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.     

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.     

Dual inhibition of cyclooxygenase and lipoxygenase by human haptoglobin: its polymorphism and relation to hemoglobin binding

Haptoglobin (Hp) binds hemoglobin (Hb) specifically and stoichiometrically. Since Hb stimulates prostaglandin (PG biosynthesis), we investigated if Hp effects arachidonic acid (AA) metabolism. The results showed that Hp (50-250 microg protein) inhibited the biosynthesis of PGs via cyclooxygenase (COX) and 12-HETE via lipoxygenase pathway in human platelets. Additional evidence was obtained by the loss of Hp inhibitory activity upon removal of Hp by affinity chromatography on hemoglobin sepharose and by inhibition of AA or bradykinin-induced bronchoconstriction in the guinea pig. Hb reduced the inhibitory effect of Hp in a concentration-related manner such that all its inhibitory activity was lost when completely bound by Hb. Of the three Hp phenotypes, Hp 1-1 showed maximum binding capacity to Hb indicating its greater protective role. These findings implicate Hp in the regulation of COX and lipoxygenase pathways and show Hp involvement in the body's endogenous defense system against inflammation. This indicates that mammals have dual defense system, i.e., a specific immune system and non-specific Hp defense system.     

Human fetal hemoglobin synthesis and its NH2-terminal acetylation in a rabbit reticulocyte lysate translational system

The biosynthesis of the acetylated (Hb FIc) and the non-acetylated (Hb F0) human fetal hemoglobin components has been examined in a cell-free translational system. The poly(A)-RNA was isolated from umbilical cord blood samples and translated in the heterologous translational system derived from rabbit reticulocyte lysates in the presence of labeled amino acid(s) or acetyl-CoA. The amount of each hemoglobin or globin chain made in the system was determined by separating the synthesis products by cation-exchange chromatographic methods. The in vitro synthesis ratios were close to the FIc/Ftotal values of the respective hemolysates. The same conclusion could be reached by determining the specific activity ratios of Hb FIc/Hb F0. Co-migration of radioactivity peaks with absorbance peaks indicated the synthesis of that hemoglobin or globin chain. Confirmation of the synthesis of true gamma 0 and gamma Ic was accomplished by high-pressure liquid chromatographic separation of 3H-labeled tryptic peptides. Each peptide corresponded well with the radioactivity peak. Labeled acetyl-group incorporation into Hb FIc and gamma IcT-1 provided direct evidence for acetylation of gamma chains in Hb FIc. The data indicate that the mRNA itself dictates whether a protein is acetylated and, if so, to what extent. The control appears to be not unique to the human red cell system.     

Preparation and in vitro antioxidant activity of kappa-carrageenan oligosaccharides and their oversulfated, acetylated, and phosphorylated derivatives

In order to study the relationship between chemical structure and properties of modified carrageenans versus antioxidant activity in vitro, kappa-carrageenan oligosaccharides were prepared through mild hydrochloric acid hydrolysis of the polysaccharide, and these were used as starting materials for the partial synthesis of their oversulfated, acetylated, and phosphorylated derivatives. The structure and substitution pattern of the oligosaccharides and their derivatives were studied using FTIR and (13)C NMR spectroscopy, and their in vitro antioxidant activities were investigated. Certain derivatives of the carrageenan oligosaccharides exhibited higher antioxidant activity than the polysaccharides and oligosaccharides in certain antioxidant systems. The oversulfated and acetylated derivatives, which scavenge superoxide radicals, the phosphorylated and low-DS acetylated derivatives, which scavenge hydroxyl radicals, and the phosphorylated derivatives, which scavenge DPPH radicals, all exhibited significant antioxidant activities in the systems examined. The effect of the molecular weight of the carrageenan on antioxidant activities, however, is not obvious from these studies.     

Study of functional properties of chemically-modified hemoglobin during circulation in the blood channel

Functional activity of the solutions of chemically modified hemoglobins (Hb) with different structure had been investigated during the replacement of acute fatal blood loss in dogs. It was found the correlation between polymerization degree of Hb derivatives and alterations of its oxygen-carrying characteristics in the process of circulation. It was shown that decline of functional activity at the prolonged terms of circulation was more expressed for macromolecular Hb derivatives with heterogeneous structure. Therefore chemically modified Hb free from high molecular weight fractions may be considered as a potential oxygen-carrying fluids, because they are capable of more effective support of the oxygen transport level in the organism.     

