Variability of haptoglobin beta-chain proteoforms.

Existing knowledge on changes of the haptoglobin (Hp) molecule suggests that it may exist in multiple proteoforms, which obviously exhibit different functions. Using two-dimensional electrophoresis (2DE) in combination with mass spectrometry and immunodetection, we have analyzed blood plasma samples from both healthy donors and patients with primary grade IV glioblastoma (GBM), and obtained a detailed composite 2DE distribution map of ß-chain proteoforms, as well as the full-length form of Hp (zonulin). Although the total level of plasma Hp exceeded normal values in cancer patients (especially patients with GBM), the presence of particuar proteoforms, detected by their position on the 2DE map, was very individual. Variability was found in both zonulin and the Hp ß-chain. The presence of an alkaline form of zonulin in plasma can be considered a conditional, but insufficient, GBM biomarker. In other words, we found that at the level of minor proteoforms of Hp, even in normal conditions, there was a high individual variability. On the one hand, this raises questions about the reasons for such variability, if it is present not only in Hp, but also in other proteins. On the other hand, this may explain the discrepancy between the number of experimentally detected proteoforms and the theoretically possible ones not only in Hp, but also in other proteins.

Development and validation of a model and nomogram for breast cancer diagnosis based on quantitative analysis of serum disease-specific haptoglobin N-glycosylation.

BACKGROUND: A better diagnostic marker is in need to distinguish breast cancer from suspicious breast lesions. The abnormal glycosylation of haptoglobin has been documented to assist cancer diagnosis. This study aims to evaluate disease-specific haptoglobin (DSHp)-ß N-glycosylation as a potential biomarker for breast cancer diagnosis. METHODS: DSHp-ß chains of 497 patients with suspicious breast lesions who underwent breast surgery were separated from serum immunoinflammatory-related protein complexes. DSHp-ß N-glycosylation was quantified by mass spectrometric analysis. After missing data imputation and propensity score matching, patients were randomly assigned to the training set (n = 269) and validation set (n = 113). Logistic regression analysis was employed in model and nomogram construction. The diagnostic performance was analyzed with receiver operating characteristic and calibration curves. RESULTS: 95 N-glycopeptides at glycosylation sites N207/N211, N241, and N184 were identified in 235 patients with benign breast diseases and 262 patients with breast cancer. DSHp-ß N-tetrafucosyl and hexafucosyl were significantly increased in breast cancer compared with benign diseases (p < 0.001 and p = 0.001, respectively). The new diagnostic model and nomogram included GN2F2, G6N3F6, GN2FS at N184, G-N&G2S2, G2&G3NFS, G2N3F, GN3 at N207/N211, CEA, CA153, and could reliably distinguish breast cancer from benign diseases. For the training set, validation set, and training and validation sets, the area under the curves (AUCs) were 0.80 (95% CI: 0.75-0.86, specificity: 87%, sensitivity: 62%), 0.77 (95% CI:0.69-0.86, specificity: 75%, sensitivity: 69%), and 0.80 (95% CI:0.76-0.84, specificity: 77%, sensitivity: 68%), respectively. CEA, CA153, and their combination yielded AUCs of 0.62 (95% CI: 0.56-0.67, specificity: 29%, sensitivity: 90%), 0.65 (95% CI: 0.60-0.71, specificity: 74%, sensitivity: 51%), and 0.67 (95% CI: 0.62-0.73, specificity: 60%, sensitivity: 68%), respectively. CONCLUSIONS: The combination of DSHp-ß N-glycopeptides, CEA, and CA153 might be a better serologic marker to differentiate between breast cancer and benign breast diseases. The dysregulated N-glycosylation of serum DSHp-ß could provide insights into breast tumorigenesis.

A novel reagent for the screening of haptoglobin-deficient blood donors.

