peroxide (H2O2), sickle-cell hemoglobin (HbS, βE6V) remains longer in a highly oxidizing ferryl form (HbFe4+=O) and induces irreversible oxidation of “hot-spot” amino acids, including βCys-93. To control the damaging ferryl heme, here we constructed three HbS variants. The first contained a redox-active Tyr in β subunits (F41Y), a substitution present in Hb Mequon; the second contained the Asp (K82D) found in the β cleft of Hb Providence; and the third had both of these β substitutions. Both the single Tyr-41 and Asp-82 constructs lowered the oxygen affinity of HbS but had little or no effects on autoxidation or heme loss kinetics. In the presence of H2O2, both rHbS βF41Y and βF41Y/K82D enhanced ferryl Hb reduction by providing a pathway for electrons to reduce the heme via the Tyr-41 side chain. MS analysis of βCys-93 revealed moderate inhibition of thiol oxidation in the HbS single F41Y variant and dramatic 3- to 8-fold inhibition of cysteic acid formation in rHbS βK82D and βF41Y/K82D, respectively. Under hypoxia, βK82D and βF41Y/K82D HbS substitutions increased the delay time by ∼250 and 600 s before the onset of polymerization compared with the rHbS control and rHbS βF41Y, respectively. Moreover, at 60 °C, rHbS βK82D exhibited superior structural stability. Asp-82 also enhanced the function of Tyr as a redox-active amino acid in the rHbS βF41Y/K82D variant. We conclude that the βK82D and βF41Y substitutions add significant resistance to oxidative stress and anti-sickling properties to HbS and therefore could be potential genome-editing targets.

Institute for Quantitative Biomedicine, Rutgers University, Piscataway, NJ 08854; 7 BioSciences Department, Rice University, Houston, TX 77005; Department of Biochemistry 8 and Molecular Biology and The Sealy Center for Structural Biology and Molecular Biophysics, 9 University of Texas Medical Branch, Galveston, TX 77555; University of Texas Southwestern 10 Medical Center, Dallas, TX, 75390; Department of Chemistry & Chemical Biology, Rutgers 11 University, Piscataway, NJ 08854; Department of Chemistry, Rice University, Houston, TX 12 77005 13 14 *Correspondence to: 15 John S. Olson, olson@rice.edu 16 George N. Phillips, Jr., georgep@rice.edu 17 18

The assembly of hetero-tetrameric HbA is a multi-faceted pathway emphasized by both quaternary structure formations via the interacting alpha and beta subunits of HbA as well as the oxygen coordinating heme (Fe(II)-protoporphyrin IX) insertion within the heme cavity of each subunit. Folding transition states at different stages of this pathway could act as precursors for misassembly of HbA, leading to hemoglobinopathies and disruption of oxygen transport within the cardiovascular system. Simultaneous circular dichroism and visible absorbance measurements of guanidine hydrochloride induced unfolding of HbA showed that the initial formation of the alpha-beta dimer interface occurs via a molten globule heterodimer state with ∼30% helical content. Reversible hemin (Fe(III)-protoporphyrin IX) binding to the melted heme pockets in this transition state results in a six coordinate, low spin iron state known as a hemichrome, which stabilizes the hetero-dimer interface. Atomic level modeling of hemin disassociation from native HbA using the Amber 2018 molecular dynamics (MD) package showed that these hemichrome transition states can also occur in both the folded alpha and beta subunits at 37 °C via hexacoordination of the iron by internal histidines within the heme cavity. These atomic level simulations enable us to characterize the transitionary nature of bond breaking and formation with metal cofactors that is beyond the resolution limits of solution spectroscopy and X-ray crystallography. Modeling studies involving thermodynamics integration are also currently being undertaken to measure free energy change for HbA tetramer interface formation, which markedly enhances heme affinity of HbA.

Global and regional releases of 14C have resulted from nuclear weapons testing activities; assessment of the chemical behavior and mechanisms of environmental transport and deposition of this radionuclide can assist remediation strategy development efforts and provide insights into global carbon cycling processes. This work reports a systematic evaluation of 14C in surface soils taken from the Nevada National Security Site. Surface soil samples are derived from above- and underground test locations, with underground test sites representing a range from near complete containment to uncontrolled radioactive releases. Only one surface soil taken from a underground test location (i.e. the Baneberry shot) shows elevated 14C concentrations (319 ± 9 pMC) in addition to elevated concentrations of 137Cs, 60Co and 152Eu above regional backgrounds. Surface soils from above-ground test locations show extremely high 14C content (~1000 to 10,000 pMC); elevated concentrations of 152Eu and 60Co for these soils are also observed, with 137Cs at or below background levels. Taken together, these data suggest that 14C in surface soils from above-ground tests is primarily derived from in-situ neutron activation of the native soil material, whereas 14C in surface soils from underground tests may be from either recondensed particulate material or soil activation.