Haptoglobin reduces lung injury associated with exposure to blood

The biological functions of the acute- phase protein haptoglobin (Hp) may be related to its ability to bind hemoglobin (Hb) or to modulate immune response. Hp is expressed at a high level in lung cells, yet its protective role(s) in the lung is not known. With the use of transgenic mice overexpressing Hp in alveolar macrophages, we demonstrated that Hp diminished Hb-induced lung injury when the lung was exposed to whole blood. In transgenic mouse lungs, Hb was more efficiently removed, and the induction of stress- responsive heme oxygenase-1 gene was significantly lower when compared with wild-type mice. At 24 h after blood treatment, the ferritin level that serves as an index for intracellular iron content was also lower in alveolar macrophages in transgenic mice than in wild-type mice. We propose that an Hp-mediated Hb catabolism process exists in alveolar macrophages. This process is likely coupled to an iron mobilization pathway and may be an efficient mechanism to reduce oxidative damage associated with hemolysis.     

Haptoglobin Preferentially Binds β but Not α Subunits Cross-Linked Hemoglobin Tetramers with Minimal Effects on Ligand and Redox Reactions

Human hemoglobin (Hb) and haptoglobin (Hp) exhibit an extremely high affinity for each other, and the dissociation of Hb tetramers into dimers is generally believed to be a prerequisite for complex formation. We have investigated Hp interactions with native Hb, αα, and ββ cross-linked Hb (ααXLHb and ββXLHb, respectively), and rapid kinetics of Hb ligand binding as well as the redox reactivity in the presence of and absence of Hp. The quaternary conformation of ββ subunit cross-linking results in a higher binding affinity than that of αα subunit cross-linked Hb. However, ββ cross-linked Hb exhibits a four fold slower association rate constant than the reaction rate of unmodified Hb with Hp. The Hp contact regions in the Hb dimer interfaces appear to be more readily exposed in ββXLHb than ααXLHb. In addition, apart from the functional changes caused by chemical modifications, Hp binding does not induce appreciable effects on the ligand binding and redox reactions of ββXLHb. Our findings may therefore be relevant to the design of safer Hb-based oxygen therapeutics by utilizing this preferential binding of ββXLHb to Hp. This may ultimately provide a safe oxidative inactivation and clearance pathway for chemically modified Hbs in circulation.     

Changes in conformation and immunological activity of ovalbumin during its modification with different acid anhydrides.

In order to probe the cause and nature of conformational changes induced by the chemical modification of amino groups in proteins, five acylated derivatives of ovalbumin namely 21% acetylated, 32% succinylated, 90% butyrated 92% succinylated, and 95% acetylated ovalbumins were prepared and their molecular and immunological properties were systematically investigated. As evidenced by the ultraviolet difference spectral, solvent perturbation, gel filtration, and viscosity data, acylation of the amino groups produced a definite conformational change in native ovalbumin whose extent was higher for higher degrees of chemical modification. The solvent pertubation data showed an exposure of 0.5 tryptophan and 3 tyrosine residues in native ovalbumin; the exposure increased to 1 tryptophan and about 5 tyrosine residues in the maximally modified proteins (i.e. 90% butyrated, 92% succinylated, and 95% acetylated ovalbumins). The Stokes radius (2.7 nm) and intrinsic viscosity (3.9 ml/g) of ovalbumin increased, respectively, to about 3.4 nm and 7.7 ml/g upon acylation of its 18 lysine residues; the intrinsic viscosity of 95% acetylated ovalbumin was 7.2 ml/g. The reduced viscosity of ovalbumin (4.2 ml/g) which remained unaltered on raising the pH to pH 11.2, increased to 7.9 ml/g on succinylation of 18 lysine residues. On raising the ionic strength from 0.15 to 1.0, the value decreased from 7.9 to 6.2 ml/g. These observations taken together with the fact that the intrinsic viscosities of 92% succinylated and 90% butyrated ovalbumins are identical, argue against the presently prevalent proposal that electrostatic effects alone are responsible for the disruption of native protein conformation during chemical modification. The immunological activity of ovalbumin towards rabbit anti-ovalbumin expectedly decreased with acylation of its amino groups but the three maximally modified ovalbumins retained 40% immunological activity. This taken along with the spectral and viscosity data showed substantial native structure (format) in the three maximally acylated derivatives. The rabbit antiserum against 95% acetylated ovalbumin did not cross-react with acetylated lysozyme and reacted poorly with the native and 92% succinylated ovalbumins suggesting that the antigenic make-up of the three maximally modified ovalbumins is different.     