BACKGROUND AND OBJECTIVES: In Japan, the prevalence of haptoglobin deficiency is approximately 1 in 4000. Haptoglobin-deficient individuals may produce anti-haptoglobin from allo-immunization, leading to serious transfusion reactions. Therefore, implementation of a consistent supply of haptoglobin-deficient fresh frozen plasma is crucial. We developed a novel reagent to facilitate large-scale identification of haptoglobin-deficient individuals as potential donors of plasma products. MATERIALS AND METHODS: We established mouse monoclonal anti-haptoglobin-producing cell lines (three clones) using the hybridoma method by immunizing mice with the haptoglobin protein. Purified antibodies were conjugated with carboxylate-modified polystyrene latex beads and used for haptoglobin measurements by the latex agglutination method using an automatic analyser (LABOSPECT008). Samples with low protein concentrations were re-examined by enzyme-linked immunosorbent assay to confirm the results. Additionally, the haptoglobin gene was amplified by polymerase chain reaction to confirm the haptoglobin deletion allele (Hpdel ). RESULTS: From February to October 2022, 7476 blood donor samples were screened. Two haptoglobin-deficient and 21 low-haptoglobin-expressing individuals were identified. Two haptoglobin-deficient donors were found homozygous for Hpdel , and 19 (90%) of the 21 low-haptoglobin-expressing individuals were heterozygous for Hpdel , which includes the first reported case of heterozygous Hpdel /HpJohnson . CONCLUSION: We developed a new reagent for the detection of haptoglobin deficiency, which is automatable and inexpensive and appears useful for large-scale screening of blood donors.

In-depth investigation of altered glycosylation in human haptoglobin associated cancer by mass spectrometry.

Serum haptoglobin (Hp), a highly sialylated biomolecule with four N-glycosylation sites, is a positive acute-phase response glycoprotein that acts as an immunomodulator. Hp has gained considerable attention due to its potential as a signature molecule that exhibits aberrant glycosylation in inflammatory disorders and malignancies. Its glycosylation can be analyzed qualitatively and quantitatively by various methods using mass spectrometry. In this review, we have provided a brief overview of Hp structure and biological function and described mass spectrometry-based techniques for analyzing glycosylation ranging from macroheterogeneity to microheterogeneity of Hp in diseases and cancer. The sugars on haptoglobin can be a sweet bridge to link the potential of cancer-specific biomarkers to clinically relevant applications.

Calorimetric Characterisation of the Binding Reaction Between Human Ferric Haemoglobins and Haptoglobin to Develop a Drug for Removal of Cell-Free Haemoglobin.

High levels of cell-free haemoglobin (Hb) may occur in plasma as a consequence of e.g., pathological haemolysis or blood transfusion. These Hb molecules can be removed from blood circulation by forming a complex with the acute-phase protein haptoglobin (Hp) and thereby can also the intrinsic toxicity of free Hb be limited. In this study it is shown that ferric HbA, HbF, HbE and HbS, respectively, all bind firmly to Hp at 25 °C. By using isothermal titration calorimetry (ITC), it is demonstrated that ferric HbF has higher affinity to Hp (Ka = 2.79 ± 0.29 ×109 M-1) compared with HbA and HbS (1.91 ± 0.24 ×109 M-1) and 1.41 ± 0.34 ×109 M-1 for HbA and HbS, respectively. In addition, the affinity constant for HbE is slightly lower than the other haemoglobins (0.47 ± 0.40 ×109 M-1). Since Hp shows a general and high affinity to all Hb variants tested, it can be concluded that Hp may be useful as a therapeutic agent for several different haemolytic conditions by intravenous injection.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Evaluation of serum haptoglobin levels and Hp1-Hp2 polymorphism in the haptoglobin gene in patients with atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is an arrhythmia that involves structural and electrophysiological abnormalities. Many of the AF-related clinical conditions are associated with an increase in inflammatory and oxidative factors. Haptoglobin (Hp) is an acute phase protein whose biological role is to promote clearance of free hemoglobin (Hb). In addition, for being considered an inflammatory marker, Hp represents a protective mechanism against the oxidative effects of Hb. The Hp1-Hp2 polymorphism at Hp locus can lead to three phenotypes related to structural and functional differences in the protein. The objective of this study were to evaluate Hp levels and Hp1-Hp2 polymorphism at Hp locus in patients with AF compared to a control group. METHODS AND RESULTS: This study included 65 patients with AF and 54 individuals without the arrhythmia. Biochemical parameters were determined using Vitros system, plasma levels of Hp were measured in serum samples by using ELISA method and polymorphisms were verified by PCR technique. Plasma Hp levels, as well as allelic and genotypic frequency, were not associated with AF. The levels of Hp also did not differ among the genotypes according to the applied models. CONCLUSIONS: The results suggest that Hp levels and Hp1-Hp2 polymorphism are not associated to AF.

