Hb Binding Research Articles

Signal Amplification in Immunoassays Using Labeling via Boronic Acid Binding to the Sugar Moiety of Immunoglobulin G: Proof of Concept for Glycated Hemoglobin

A novel electrochemical immunoassay based on the multiple affinity labeling of the indicator antibody with an electro‐active tag is presented. The concept is illustrated for the determination of the glycated hemoglobin HbA1c in hemoglobin samples. Hemoglobin is adsorbed to the surfactant‐modified surface of a piezoelectric quartz crystal. Whereas the quartz crystal nanobalance is used to validate the total Hb binding, the HbA1c on the sensor surface is recognized by an antibody and quantified electrochemically after the sugar moieties of the antibody have been labeled in‐situ with ferroceneboronic acid. The sensitivity of this sensor is about threefold higher than the sensitivity of a hemoglobin sensor, where the ferroceneboronic acid is bound directly to HbA1c.

Development of a biosensor for glycated hemoglobin

The development of an electrochemical piezoelectric sensor for the detection of glycated hemoglobin is presented. The total hemoglobin (Hb) content is monitored with a mass-sensitive quartz crystal modified with surfactants, and the glycated fraction of the immobilized Hb is determined by subsequent voltammetric measurement of the coupled ferroceneboronic acid. Different modifications of the sensor were tested for their hemoglobin binding ability. Deoxycholate (DOCA) was found to be the most suitable among the examined modifiers. Piezoelectric quartz crystals with gold electrodes were modified with DOCA by covalent binding to a pre-formatted 4-aminothiophenol monolayer. The properties of the Hb binding to DOCA and the pH effect on this interaction were studied. In the proposed assay for glycated hemoglobin at first an Hb sample is incubated with ferroceneboronic acid (FcBA), which binds to the fructosyl residue of the glycated Hb. Then this preincubated Hb sample is allowed to interact with the DOCA-modified piezoelectric quartz crystal. The binding is monitored by quartz crystal nanobalance (QCN). The amount of FcBA present on the sensor surface is determined by square wave voltammetry. The binding of FcBA results in well-defined peaks with an E 0′ of +200 mV versus Ag/AgCl (1 M KCl). The peak height depends on the degree of glycated Hb in the sample ranging from 0% to 20% of total Hb. The regeneration of the sensing surface is achieved by pepsin digestion of the deposited Hb. Thus the sensor can be re-used more than 30 times.

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.

Assignment of the Binding Site for Haptoglobin on Apolipoprotein A-I

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Binding and cleavage studies of two proteins intercalated at the galleries of α-zirconium phosphate

Met hemoglobin (Hb) and lysozyme are intercalated in the galleries of α-Zr(IV)phosphate (α-ZrP), and the resulting bioactive materials have been characterized using scanning electron microscopy, calorimetry and chemical footprinting methods. Microscopy revealed that the inorganic material crystallizes as nanoscopic discs of 600 nm in diameter, and that the layered structure is retained when proteins are intercalated in the galleries. Isothermal titration calorimetric studies show that binding of Hb to α-ZrP at 25 °C is strongly exothermic (Δ H = −31.0 ± 2.5 kcal/mol) with a decrease in entropy (Δ S = −77 ± 8 e.u.), while a model system such as Mg(II) binding to α-ZrP was endothermic (Δ H 1 = 2.4 ± 0.03 kcal/mol, Δ H 2 = 4.3 ± 0.3 kcal/mol). These differences in the binding thermodynamics of the two guests clearly indicate that a simple electrostatic model for the binding process is not adequate to explain Hb binding to α-ZrP. The protein-solid composites are further characterized in chemical footprinting studies. PolyammineCo(III) complexes cleave Hb and lysozyme at specific sites, and these have been used as probes to examine protein-solid contact regions. Protein cleavage studies indicated that Hb bound to α-ZrP is cleaved at four specific locations, and that these sites are accessible to the Co(III) reagents. Such access strongly suggests that the Hb tetramer is oriented in the galleries with its long axis parallel to the galleries. This conclusion is consistent with the powder X-ray diffraction (XRD) and activity studies reported earlier. Most likely this is one of the significant orientations of Hb at the galleries while other orientations are also possible. Similar footprinting studies with lysozyme bound in the galleries indicated that lysozyme is cleaved predominantly at a single site, in good yield (28%). Current results support the idea that chemical footprinting methods, when coupled with powder XRD and activity studies, are useful to examine specific orientations of the proteins at solid surfaces.