Iodine environmental measurement programs are in need of new materials with certified I-129 activity. Frequently I-129 measurements are validated in the literature using the standard material IAEA-375, Chernobyl soil, which is the only soil/sediment material with a recommended I-129 activity. IAEA-375 has not been available for purchase since 2010. This study is an extension of previous work at INL to include four additional standard materials that are commercially available (NIST materials: RM 8704, Buffalo River sediment, SRM 2710a, Montana I soil, and IAEA materials: SL-1, lake sediment, IAEA-385, Irish Sea sediment). These materials have certified or recommended activities for a variety of radionuclides but not for I-129. This paper reports a comparison of accelerator mass spectrometry (AMS) and thermal ionization mass spectrometry (TIMS) data for I-129 activity, as well as the I-129/127 ratios for these standards to assist in identifying a suitable alternative for IAEA-375. Two independent chemical separation and mass spectrometric analysis techniques have been applied in an effort to corroborate the data. Both methods were validated via analyses of IAEA-375 for I-129 and show good agreement with the recommended activity of 1.7 x 10(-3) Bq kg(-1) for I-129; (1.6 x 10(-3) Bq kg(-1) by AMS and 1.8 x 10(-3) Bq kg(-1) by TIMS) with both sets of results within the 95% confidence interval of the recommended value.

This article reviews the key biochemical mechanisms that govern O2 transport, NO scavenging, and oxidative degradation of acellular hemoglobin (Hb) and how these ideas have been used to try to develop strategies to engineer safer and more effective hemoglobin-based oxygen carriers (HBOCs). Significant toxicities due to acellular Hb have been observed after the administration of HBOCs or after the lysis of red cells, and include rapid clearance and kidney damage due to dissociation into dimers, haptoglobin binding, and macrophage activation; early O2 release leading to decreased tissue perfusion in capillary beds; interference with endothelial and smooth muscle signaling due to nitric oxide (NO) scavenging; autooxidization of heme iron followed by production of reactive oxygen species; and iron overload symptoms due to hemin loss, globin denaturation, iron accumulation, and further inflammation. Protein engineering can be used to mitigate some of these side effects, but requires an in-depth mechanistic understanding of the biochemical and biophysical features of Hb that regulate quaternary structure, O2 affinity, NO dioxygenation, and resistance to oxidation, hemin loss, and unfolding.

A variety of genetic techniques have been devised to determine cell lineage relationships during tissue development. Some of these systems monitor cell lineages spatially and/or temporally without regard to gene expression by the cells, whereas others correlate gene expression with the lineage under study. The GAL4 Technique for Real-time and Clonal Expression (G-TRACE) system allows for rapid, fluorescent protein-based visualization of both current and past GAL4 expression patterns and is therefore amenable to genome-wide expression-based lineage screens. Here we describe the results from such a screen, performed by undergraduate students of the University of California, Los Angeles (UCLA) Undergraduate Research Consortium for Functional Genomics (URCFG) and high school summer scholars as part of a discovery-based education program. The results of the screen, which reveal novel expression-based lineage patterns within the brain, the imaginal disc epithelia, and the hematopoietic lymph gland, have been compiled into the G-TRACE Expression Database (GED), an online resource for use by the Drosophila research community. The impact of this discovery-based research experience on student learning gains was assessed independently and shown to be greater than that of similar programs conducted elsewhere. Furthermore, students participating in the URCFG showed considerably higher STEM retention rates than UCLA STEM students that did not participate in the URCFG, as well as STEM students nationwide.

Sample preparation for Accelerator Mass Spectrometry (AMS) analysis of I-129 from environmental samples typically involves isolation and purification of the iodine followed by precipitation as silver iodide. The silver iodide is mixed with silver or niobium powder as a binder and medium for electrical conductivity and approximately 3 mg of the mixture pressed into a cathode for AMS analysis. Electrodeposited silver iodide on a silver clad niobium 50 mu m diameter wire provides an attractive alternative to precipitation. Six inches of wire and electrodeposited silver iodide are easier to handle when pressing cathodes and minimizes possible cross contamination between samples. Although electrodeposition onto pure silver wires has been recently reported, the usage of silver clad niobium wires could offer an additional advantage. The use of niobium in the silver iodide matrix has been reported to result in higher intensity and long term stability of the iodide ion current. This paper presents a comparison of electrodeposition methods using silver and silver clad niobium wires to the traditional precipitated silver iodide mixed with silver and niobium powders. The comparison was done using materials with I-129/127 ratios having nominal values of 5 x 10(-)(10) and 5 x 10(-11). For each I-129/127 ratio material, eight samples were analyzed for each preparation method on three days, with seven replicate AMS measurements per sample. For each ratio, the measurements of the separate days were combined for each method, yielding 168 measurements per method. An analysis of variance indicated minor statistical differences between the precipitated and electro-deposited materials, with the pooled standard error of the means being 0.2% for both similar to 5 x 10(-10) and similar to 5 x 10(-11) ratios.