Rapid Evaluation of the Extent of Haptoglobin Glycosylation Using Orthogonal Intact-Mass MS Approaches and Multivariate Analysis.

Intact-mass measurements are becoming increasingly popular in mass spectrometry (MS) based protein characterization, as they allow the entire complement of proteoforms to be evaluated within a relatively short time. However, applications of this approach are currently limited to systems exhibiting relatively modest degrees of structural diversity, as the high extent of heterogeneity frequently prevents straightforward MS measurements. Incorporation of limited charge reduction into electrospray ionization (ESI) MS is an elegant way to obtain meaningful information on most heterogeneous systems, yielding not only the average mass of the protein but also the mass range populated by the entire complement of proteoforms. Application of this approach to characterization of two different phenotypes of haptoglobin (1-1 and 2-1) provides evidence of a significant difference in their extent of glycosylation (with the glycan load of phenotype 2-1 being notably lighter) despite a significant overlap of their ionic signals. More detailed characterization of their glycosylation patterns is enabled by the recently introduced technique of cross-path reactive chromatography (XP-RC) with online MS detection, which combines chromatographic separation with in-line reduction of disulfide bonds to generate metastable haptoglobin subunits. Application of XP-RC to both haptoglobin phenotypes confirms that no modifications are present within their light chains and provides a wealth of information on glycosylation patterns of the heavy chains. N-Glycosylation patterns of both haptoglobin phenotypes were found to be consistent with bi- and triantennary structures of complex type that exhibit significant level of fucosylation and sialylation. However, multivariate analysis of haptoglobin 1-1 reveals higher number of the triantennary structures, in comparison to haptoglobin 2-1, as well as a higher extent of fucosylation. The glycosylation patterns deduced from the XP-RC/MS measurements are in agreement with the conclusions of the intact-mass analysis supplemented by limited charge reduction, suggesting that the latter technique can be employed in situations when fast assessment of protein heterogeneity is needed (e.g., process analytical technology applications).

Hydroxyl radical footprinting analysis of a human haptoglobin-hemoglobin complex.

Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) - haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. FPOP footprinting using a timsTOF Pro mass spectrometer revealed structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3%. The most affected residues upon complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpß. The data allowed determination of amino acids directly involved in Hb - Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb ßTrp37 and Hp ßPhe292 was not confirmed by our data. Data are available via ProteomeXchange with identifier PXD021621.

Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production.

Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates.

Kinetic inequivalence between α and ß subunits of ligand dissociation from ferrous nitrosylated human haptoglobin:hemoglobin complexes. A comparison with O2 and CO dissociation.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) by trapping the αß dimers of Hb in the bloodstream. In turn, the Hp:Hb complexes display Hb-like reactivity. Here, the kinetics of NO dissociation from ferrous nitrosylated Hp:Hb complexes (i.e., Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO, respectively) are reported at pH 7.0 and 20.0 °C. NO dissociation from Hp:Hb(II)-NO complexes has been followed by replacing NO with CO. Denitrosylation kinetics of Hp1-1:Hb(II)-NO and Hp2-2:Hb(II)-NO are biphasic, the relative amplitude of the fast and slow phase being 0.495 ± 0.015 and 0.485 ± 0.025, respectively. Values of koff(NO)1 and koff(NO)2 (i.e., (6.4 ± 0.8) × 10-5 s-1 and (3.6 ± 0.6) × 10-5 s-1 for Hp1-1:Hb(II)-NO and (5.8 ± 0.8) × 10-5 s-1 and (3.1 ± 0.6) × 10-5 s-1 for Hp2-2:Hb(II)-NO) are unaffected by allosteric effectors and correspond to those reported for the α and ß subunits of tetrameric Hb(II)-NO and isolated α(II)-NO and ß(II)-NO chains, respectively. This highlights the view that the conformation of the Hb α1ß1 and α2ß2 dimers matches that of the Hb high affinity conformation. Moreover, the observed functional heterogeneity reflects the variation of energy barriers for the ligand detachment and exit pathway(s) associated to the different structural arrangement of the two subunits in the nitrosylated R-state. Noteworthy, the extent of the inequivalence of α and ß chains is closely similar for the O2, NO and CO dissociation in the R-state, suggesting that it is solely determined by the structural difference between the two subunits.

