Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma

CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages.The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal some interesting perspectives relating to the suggested anti-inflammatory properties of the receptor and the Hp phenotypes.     

Oleacein enhances anti-inflammatory activity of human macrophages by increasing CD163 receptor expression

BackgroundOleacein (dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol; 3,4-DHPEA-EDA) have been proven to possess antioxidant and anti-inflammatory activity.PurposeIn this study, we examined whether oleacein could increase CD163 and IL-10 receptor expression as well as HO-1 intracellular secretion in human macrophages.MethodsEffect of oleacein (10 and 20 μmol/l) or oleacein together with complexes of haemoglobin (Hb) and haptoglobin 1-1 (Hp11) or haptoglobin 2-2 (Hp22) on expression of IL-10 and CD163 receptor was determined by Flow Cytometry. Expression of CD163mRNA was measured by real-time quantitative RT-PCR. Heme oxygenase 1 (HO-1) intracellular secretion in macrophages was investigated by enzyme-linked immunosorbent assay (ELISA).ResultsOleacein (OC) together with complexes HbHp11 or HbHp22 stimulated the expression of CD163 (30-100-fold), IL-10 (170-300-fold) and HO-1 secretion (60-130-fold) after 5 days of coincubation. The 2-fold (24 h), 4-fold (48 h) increase of CD163 mRNA level and its final (72 h) decrease was also observed.ConclusionOur results suggested that oleacein enhances anti-inflammatory activity of complexes haemoglobin with haptoglobin 1-1 and 2-2 and could play a potential role in the prevention of inflammatory disease related to atherosclerosis.     

Molecular characterization of the haptoglobin.hemoglobin receptor CD163. Ligand binding properties of the scavenger receptor cysteine-rich domain region

CD163 is the macrophage receptor for endocytosis of haptoglobin.hemoglobin complexes. The extracellular region consisting of nine scavenger receptor cysteine rich (SRCR) domains also circulates in plasma as a soluble protein. By ligand binding analysis of a broad spectrum of soluble CD163 truncation variants, the amino-terminal third of the SRCR region was shown to be crucial for the binding of haptoglobin.hemoglobin complexes. By Western blotting of the CD163 variants, a panel of ten monoclonal antibodies was mapped to SRCR domains 1, 3, 4, 6, 7, and 9, respectively. Only the two antibodies binding to SRCR domain 3 exhibited effective inhibition of ligand binding. Furthermore, analysis of purified native CD163 revealed that proteolytic cleavage in SRCR domain 3 inactivates ligand binding. Calcium protects against cleavage in this domain. Analysis of the calcium sensitivity of ligand binding to CD163 demonstrated that optimal ligand binding requires physiological plasma calcium concentrations, and an immediate ligand release occurs at the low calcium concentrations measured in acidifying endosomes. In conclusion, SRCR domain 3 of CD163 is an exposed domain and a critical determinant for the calcium-sensitive coupling of haptoglobin.hemoglobin complexes.     

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.     

CD163 Binding to Haptoglobin-Hemoglobin Complexes Involves a Dual-point Electrostatic Receptor-Ligand Pairing

Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.     

A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobin-hemoglobin complex

Haptoglobin and haptoglobin-related protein are homologous hemoglobin-binding proteins consisting of a complement control repeat (alpha-chain) and a serine protease domain (beta-chain). Haptoglobin-hemoglobin complex formation promotes high affinity binding of hemoglobin to the macrophage scavenger receptor CD163 leading to endocytosis and degradation of the haptoglobin-hemoglobin complex. In contrast, complex formation between haptoglobin-related protein and hemoglobin does not promote high affinity interaction with CD163. To define structural components of haptoglobin important for CD163 recognition, we exploited this functional difference to design and analyze recombinant haptoglobin/haptoglobin-related protein chimeras complexed to hemoglobin. These data revealed that only the beta-chain of haptoglobin is involved in receptor recognition. Substitution of 4 closely spaced amino acid residues of the haptoglobin beta-chain (valine 259, glutamate 261, lysine 262, and threonine 264) abrogated the high affinity receptor binding. The 4 residues are encompassed by a part of the primary structure not present in other serine protease domain proteins. Structural modeling based on the well characterized serine protease domain fold suggests that this sequence represents a loop extension unique for haptoglobin and haptoglobin-related protein. A synthetic peptide representing the haptoglobin loop sequence exhibited a pronounced inhibitory effect on receptor binding of haptoglobin-hemoglobin.     