Synthesis and characterization of a hemoglobin-ribavirin conjugate for targeted drug delivery

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.     

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.     

Peroxidase Activity of Hemoglobin��Haptoglobin Complexes: COVALENT AGGREGATION AND OXIDATIVE STRESS IN PLASMA AND MACROPHAGES*

As a hemoprotein, hemoglobin (Hb) can, in the presence of H₂O₂, act as a peroxidase. In red blood cells, this activity is regulated by the reducing environment. For stroma-free Hb this regulation is lost, and the potential for Hb to become a peroxidase is high and further increased by inflammatory cells generating superoxide. The latter can be converted into H₂O₂ and feed Hb peroxidase activity. Haptoglobins (Hp) bind with extracellular Hb and reportedly weaken Hb peroxidase activity. Here we demonstrate that: (i) Hb peroxidase activity is retained upon binding with Hp; (ii) in the presence of H₂O₂, Hb·Hp peroxidase complexes undergo covalent cross-linking; (iii) peroxidase activity of Hb·Hp complexes and aggregates consumes reductants such as ascorbate and nitric oxide; (iv) cross-linked Hb·Hp aggregates are taken up by macrophages at rates exceeding those for noncovalently cross-linked Hb·Hp complexes; (v) the engulfed Hb·Hp aggregates activate superoxide production and induce intracellular oxidative stress (deplete endogenous glutathione and stimulate lipid peroxidation); (vi) Hb·Hp aggregates cause cytotoxicity to macrophages; and (vii) Hb·Hp aggregates are present in septic plasma. Overall, our data suggest that under conditions of severe inflammation and oxidative stress, peroxidase activity of Hb·Hp covalent aggregates may cause macrophage dysfunction and microvascular vasoconstriction, which are commonly seen in severe sepsis and hemolytic diseases.As a hemoprotein, Hb,² in the presence of oxidizing equivalents such as H₂O₂, can act as a peroxidase with very high oxidizing potential (1). In red blood cells, this potentially dangerous activity is strictly regulated by the reducing environment and the lack of oxidizing equivalents. The inadvertently appearing ferric forms of Hb are short-lived, and the hemoprotein is effectively converted into ferro-Hb (deoxy-Hb) by metHb reductase (2). Normally, less than 2% of total Hb exists in the form of MetHb because the rate of Hb reduction is far greater than its oxidation (2). For stroma-free Hb, however, this intracellular regulation is lost, and the likelihood for Hb to act as a peroxidase is high. This possibility is markedly increased in the course of severe inflammation (e.g. in sepsis) by the generation of superoxide radicals by immune cells. The latter can be spontaneously or catalytically (by extracellular superoxide dismutase) converted into H₂O₂, a fuel for Hb peroxidase activity. In line with this, several clinical and experimental investigations have established that lethality in sepsis is increased in the setting of hemolysis (3–5).Circulating haptoglobin (Hp) provides an important endogenous defense against the toxic effects of Hb (6, 7). The major biological function of this abundant plasma protein is binding and recycling of stroma-free Hb via the macrophage CD163 receptor-mediated pathway (8, 9). It has been proposed that Hp possesses antioxidant activity and diminishes oxidative stress induced by stroma-free Hb (10–12). The antioxidant action of Hp toward Hb has been associated, at least in part, with weakening of its peroxidase activity (10) or preventing oxidation and cross-linking of Hb (7).Previous work has demonstrated that peroxidase activity of different hemoproteins, including Hb, can induce protein self-oxidation leading to covalent cross-linking and aggregation (13–16). Whether these hetero-oligomeric covalent aggregates retain the peroxidase activity is unknown. If the aggregates retain peroxidase activity, they may continue to be a source of oxidative stress both in circulation as well as in phagocytizing cells involved in their clearance such as macrophages.In the current work, we determined the extent to which: (i) Hb peroxidase activity is decreased by binding with Hp; (ii) peroxidase activity of Hb·Hp complexes initiates cross-linking into covalent hetero-oligomers; (iii) peroxidase activity of Hb·Hp complexes and aggregates utilizes nitric oxide (NO^•); (iv) Hb·Hp aggregates are taken up by macrophages as compared with noncovalent Hb·Hp complexes; and (v) the engulfed Hb·Hp aggregates induce oxidative stress and cytotoxicity. Here, we report that Hb·Hp complexes and aggregates are potent peroxidases capable of inducing oxidative stress in both plasma and macrophages. We further demonstrate the presence of Hb·Hp aggregates in septic plasma.     