[Clinical and biological features of haptoglobin phenotypes]. / Phénotypes d'haptoglobine : impacts cliniques et biologiques.

Haptoglobin is a late positive acute phase protein of inflammation. Haptoglobin binds to free hemoglobin released from erythrocytes during intravascular hemolysis to form a complex which is removed shortly. Other properties like inhibition of oxidative stress and prostaglandin synthesis have been described. Three main phenotypes of haptoglobin have been identified: Hp1-1, Hp2-1, Hp2-2, which may have an impact in different diseases such as cardiovascular or infectious diseases. Haptoglobins of different phenotypes can be separated by capillary electrophoresis. They may induce a split of the alpha 2-globulin zone in the electrophoretic pattern. Hp1-1 and Hp2-1 phenotypes induce an important and a moderate split of the α2 globulin zone, respectively, whereas Hp2-2 does not. In vitro hemolysis and migration of a monoclonal component (i.e. immunoglobulin free light chain) may also induce a split of the alpha 2-globulin zone. In daily practice, Hp2-1 or Hp1-1 phenotypes could be notified in the electrophoresis report to alert the clinician about the possible physiopathological consequences.

Haptoglobin: From hemoglobin scavenging to human health.

Haptoglobin (Hp) belongs to the family of acute-phase plasma proteins and represents the most important plasma detoxifier of hemoglobin (Hb). The basic Hp molecule is a tetrameric protein built by two α/ß dimers. Each Hp α/ß dimer is encoded by a single gene and is synthesized as a single polypeptide. Following post-translational protease-dependent cleavage of the Hp polypeptide, the α and ß chains are linked by disulfide bridge(s) to generate the mature Hp protein. As human Hp gene is characterized by two common Hp1 and Hp2 alleles, three major genotypes can result (i.e., Hp1-1, Hp2-1, and Hp2-2). Hp regulates Hb clearance from circulation by the macrophage-specific receptor CD163, thus preventing Hb-mediated severe consequences for health. Indeed, the antioxidant and Hb binding properties of Hp as well as its ability to stimulate cells of the monocyte/macrophage lineage and to modulate the helper T-cell type 1 and type 2 balance significantly associate with a variety of pathogenic disorders (e.g., infectious diseases, diabetes, cardiovascular diseases, and cancer). Alternative functions of the variants Hp1 and Hp2 have been reported, particularly in the susceptibility and protection against infectious (e.g., pulmonary tuberculosis, HIV, and malaria) and non-infectious (e.g., diabetes, cardiovascular diseases and obesity) diseases. Both high and low levels of Hp are indicative of clinical conditions: Hp plasma levels increase during infections, inflammation, and various malignant diseases, and decrease during malnutrition, hemolysis, hepatic disease, allergic reactions, and seizure disorders. Of note, the Hp:Hb complexes display heme-based reactivity; in fact, they bind several ferrous and ferric ligands, including O2, CO, and NO, and display (pseudo-)enzymatic properties (e.g., NO and peroxynitrite detoxification). Here, genetic, biochemical, biomedical, and biotechnological aspects of Hp are reviewed.

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin.

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

Tangential flow filtration of haptoglobin.

Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 µm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.

Kinetics of cyanide and carbon monoxide dissociation from ferrous human haptoglobin:hemoglobin(II) complexes.

Haptoglobin (Hp) counterbalances the adverse effects of extra-erythrocytic hemoglobin (Hb) trapping the αß dimers of Hb. In turn, the Hp:Hb complexes display heme-based reactivity. Here, the kinetics of cyanide and carbon monoxide dissociation from ferrous-ligated Hp:Hb complexes are reported at pH 7.0 and 20.0 °C. Cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb-CN- has been followed upon the dithionite-mediated conversion of ferric to ferrous-ligated Hp:Hb complexes. Values of kon for the dithionite-mediated reduction of Hp1-1:Hb(III)-CN- and Hp2-2:Hb(III)-CN- are (7.3 ± 1.1) × 106 M-1 s-1 and (6.2 ± 1.0) × 106 M-1 s-1, respectively. Values of the first-order rate constant (i.e., h) for cyanide dissociation from Hp1-1:Hb(II)-CN- and Hp2-2:Hb(II)-CN- are (1.2 ± 0.2) × 10-1 s-1 and (1.3 ± 0.2) × 10-1 s-1, respectively. CO dissociation from Hp:Hb(II)-CO complexes has been followed by replacing CO with NO. Values of the first-order rate constant (i.e., l) for CO dissociation from Hp1-1:Hb(II)-CO are (1.4 ± 0.2) × 10-2 s-1 and (6.2 ± 0.8) × 10-3 s-1, and those from Hp2-2:Hb(II)-CO are (1.3 ± 0.2) × 10-2 s-1 and (7.3 ± 0.9) × 10-3 s-1. Values of kon, h, and l correspond to those reported for the R-state of tetrameric Hb and isolated α and ß chains. This highlights the view that the conformation of the Hb αß-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer. Furthermore, unlike ferric Hb(III), ligated ferrous Hb(II) does not show an assembly-linked structural change.