Corticosteroid enhances heme oxygenase-1 production by circulating monocytes by up-regulating hemoglobin scavenger receptor and amplifying the receptor-mediated uptake of hemoglobin-haptoglobin complex

This study examined the relationship between steroid treatment and CD163-mediated downstream pathways linked to inflammatory resolution. Twelve patients referred for congenital heart disease surgery were divided into two groups based on the severity of intravascular hemolysis during cardiopulmonary bypass surgery. Patients with severe intravascular hemolysis were administered haptoglobin during the procedure. Flow cytometry indicated a peak in monocyte CD163 expression on post-operative day 1 in both groups. Enhanced and prolonged heme oxygenase-1 (HO-1) mRNA expression levels were observed in patients who received haptoglobin. Binding of hemoglobin-haptoglobin complex (Hb/Hp) to CD163 resulted in significant induction of HO-1 by peripheral blood mononuclear cells after exposure to dexamethasone prior to culture. This effect was significantly inhibited by anti-CD163 antibody. Our results demonstrated up-regulation of CD163 expression on the monocyte surface by steroid treatment. Steroid treatment was suggested to facilitate CD163-mediated endocytosis of hemoglobin to monocytes/macrophages and thereby induce acceleration of HO-1 synthesis.     

Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin

Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein–hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein–hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin–hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin–hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele.     

Haptoglobin-hemoglobin binding involves the heavy chain of haptoglobin and the α and β chains of hemoglobin

Previous studies from this laboratory have demonstrated unambiguously that the isolated β chain of human adult hemoglobin binds human haptoglobin (Hp). In the present work, the ability of the isolated subunits of haptoglobin and hemoglobin to form complexes is investigated. In quantitative radiometric adsorbent titrations, the H chain of haptoglobin bound to hemoglobin whereas the L chain had no binding activity. Also, the H chain of haptoglobin bound to the isolated α and β subunits of hemoglobin, but its binding to the α or β chain was less than the binding it exhibits to hemoglobin. The isolated L chain was able to reassociate with the H chain to form a complex that binds to hemoglobin or its subunits. Although the L chains had no binding activity, its association with the H chain increased the binding of the latter to Hb or its isolated α and β subunits suggesting a more indirect role for the L chain in haptoglobin-hemoglobin interactions.     

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes

Abstract

The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin – an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.     

Haptoglobin polymorphism--not only a genetic marker]

The biological activities of the haptoglobin polymorphism are controlled by continuous DNA sequences coding for the HP alpha and Hp beta polypeptide chains and forming with a linked Hp related gene the haptoglobin gene complex on chromosome 16. Probably, this DNA domain originates from the gene family of the serine proteases after having lost the informations for the proteolytic functions. Instead of this, the haptoglobins have acquired other qualities, among them the hemoglobin binding capacity, inserted into the Hp beta chain. The Hp polymorphism is constituted by the evolutionary progressive DNA sequences for the Hp alpha chains, which probably have activation functions. The haptoglobins display immunoregulative abilities, which can be immunosuppressive by inhibition of the lymphocyte reactivity or immunoinductive by influencing the IgM biosynthesis, adapted to the functional requirements. In this field, Hp 2-2 has a stronger effect than the two other Hp types. Moreover, the haptoglobins inhibit the prostaglandin synthesis and protect against harmful oxidation processes. These qualities are based on the hemoglobin binding ability and can be realized by Hp 1-1 with the comparatively highest efficacy. Further on, the haptoglobins are protease inhibitors. Finally, Hp 2-2 is associated with higher albumin and ceruloplasmin serum levels than Hp 2-1 and Hp 1-1. Evidently, the haptoglobins are inserted into a widely ramified network of biological functions. The selective advantages and disadvantages of the Hp polymorphism are noticeable under pathological conditions in case of malignant tumors, inflammations, autoimmune diseases, allergic illness, affective psychoses and affective lability favouring addiction.     