Haptoglobin Phenotype and Gestational Diabetes

Haptoglobin (Hp), an Hb-binding plasma protein, exists in two major allelic variants. Hp1 has higher Hb binding and antioxidant capacity compared with Hp2. Individuals with Hp1 exhibit a lower incidence of angiopathies. Gestational diabetes mellitus (GDM) is an early manifestation of type 2 diabetes in pregnant women. It is usually confined to the time of gestation, but carries an increased risk to develop type 2 diabetes later in life. From consecutive Caucasian pregnant women (n = 250) referred for oral glucose tolerance testing, the Hp phenotype was determined. Significance of distribution and odds ratios (ORs) associated with Hp phenotype were calculated for women with GDM (n = 110) and women with normal glucose tolerance (n = 140). -Frequency of GDM in Hp phenotype classes increased with the number of Hp2 alleles (P < 0.001). ORs for GDM in women heterozygous and homozygous for Hp2 were 2.7 (95% CI 1.06-6.84) and 4.2 (1.67-10.55), respectively. Hp phenotype is an apparent risk factor for the development of GDM in our study population. This might be due to the low antioxidative potential of Hp2 compared with Hp1.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae.

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of approximately 105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for -35 and -10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

Identification of functionally important regions of a haemoglobin receptor from Neisseria meningitidis.

The HmbR outer-membrane receptor enables Neisseria meningitidis to use haemoglobin (Hb) as a source of iron. This protein functions by binding Hb, removing haem from it, and releasing the haem into the periplasm. Functionally important HmbR receptor domains were discerned using a series of HmbR deletions and site-directed mutations. Mutations exhibiting similar defective phenotypes in N. meningitidis fell into two groups. The first group of mutations affected Hb binding and were located in putative extracellular loops (L) L2 (amino acid residues (aa) 192-230) and L3 (aa 254-284). The second group of mutations resulted in a failure to utilize Hb but proficiency in Hb binding was retained. These mutations localized to the putative extracellular loops L6 (aa 420-462) and L7 (aa 486-516). A highly conserved protein motif found in all haem/Hb receptors, within putative extracellular loop L7 of HmbR, is essential for Hb utilization but not required for Hb binding. This finding suggests a mechanistic involvement of this motif in haem removal from Hb. In addition, an amino-terminal deletion in the putative cork-like domain of HmbR affected Hb usage but not Hb binding. This result supports a role of the cork domain in utilization steps that are subsequent to Hb binding.

Cross-linked Hemoglobin Converts Endotoxically Inactive Pentaacyl Endotoxins into a Physiologically Active Conformation

The interaction of purified alpha alpha cross-linked hemoglobin (alpha alpha Hb) with a pentaacylated mutant lipopolysaccharide (pLPS) and the corresponding lipid A (pLA) was studied biophysically and the effects correlated with data from biological assays, i.e. cytokine induction (tumor necrosis factor-alpha) in human mononuclear cells and the Limulus amebocyte lysate assay. Fourier transform infrared spectroscopic and Zeta-Sizer experiments indicated an electrostatic as well as a non-electrostatic binding of alpha alpha Hb to the hydrophilic and to the hydrophobic moieties of the endotoxins with an increase of the inclination angle of the pLA backbone, with respect to the membrane surface, from 25 degrees to more than 50 degrees. Small angle synchrotron radiation x-ray diffraction measurements indicated a reorientation of the lipid A aggregates from a multilamellar into a cubic structure as a result of alpha alpha Hb interaction. Thus, in the absence of alpha alpha Hb, the molecular shape of the pentaacyl samples was cylindrical with a moderate inclination of the diglucosamine backbone, whereas, in the presence of the protein, the shape was conical, and the inclination angle was high. The cytokine-inducing capability in human mononuclear cells, negligible for the pure pentaacylated compounds, increased markedly in the presence of alpha alpha Hb in a concentration-dependent manner. In the Limulus assay, the pentaacylated samples were active a priori, and their activity was enhanced following binding to alpha alphaHb, at least at the highest protein concentrations. The data can be understood in the light of a reaggregation of the endotoxins because of alpha alpha Hb binding, with the endotoxin backbones then readily accessible for serum and membrane proteins. By using fluorescence resonance energy transfer spectroscopy, an uptake of the endotoxin-Hb complex into phospholipid liposomes was observed, which provides a basis for cell activation.