The human protein haptoglobin inhibits IsdH-mediated heme-sequestering by Staphylococcus aureus.

Iron is an essential nutrient for all living organisms. To acquire iron, many pathogens have developed elaborate systems to steal it from their hosts. The iron acquisition system in the opportunistic pathogen Staphylococcus aureus comprises nine proteins, called iron-regulated surface determinants (Isds). The Isd components enable S. aureus to extract heme from hemoglobin (Hb), transport it into the bacterial cytoplasm, and ultimately release iron from the porphyrin ring. IsdB and IsdH act as hemoglobin receptors and are known to actively extract heme from extracellular Hb. To limit microbial pathogenicity during infection, host organisms attempt to restrict the availability of nutrient metals at the host-pathogen interface. The human acute phase protein haptoglobin (Hp) protects the host from oxidative damage by clearing hemoglobin that has leaked from red blood cells and also restricts the availability of extracellular Hb-bound iron to invading pathogens. To investigate whether Hp serves an additional role in nutritional immunity through a direct inhibition of IsdH-mediated iron acquisition, here we measured heme extraction from the Hp-Hb complex by UV-visible spectroscopy and determined the crystal structure of the Hp-Hb-IsdH complex at 2.9 Å resolution. We found that Hp strongly inhibits IsdH-mediated heme extraction and that Hp binding prevents local unfolding of the Hb heme pocket, leaving IsdH unable to wrest the heme from Hb. Furthermore, we noted that the Hp-Hb binding appears to trap IsdH in an initial state before heme transfer. Our findings provide insights into Hp-mediated IsdH inhibition and the dynamics of IsdH-mediated heme extraction.

Ligand-dependent inequivalence of the α and ß subunits of ferric human hemoglobin bound to haptoglobin.

Haptoglobin (Hp) prevents extra-erythrocytic hemoglobin- (Hb-)mediated damage. Hp binds αß dimers of Hb, displaying heme-based reactivity. Here, kinetics and thermodynamics of cyanide, thiocyanate and imidazole binding to ferric human Hb (Hb(III)), Hb(III) dimers complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(III) and Hp2-2:Hb(III), respectively), and α(III) and ß(III) chains are reported and analyzed in parallel with fluoride and azide binding properties (at pH 7.0 and 20.0 °C). Cyanide and fluoride bind to Hb(III), Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and ß(III) with a simple behavior. In contrast, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) follows a two-step process, whereas ligand binding to α(III) and ß(III) chains follows a simple behavior. However, azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) is characterized by a simple equilibrium, reflecting the compensation of kinetic parameters. The fast and the slow step of azide, thiocyanate and imidazole binding to Hb(III), Hp1-1:Hb(III) and Hp2-2:Hb(III) mirror the ligand binding properties of the ß(III) and α(III) chains, respectively. Values of kinetic and thermodynamic parameters for binding of ferric ligands to Hp1-1:Hb(III) and Hp2-2:Hb(III) match very well with those obtained for ligation of Hb(III) and α(III) and ß(III) chains, confirming the ligand-dependent kinetic inequivalence of α(III) and ß(III) subunits. However, a variation between tetrameric Hb(III) on one side and Hp1-1:Hb(III), Hp2-2:Hb(III), α(III), and ß(III) on the other side for the rate-limiting step (likely referable to the dissociation of heme-coordinated H2O from the heme-Fe(III) atom) suggests a structural change(s) upon dimers to tetramer assembly in Hb(III).