Haptoglobin and CD163: captor and receptor gating hemoglobin to macrophage lysosomes

The plasma protein haptoglobin and the endocytic hemoglobin receptor HbSR/CD163 are key molecules in the process of removing hemoglobin released from ruptured erythrocytes. Hemoglobin in plasma is instantly bound with high affinity to haptoglobin--an interaction leading to the recognition of the complex by HbSR/CD163 and endocytosis in macrophages. The haptoglobin-dependent HbSR/CD163 scavenging system for hemoglobin clearance prevents toxic effects of hemoglobin in plasma and kidney and explains the decrease in the haptoglobin plasma concentration in patients with accelerated hemolysis. The HbSR/CD163 activity may be of quantitative importance for iron uptake in macrophages in general and for some iron-associated pathological processes, e.g. the atherogenesis-promoting oxidation of LDL leading to foam cell formation and apoptosis in the vessel wall.     

Haptoglobin phenotypes differ in their ability to inhibit heme transfer from hemoglobin to LDL

LDL oxidation plays a pivotal role in atherosclerosis. Excellular hemoglobin (Hb) is a trigger of LDL oxidation. By virtue of its ability to bind hemoglobin, haptoglobin (Hp) serves as an antioxidant. Oxidation of LDL by hemoglobin was analyzed to occur by heme displacement from methemoglobin lodged in LDL. The LDL-associated heme is disintegrated, and iron inserted this way in LDL triggers formation of lipid peroxides. The genetic polymorphism of haptoglobin was found to be a risk factor in the pathogenesis of atherosclerosis. Individuals with Hp2-2 have more vascular incidences as compared to those with Hp1-1. In the current study, oxidation of LDL by metHb was carried out at physiological pH without addition of external peroxides. Hb-derived oxidation of lipids and protein was found to be practically inhibited by Hp1-1 but only partially by Hp2-2. Heme transfer from metHb to LDL was almost completely omitted by Hp1-1 and only partially by Hp2-2. We concluded that partial heme transfer from the Hb-Hp2-2 complex to LDL is the reason for oxidation of LDL lipids as well as protein. These findings provide a molecular basis for Hp2-2 atherogenic properties.     

Human haptoglobin structure and function--a molecular modelling study

Hemoglobin is the most prominent protein in blood, transporting O(2) and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer alpha-chain. Haptoglobin consists of a dimer of alphabeta-chains covalently linked by a disulphide bond between the Cys15 residue of each alpha-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s).     

Molecular Characterization of Buffalo Haptoglobin: Sequence Based Structural Comparison Indicates Convergent Evolution Between Ruminants and Human

Haptoglobin (Hp) protein has high affinity for hemoglobin (Hb) binding during intravascular hemolysis and scavenges the hemoglobin induced free radicals. Earlier reports indicate about uniqueness of Hp molecule in human and cattle, but in other animals, it is not much studied. In this paper, we characterized buffalo Hp molecule and determined its molecular structure, evolutionary importance, and tissue expression. Comparative analysis and predicted domain structure indicated that the buffalo Hp has an internal duplicated region in α-chain only similar to an alternate Hp2 allele in human. This duplicated part encoded for an extra complement control protein CCP domain. Phylogenetic analysis revealed that buffalo and other ruminants were found to group together separated from all other non-ruminants, including human. The key amino acid residues involved in Hp and Hb as well as Hp and macrophage scavenger receptor, CD163 interactions in buffalo, depicted a significant variation in comparison to other non-ruminant species. Constitutive expression of Hp was also confirmed across all the vital tissues of buffalo, for the first time. Results revealed that buffalo Hp is both structurally and functionally conserved, having internal duplication in α-chain similar to human Hp2 and other ruminant species, which might have evolved separately as a convergent evolutionary process. Furthermore, the presence of extra Hp CCP domain possibly in all ruminants may have an effect during dimerization of molecule in these species.     