The binding of haptoglobin to apolipoprotein AI: influence of hemoglobin and concanavalin A.

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin (Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in heavy contamination of the Hp preparation by apolipoprotein AI (ApoAI), and partial loss of Hb binding activity. Hp, separated from ApoAI, was recovered by elution with glycine-HCl at pH 3. Complexes of the isolated protein with Hb or ApoAI were detected by enzyme-linked immunosorbent assay (ELISA). Competition between the two ligands in their interaction with Hp was observed. Concanavalin A (ConA), which binds the Hp carbohydrate chains, did not influence Hp binding to ApoAI. These results suggest that changes in the plasma levels of ApoAI or Hb affect the Hp role in regulating the reverse transport of cholesterol or preventing Hb-dependent oxidative damage.

Point mutations in HpuB enable gonococcal HpuA deletion mutants to grow on hemoglobin.

Neisseria gonorrhoeae ordinarily requires both HpuA and HpuB to use hemoglobin (Hb) as a source of iron for growth. Deletion of HpuA resulted in reduced Hb binding and failure of growth on Hb. We identified rare Hb-utilizing colonies (Hb(+)) from an hpuA deletion mutant of FA1090, which fell into two phenotypic classes. One class of the Hb(+) revertants required expression of both TonB and HpuB for growth on Hb, while the other class required neither TonB nor HpuB. All TonB/HpuB-dependent mutants had single amino acid alterations in HpuB, which occurred in clusters, particularly near the C terminus. The point mutations in HpuB did not restore normal Hb binding. Human serum albumin inhibited Hb-dependent growth of HpuB point mutants lacking HpuA but did not inhibit growth when expression of HpuA was restored. Thus, HpuB point mutants internalized heme in the absence of HpuA despite reduced binding of Hb. HpuA facilitated Hb binding and was important in allowing use of heme from Hb for growth.

Effects of Exercise on Plasma Haptoglobin Composition in Control and Splenectomized Thoroughbred Horses.

In splenectomized Thoroughbred horses (Group S), the plasma haptoglobin (Hp) concentration was increased by the operation but thereafter significantly decreased to a lower level than before the surgery. The red cell haemolysis rate in a hypotonic salt solution and haemoglobin (Hb) binding Hp after exercise in Group S were significantly lower than in control horses (Group C). These results suggest that: 1) the spleen accelerates the fragility of the red cell membrane; 2) osmotic sensitive red cells are easy to haemolyze during exercise; and 3) the haemolysis during exercise increases the Hb-binding Hp level in plasma and then accelerates the synthesis of Hp in liver. Therefore, plasma Hp is a clinical indicator of red cell membrane fragility and haemolysis in a blood vessel during exercise.

Interactions of haemoglobin with the Neisseria meningitidis receptor HpuAB: the role of TonB and an intact proton motive force.