Genetic markers in the blood of three species of tamarins (Saguinus mystax, S. labiatus and S. oedipus)

Alkaline phosphatase (Alp), esterase-I (Es-I), esterase-II (Es-II), carbonic anhydrase (CA), cell esterase (cEs), esterase-D (Es-D), isocitrate dehydrogenase (ICD), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (PGD), tetrazolium oxidase (To), ceruloplasmin (Cp), Haptoglobin (Hp) and hemoglobin (Hb) in 58-75 samples of three species of tamarins (Saguinus mystax, S. labiatus and S. oedipus) were detected by means of horizontal starch gel electrophoresis. Two types (Es-I 1 and Es-I 2) for Es-I, four types (Es-II 1, Es-II 2, Es-II 3 and Es-II 2-3) for Es-II, three types (cEs 1, cEs 2 and cEs 1-2) for cEs, three types (PGD 1, PGD 2 and PGD 1-2) for PGD, two types (To 1 and To 2) for To, and three types (Hp 3, Hp 1-3 and Hp 2-3) for Hp were observed. However, Alp, CA, Es-D, ICD, MDH, Cp and Hb were monomorphic. In the S. mystax, no Es-II or PGD variants were observed. No Es-II variant was seen in the S. oedipus. Gene frequencies of cEs, PGD and Hb were biased in the three species. It is concluded that six polymorphic loci are useful as genetic markers for a species or individual.     

Different target specificities of haptoglobin and hemopexin define a sequential protection system against vascular hemoglobin toxicity

Free hemoglobin (Hb) triggered vascular damage occurs in many hemolytic diseases, such as sickle cell disease, with an unmet need for specific therapeutic interventions. Based on clinical observations the Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe intravascular hemolysis. In this study we present a mechanistic rationale for this paradigm based on a combined biochemical and cell biological approach directed at understanding the unique roles of Hp and Hx in Hb detoxification. Using a novel in vitro model of Hb triggered endothelial damage, which recapitulates the well-characterized pathophysiologic sequence of oxyHb(Fe²⁺) transformation to ferric Hb(Fe³⁺), free heme transfer from ferric Hb(Fe³⁺) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions ultimately lead to endothelial damage characterized by a specific gene expression pattern with reduced cellular ATP and monolayer disintegration. Quantitative analysis of key chemical and biological parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe²⁺) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protection defines a highly efficient backup scavenger system during conditions of large excess of free oxyHb(Fe²⁺) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.     