Idaho National Laboratory (INL) is a nuclear research facility located in southeastern Idaho, USA; over the course of its operational history, INL has operated 52 reactors and 1 reactor fuel reprocessing facility. To determine the extent to which previous nuclear operations at INL have impacted local environmental carbon-14 (C-14) concentrations, tree and soil samples from the INL desert and surrounding areas were collected, combusted, and analyzed by Accelerator Mass Spectrometry (AMS). Transport models of the plumes from the Advanced Test Reactor Facility (ATR) and the Idaho Chemical Processing Plant (ICPP) suggest the historic annual integrated plume distributions from each of these source terms was most likely in the northeast-southwest direction, with very little ground contact from stack emissions in the immediate vicinity of the facilities and maximum estimated ground contact arising similar to 400-1000 m to the northeast/southwest from each facility. C-14 data from annual growth ring data from trees located immediately adjacent to the ATR, INL's Central Facilities Area (CFA), and Mud Lake, Idaho (ML) are in statistical agreement with regional nuclear weapons testing fallout backgrounds. Surface soil samples taken near a low level radioactive disposal facility and downwind from the ICPP show percent modern carbon (pMC) values ranging from 28 +/- 2 to 92 +/- 4, suggesting a mixture of aged and modern background carbon containing materials. Taken together, these data suggest that C-14 in the INL region is predominantly derived from a mixture of aged and modern background sources (e.g. nuclear weapons testing fallout), with insignificant contributions from INL source terms.

Enriched isotope standards are required for routine nuclear fuel burnup and other radiochemical analyses. In 2017 Idaho National Laboratory (INL) generated isotope standard solutions containing enriched 85Rb, 136Ce, 142Nd, and 150Nd. Oxide and chloride forms of each enriched isotope were obtained from the Oak Ridge Isotope Production program, dissolved, and quantified by isotope dilution-multi collector mass spectrometry. Quantification of the isotope solutions was performed independently by two separate laboratories at INL: the Analytical Laboratory (AL) at the Material and Fuels Complex (MFC) and the Ultra-trace Analytical Laboratory (UAL) in Idaho Falls. Concentration measurement results for all solutions and 142Nd isotope ratio values reported by both laboratories are in quantitative agreement with each other, while isotope ratio values reported for 150Nd are observed to have an apparent ~0.8% bias between the results of each laboratory. Data for 85Rb/87Rb isotope ratio values for UAL TIMS measurements and AL MC-ICP-MS measurements are in quantitative agreement with each other, while those of UAL MC-ICP-MS are observed to be much higher due to the existence of unidentified polyatomic interferences in the 84-88 m/z range for this instrument. Concentration and isotope ratio values for these isotope tracer solutions, along with detailed description of the analytical and data analysis methods utilized, are reported herein.

Determining mechanisms for the autooxidation of hemoglobin is required for understanding and treating unstable hemoglobinopathies and for developing more stable hemoglobin based O2 carriers. Previous studies suggested significant differences in autooxidation rates of α and β subunits. We used an azide reaction assay to measure the concentrations of ferric α and β chains at different time points during autooxidation. Our results showed no differences between the subunits. To obtain more accurate time courses for autooxidation, we deconvoluted observed spectra into the decay of HbO2, metHb appearance, hemichrome generation, and increases in turbidity due to hemin loss and apoprotein precipitation. The time courses for HbO2 decay at high concentrations (≥ 100µM heme) accelerate implying cooperative autooxidation, where as at low concentrations (≤ 10uM) the time courses are biphasic. These results suggest that the biphasic time courses at low hemoglobin are due to differences between tetramers and dimers. We have also measured autooxidation rates for a recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-α subunit. This hemoglobin shows a monophasic time course for autooxidation at both high and low protein concentrations, and the azide binding assay showed equal amounts of ferric α and β subunits. We have also examined recombinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed that the rate of autooxidation shows a bell-shaped dependence on oxygen concentration and increases markedly as the pH is decreased. Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert A. Welch Foundation.

Removal of heme from human hemoglobin (Hb) results in formation of an apoglobin heterodimer. Titration of this apodimer with guanidine hydrochloride (GdnHCl) leads to biphasic unfolding curves indicating two distinct steps. Initially, the heme pocket unfolds and generates a dimeric intermediate in which ∼50% of the original helicity is lost, but the α1β1 interface is still intact. At higher GdnHCl concentrations, this intermediate dissociates into unfolded monomers. This structural interpretation was verified by comparing GdnHCl titrations for adult human hemoglobin A (HbA), recombinant fetal human hemoglobin (HbF), recombinant Hb cross-linked with a single glycine linker between the α chains, and recombinant Hbs with apolar heme pocket mutations that markedly stabilize native conformations in both subunits. The first phase of apoHb unfolding is independent of protein concentration, little affected by genetic cross-linking, but significantly shifted toward higher GdnHCl concentrations by the stabilizing distal pocket mutations. The second phase depends on protein concentration and is shifted to higher GdnHCl concentrations by genetic cross-linking. This model for apoHb unfolding allowed us to quantitate subtle differences in stability between apoHbA and apoHbF, which suggest that the β and γ heme pockets have similar stabilities, whereas the α1γ1 interface is more resistant to dissociation than the α1β1 interface.