We have characterized the interaction of the Neisseria meningitidis TonB-dependent receptor HpuAB with haemoglobin (Hb). Protease accessibility assays indicated that HpuA and HpuB are surface exposed, HpuB interacts physically with HpuA, and TonB energization affects the conformation of HpuAB. Binding assays using [125I]-Hb revealed that the bipartite receptor has a single binding site for Hb (Kd 150 nM). Competitive binding assays using heterologous Hbs revealed that HpuAB Hb recognition was not species specific. The binding kinetics of Hb to HpuAB were dramatically altered in a TonB- mutant and in wild-type meningococci treated with the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that TonB and an intact proton motive force are required for normal Hb binding and release from HpuAB. Our results support a model in which both HpuA and HpuB are required to form a receptor complex in the outer membrane with a single binding site, whose structure and ligand interactions are significantly affected by the TonB-mediated energy state of the receptor.

Detection of HbA 1c by boronate affinity immunoassay using bacterial magnetic particles

We have developed a boronate affinity immunoassay system using m-aminophenylboronic acid (mAPB) coupling to bacterial magnetic particles (BMPs). Homobifunctional crosslinker, Bis-(succcimidyl)suberate (BS3), was employed for preparation of mAPB-BMPs conjugates (mAPB-BMPs). Quantities of HbA 1c on mAPB-BMPs were evaluated based on luminescence from alkaline phosphatase-conjugated anti-Hb antibody (ALP–antibody) binding to HbA 1c on the BMP surface. The binding of HbA 1c to mAPB-BMPs occurred gradually and was almost completed within 10 mm. The coupling reaction is enhanced due to static electric interaction between the positive charges on HbA 1c and negative charges on BMPs. The amount of HbA 1c binding to mAPB-BMPs increased with increasing sodium chloride concentrations in the range of 0–100 mM. However, the amount of Hb binding to mAPB-BMPs also increased in high concentration of sodium chloride. The Hb binding to mAPB-BMPs was detached from mAPB-BMPs when Hb–mAPB-BMPs were washed with low salt buffer. This indicates that Hb is nonspecifically adsorbed onto the surface of mAPB-BMPs in high concentration of sodium chloride. These results suggest that selective separation of HbA 1c using mAPB-BMPs can be achieved with these conditions. A dose–response curve was obtained between luminescence intensity and HbA 1c concentration using a fully automated boronate affinity immunoassay. A linear relationship between luminescence intensity and HbA 1c concentration was obtained in the range of 10–10 4 ng/ml.

Interaction of hemoglobin with enterobacterial lipopolysaccharide and lipid A. Physicochemical characterization and biological activity.

The interaction of hemoglobin (Hb) with endotoxins [i.e. with enterobacterial deep rough mutant lipopolysaccharide (LPS) Re and the "endotoxic principle" of LPS, lipid A] was investigated using a variety of physical techniques and with two biological assays, tumor necrosis factor (TNF)-alpha induction in human mononuclear cells and the Limulus amebocyte lysate (LAL) assay. Fourier-transform IR-spectroscopic experiments indicate nonelectrostatic binding to the hydrophobic moiety with a slight rigidification of the lipid A acyl chains, and an increase in the inclination of the lipid A backbone with respect to the membrane surface from 35 degrees to more than 40 degrees due to Hb binding, but no change of the predominantly alpha-helical secondary structures of Hb due to LPS binding. From isothermal titration calorimetry, the molar [Hb] : [endotoxin] binding ratio lies between 1 : 3 and 1 : 5 molar. Synchrotron radiation X-ray diffraction measurements indicate a reorientation of the lipid A aggregates from one cubic structure to another, the final structure belonging to space group Q224. The LPS-induced TNF-alpha production of mononuclear cells is enhanced by Hb, whereas in the LAL assay an LPS concentration-dependent increase or decrease was observed. Although a detailed mechanism of action cannot be given, the enhancement of LPS bioactivity can be understood in the light of the previously presented conformational concept; Hb induces an increase in the conical shape of the lipid A moiety of LPS, higher cross-section of the hydrophobic than the hydrophilic part, and of the inclination angle of the diglucosamine backbone with respect to the direction of the acyl chains.

Urinary metabolites and haemoglobin adducts as biomarkers of exposure to 1,3-butadiene: a basis for 1,3-butadiene cancer risk assessment.