Complexity of a complex trait locus: HP, HPR, haemoglobin and cholesterol

HP and HPR are related and contiguous genes in strong linkage disequilibrium (LD), encoding haptoglobin and haptoglobin-related protein. These bind and chaperone free Hb for recycling, protecting against oxidation. A copy number variation (CNV) within HP (Hp1/Hp2) results in different possible haptoglobin complexes which have differing properties. HPR rs2000999 (G/A), identified in meta-GWAS, influences total cholesterol (TC) and LDL-cholesterol (LDL-C). We examined the relationship between HP CNV, HPR rs2000999, Hb, red cell count (RCC), LDL-C and TC in the British Women's Heart and Health Study (n=2779 for samples having CNV, rs2000999, and phenotypes). Analysing single markers by linear regression, rs2000999 was associated with LDL-C (β=0.040 mmol/L, p=0.023), TC (β=-0.040 mmol/L, p=0.019), Hb (β=-0.044 g/dL, p=0.028) and borderline with RCC (β=-0.032 × 10(12)/L, p=0.066). Analysis of CNV by linear regression revealed an association with Hb (Hp1 vs Hp2, β=0.057 g/dL, p=0.004), RCC (β=0.045 × 10(12)/L, p=0.014), and showed a trend with LDL-C and TC. There were 3 principal haplotypes (Hp1-G 36%; Hp2-G 45%; Hp2-A 18%). Haplotype comparisons showed that LDL-C and TC associations were from rs2000999; Hb and RCC associations derived largely from the CNV. Distinct genotype-phenotype effects are evident at the genetic epidemiological level once LD has been analysed, perhaps reflecting HP-HPR functional biology and evolutionary history. The derived Hp2 allele of the HP gene has apparently been subject to malaria-driven positive selection. Haptoglobin-related protein binds Hb and apolipoprotein-L, i.e. linking HPR to the cholesterol system; and the HPR/apo-L complex is specifically trypanolytic. Our analysis illustrates the complex interplay between functions and haplotypes of adjacent genes, environmental context and natural selection, and offers insights into potential use of haptoglobin or haptoglobin-related protein as therapeutic agents.     

Mechanism-based partial inactivation of glutathione S-transferases by nitroglycerin: tyrosine nitration vs sulfhydryl oxidation.

Liver glutathione-S-transferases (GSTs) are responsible for the detoxification of electrophiles, and specifically for the metabolism of orally administered organic nitrates such as nitroglycerin (NTG). Recent studies showed that reactive nitrogen species produced by tetranitromethane (TNM), peroxynitrite, or the myeloperoxidase/H2O2/nitrite system can inactivate GST. It is not known whether NTG can similarly inactivate liver GSTs, and if shown, by what mechanism(s). We incubated purified GSTs with NTG, S-nitroso-N-acetylpenicillamine (SNAP), TNM, or vehicle (5% dextrose, D5W), followed by determination of GST activity. Incubation of GST with NTG and TNM caused significant decreases in GST activity whereas no changes were observed with SNAP or D5W. The relative GST activity (vs preincubation) was 73+/-14% for NTG, 37+/-8% for TNM, 98+/-13% for SNAP, and 98+/-9% for D5W, respectively. Exogenous glutathione (GSH) prevented both NTG- and TNM-induced changes in GST activity, consistent with the observed oxidative modification of GST, such as -SH oxidation and dimerization of oxidized GST. In contrast, NTG and TNM exhibited substantial differences in their ability to nitrate tyrosine (TYR) sites in GST. These results demonstrated that NTG can reduce the activity of its own metabolizing enzyme such as GST and this inhibitory effect of NTG was unlikely to be mediated through NO, as such, since SNAP had no effect on GST activity. The partial inactivation of GST by NTG appeared to involve -SH oxidation, but not TYR nitration. These findings provided the first evidence of mechanism-based protein inactivation by NTG, and may lend insight into the hepatic metabolism of NTG and other organic nitrates after repeated oral exposure.     

Undecagold labeling of a glycoprotein: STEM visualization of an undecagoldphosphine cluster labeling the carbohydrate sites of human haptoglobin-hemoglobin complex