Development of a dichloroacetic acid-hemoglobin conjugate as a potential targeted anti-cancer therapeutic

This work focuses on conjugating the anti-cancer drug dichloroacetic acid (DCA) to the monocyte/macrophage targeting protein hemoglobin (Hb). The DCA-Hb conjugate carries approximately 12 DCA molecules per Hb tetramer, and binds to haptoglobin (Hp) forming stable DCA-Hb-Hp complexes, in a similar manner to unmodified Hb. The results of this study show that DCA-Hb-Hp is taken up by the monocytic cancer cell line THP-1, where it depolarizes the mitochondrial membrane potential, thereby inhibiting cancerous cell growth at a comparable level to free DCA. Taken together, the results of this study show promise for the use of the DCA-Hb conjugate as a potential therapeutic to treat monocytic leukemia.     

Liposomes surface conjugated with human hemoglobin target delivery to macrophages

Current strategies to deliver therapeutic molecules to specific cell and tissue types rely on conjugation of antibodies and other targeting ligands directly to the therapeutic molecule itself or its carrier. This work describes a novel strategy to deliver therapeutic molecules into macrophages that takes advantage of the native hemoglobin (Hb) scavenging activity of plasma haptoglobin (Hp) and the subsequent uptake of the Hb-Hp complex into macrophages via CD163 receptor-mediated endocytosis. The drug delivery system described in this work consists of Hb decorated liposomes that can encapsulate any therapeutic molecule of interest, in this case the model fluorescent dye calcein was used in this study. The results of this study clearly demonstrate that this delivery system is specific towards macrophages and demonstrates the feasibility of using this approach in targeted drug delivery.     

Studies on free or haptoglobin-bound hemoglobin and derivatives (semihemoglobins and porphyrinated semihemoglobins). Some aspects of their peroxidatic activity.

The peroxidatic activity of hemoglobin (Hb) is known to be enhanced when this hemoprotein is bound to haptoglobin (Hp). The peroxidatic reaction (H2O2, guaiacol as donor) has been kinetically studied (Steady-state) in the presence of free or rabbit-haptoglobin bound human hemoglobin and some of its derivatives, all in ferricyano-form. With free Hb+ CN, we observed linearity of Lineweaver and Burk plots in a wide range of concentrations, the donor's behaviour was therefore assumed to obey the Michaelis-Menten mechanism. When Hp-Hb+ CN is the enzyme, the donor's behaviour is more complicated, analysis shows the existence of two kinds of donor's binding sites. The possibility whether this behaviour might correspond to the intrinsic properties of Hb chains, as revealed after combination with Hp, was examined. The peroxidatic activity of free and Hp-bound alpha and beta chains of Hb were studied. The alpha chains of Hb combine with Hp whereas the beta chains fail to do so. In order to make useful comparisons, the peroxidatic activity of Hp-bound alpha and beta chains were studied by the use of Hp-semihemoglobin complexes where the semihemoglobins carried heme on only one type of chain (alpha or beta). Results did not show an evident correlation between the activities of the two free or bound types of chains and those of the two classes of binding sites revealed in Hp-Hb+ CN. Moreover, it appeared that the heme-free complementary chain might influence the activity of the heme-carrying alpha or beta chain in semihemoglobins and Hp-semihemoglobin complexes. The binding or protoporphyrin on free and Hp-bound semihemoglobins leads to species which exhibit structures close to that of Hb and Hp-Hb complex respectivley. Results of studies on these derivatives brought up new interesting data : when the porphyrin ring alone is bound to the heme deficient chains (alpha or beta), in Hp-semihemoglobin complexes, the same peculiar behaviour, already observed with Hp-Hb complex, is found again. The structural implications of these results are discussed.     

Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Some suggested polymer subunit compositions.

1. Some of the individual members of the polymeric series of proteins from human haptoglobin types 2-1 and 2-2 were isolated by gel electrophoresis. By reacting this purified material with less than an equivalent amount of hemoglobin and analyzing the result by electrophoresis, the number of haptoglobin-hemoglobin complexes could be clearly counted. For the haptoglobin 2-1 series, the number of complexes formed was n+1, where n is the serial order, in decreasing electrophoretic mobility, of the haptoglobin polymeric form used. For the haptoglobin 2-2 series, the number of complexes was n+2. 2. For the first three members of haptoglobin 2-1 series, the haptoglobin-hemoglobin composition of the complexes was estimated from scans of the unstained gels. The data indicated that this series consists of 2,3,4... alpha beta haptoglobin subunits, each of which can combine with an alpha beta subunit of hemoglobin.