Since 1,3-butadiene (BD) is a suspected human carcinogen, exposure to BD should be minimised and controlled. This study aimed at comparing the suitability of biomarkers for low levels of exposure to BD, and at exploration of the relative pathways of human metabolism of BD for comparison with experimental animals. Potentially sensitive biomarkers for BD are its urinary metabolites 1,2-dihydroxybutyl mercapturic acid (DHBMA, also referred to as MI) and 1- and 2-monohydroxy-3-butenyl mercapturic acid (MHBMA, also referred to as MII) and its haemoglobin (Hb) adducts 1- and 2-hydroxy-3-butenyl valine (MHBVal). In two field studies in BD-workers, airborne BD, MHBMA, DHBMA and MHBVal were determined. MHBMA proved more sensitive than DHBMA for monitoring recent exposures to BD and could measure 8-h time weighted average exposures as low as 0.13 ppm (0.29 mg/m 3). The sensitivity of DHBMA was restricted by relatively high natural background levels in urine, of which the origin is currently unknown. MHBVal proved a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm (0.77 mg/m 3). Statistically significant relationships were found between either MHBMA or DHBMA and 8-h airborne BD levels, and between MHBVal adducts and average airborne BD levels over 60 days. The data showed a much higher rate of hydrolytic metabolism of BD in humans compared to animals, which was reflected in a much higher DHBMA/(MHBMA+DHBMA) ratio, and in much lower levels of MHBVal in humans, confirming in vitro results. Assuming a genotoxic mechanism, the data of this study coupled with our recent data on DNA and Hb binding in rodents, suggest that the cancer risk for humans from exposure to BD will be less than for the rat, and much less than for the mouse.

Hemoglobin enhances tissue factor expression on human malignant cells.

Tissue Factor (TF) is a transmembrane glycoprotein that complexes with factor VII/activated factor VII to initiate blood coagulation. TF may be expressed on the surface of various cells including monocytes and endothelial cells. Over-expression of TF in human tumor cell lines promotes metastasis. We recently showed that hemoglobin (Hb) forms a specific complex with TF purified from human malignant melanoma cells and enhances its procoagulant activity (PCA). To further study this interaction, we examined the effect of Hb on the expression of TF on human malignant (TF+) cells and KG1 myeloid leukemia (TF-) cells. Human melanoma A375 and J82 bladder carcinoma cells, which express TF at moderate and relatively high levels, respectively, were incubated with varying concentrations (0-1.5 mg/ml) of Hb. After washing, cells were analyzed for Hb binding and TF expression using flow cytometry and confocal microscopy. Hb bound to the cells in a concentration-dependent manner, and increased both TF expression and PCA. The human A375 malignant melanoma cells incubated with Hb (1 mg/ml) expressed up to six times more TF antigen than cells without Hb. This increase in TF expression and PCA of intact cells incubated with Hb was significantly inhibited by cycloheximide at a concentration of 10 microg/ml (P < 0.01). An increase in total cellular TF antigen content was demonstrated by specific immunoassay. In contrast, Hb (5 mg/ml) did not induce TF expression and PCA on KG1 cells as determined by flow cytometry and TF (FXAA) activity. We conclude that Hb specifically binds to TF-bearing malignant cells and increases their PCA. This effect seems to be at least partly due to de novo synthesis of TF and increased surface expression. However, the exact mechanism by which Hb binds and upregulates TF expression remains to be determined.

Oxygen binding and its allosteric control in hemoglobin of the pulmonate snail, Biomphalaria glabrata.