A new type of label for electron microscopy has been introduced recently which consists of 11 gold atoms in a compact stable cluster with an organic shell composed of primary amine-substituted phosphine ligands. The radius of the cluster is about 10 A. The (phosphine ligand) amines can be derivatized or allowed to react directly forming covalent bonds to specific sites of other molecules. This report describes the specific labeling of carbohydrate moietis on the glycoprotein human haptoglobin (Hp) in the haptoglobin-hemoglobin complex (Hp X Hb). The Hp X Hb complex is easily recognized in the EM as a barbell-shaped molecule. Only the Hp portion contains carbohydrate (eight carbohydrate chains per Hp X Hb). The carbohydrate moieties of the Hp X Hb complex were oxidized by sodium periodate to produce aldehydes. The primary amines on the undecagold cluster were allowed to react with the aldehyde residues to produce Schiff's base linkages which were subsequently reduced with sodium borohydride. Micrographs obtained on the Brookhaven National Laboratory high-resolution scanning transmission electron microscope (STEM) showed the undecagold label to be localized in a region known to be occupied by the heavy chains of haptoglobin. The amount of labeling was found to be two to four gold clusters per molecule when excess label was reacted. The variation in position of the label is discussed and may be due to flexibility of the carbohydrate chains. Control experiments ruled out nonspecific binding of the gold cluster to the Hp X Hb. The high chemical specificity of the reaction and the high resolution of the gold cluster should make this new label of widespread value in studies of other glycoproteins or carbohydrate-bearing molecules.     

In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta)

The antioxidant activity of natural ulvan and its derivatives (acetylated and benzoylated ulvans) in vitro was determined, including scavenging activity against superoxide and hydroxyl radicals, reducing power, and chelating ability. Obvious differences in antioxidant activity between natural ulvan and its derivatives were observed, moreover, the antioxidant activity of acetylated and benzoylated ulvans was stronger than that of natural ulvan.     

Hemoglobin-induced lung vascular oxidation,inflammation, and remodeling contribute to the progression of hypoxic pulmonary hypertension and is attenuated in rats with repeated-dose haptoglobin administration

Haptoglobin (Hp) is an approved treatment in Japan for trauma, burns, and massive transfusion-related hemolysis. Additional case reports suggest uses in other acute hemolytic events that lead to acute kidney injury. However, Hp’s protective effects on the pulmonary vasculature have not been evaluated within the context of mitigating the consequences of chronic hemoglobin (Hb) exposure in the progression of pulmonary hypertension (PH) secondary to hemolytic diseases. This study was performed to assess the utility of chronic Hp therapy in a preclinical model of Hb and hypoxia-mediated PH. Rats were simultaneously exposed to chronic Hb infusion (35 mg per day) and hypobaric hypoxia for 5 weeks in the presence or absence of Hp treatment (90 mg/kg twice a week). Hp inhibited the Hb plus hypoxia-mediated nonheme iron accumulation in lung and heart tissue, pulmonary vascular inflammation and resistance, and right-ventricular hypertrophy, which suggests a positive impact on impeding the progression of PH. In addition, Hp therapy was associated with a reduction in critical mediators of PH, including lung adventitial macrophage population and endothelial ICAM-1 expression. By preventing Hb-mediated pathology, Hp infusions: (1) demonstrate a critical role for Hb in vascular remodeling associated with hypoxia and (2) suggest a novel therapy for chronic hemolysis-associated PH.     

Hemoglobin-binding site on human haptoglobin. Identification of lysyl residues participating in the binding.

The locus of human haptoglobin (Hp) molecules that interacts with human hemoglobin (Hb) was examined by the differential labeling technique. First, amino groups of Hp were extensively labeled with ethyl acetimidate (EAI) either in the free state (experiment A) or in the equimolar complex with Hb (experiment B). Each of the labeled Hp samples was reduced and S-carboxymethylated, and the remaining amino groups were then allowed to react with trinitrobenzenesulfonic acid (TNBS) under denatured conditions. Only 0.6 mol/mol of the trinitrophenyl (TNP) group was introduced into the heavy chain of Hp in experiment A, whereas 2.5 mol/mol of TNP groups were into the same chain in experiment B. The extent of TNP modification for the light chain was very low in either of the experiments. The amino acid residues carrying TNP groups in the heavy chain were identified by the peptide mapping procedure. They were Ile1, Lys136, and Lys218 in experiment B, and only Ile1 in experiment A. These findings suggest that epsilon-amino groups of Lys136 and Lys218 in the heavy chain are both situated within the Hb-binding locus of Hp, and that the alpha-amino group of Ile1 is buried inside the molecule either in the presence or absence of Hb.