Pulmonate snails that experience extreme variations in gas tensions and temperatures possess extracellular, high-molecular mass ( approximately 1.7 x 10(6) Da) hemoglobins (Hbs) that are little known as regards oxygenation and allosteric characteristics. Biomphalaria glabrata hemolymph exhibits a high O2 affinity (half-saturation O2 tension = 6.1 mmHg; pH 7.7, 25 degreesC), pronounced Bohr effect (Bohr factor = -0.5), and pH-dependent cooperativity (Hill's cooperativity coefficient at half-saturation = 1.1-2.0). Divalent cations increase O2 affinity, Ca2+ exerting greater effect than Mg2+. Analyses in terms of the Monod-Wyman-Changeux model indicate novel O2 affinity control mechanisms. In contrast to vertebrate Hb, where organic phosphates and protons lower affinity via decreased O2 association equilibrium constant of Hb in low-affinity state (KT), and to extracellular annelid Hbs, where protons and cations primarily modulate O2 association equilibrium constant of Hb in high-affinity state (KR), in B. glabrata Hb, the Bohr effect is mediated predominantly via KR and the cation effect via KT, reflecting preferential, oxygenation-linked proton binding to oxygenated Hb and cation binding to deoxygenated Hb. CO2 has no specific (pH independent) effect. Nonlinear van't Hoff plots show temperature dependence of the overall heats of oxygenation, indicating oxy-deoxy heat capacity differences. The findings are related to possible physiological significance in pond habitats.

Difference in Hemoglobin-Binding Ability of Polymers Among Haptoglobin Phenotypes

Langlois and Delanghe (1) described several functional differences between haptoglobin phenotypes in this journal. They reviewed the functional properties of haptoglobin phenotypes such as ability for hemoglobin (Hb) binding, antioxidative capacity, and inhibition of prostaglandin synthesis, which were strong in Hp-1, intermediate in Hp2–1, and weak in Hp2–2. However, we do not agree with their results about the ability for Hb binding. Hp2–1 and Hp2–2 phenotypes form many polymers, and analysis of the functional properties of each of the isolated polymer proteins had been very difficult (2). We designed new analytical methods such as crossed Hb electrophoresis (3) and two-dimensional affinity electrophoresis, using Hb (4) to identify the Hb-binding ability of each of the haptoglobin polymer proteins of Hp2–1 and Hp2–2 phenotypes. From the results of crossed Hb electrophoresis, we found that a haptoglobin molecule combined with equimolar amounts of Hb to form at least a hexamer. We confirmed by two-dimensional affinity electrophoresis that the larger polymer of the Hp2–2 phenotype had a lower affinity to Hb, but a Hp2–1 polymer of very … aAuthor for correspondence.

Effects of pig hemoglobin binding on some physical and biological properties of Actinobacillus pleuropneumoniae lipopolysaccharides

Binding of pig hemoglobin (Hb) to Actinobacillus pleuropneumoniae lipopolysaccharide (LPS), either extracted or present at the surface of whole cells, was studied. After a short incubation period, pig Hb seemed to cover the bacterial cell surface and enhanced the cells' contrast when examined by transmission electron microscopy (TEM). Energy-dispersive X-ray spectroscopy analysis showed that the amount of elemental iron detected was increased when cells of A. pleuropneumoniae were incubated with pig Hb. Coating with pig Hb, however, did not interfere with the accessibility of O- and capsular antigens to antibodies on the bacterial cell surface. Binding of pig Hb and polymyxin B to lipid A of A. pleuropneumoniae was confirmed with a fluorescent probe (dansylcadaverine) displacement assay. The binding of pig Hb to extracted LPS resulted in a disaggregation of LPS as observed by TEM after negative staining. Additional evidence for a direct physical interaction between pig Hb and A. pleuropneumoniae LPS was demonstrated by a shift in the sedimentation velocity of LPS-Hb complexes determined by sucrose gradient centrifugation. Pig Hb binding to extracted LPS or to bacterial cells resulted in a significant decrease of chromogenic Limulus amebocyte lysate activation. Finally, the capacity of extracted LPS to induce NO2-in the presence of pig Hb was tested by using cell line J774 and determined by the Greiss' reaction. LPS alone induced, as expected, NO2- production, whereas the presence of pig Hb significantly reduced NO2-production by murine macrophages. Taken together, our results indicate that binding of pig Hb affected some physical and biological properties of A. pleuropneumoniae LPS.