Immunoassay of haemoglobin-acrylonitrile adduct in rat as a biomarker of exposure

Acrylonitrile (AN) is a rat carcinogen. Human exposure may come from chemical industries and smoking. A haemoglobin adduct of acrylonitrile (Hb-AN) has been used as a biomarker of exposure by means of gas chromatography-mass spectrometry (GC-MS) analysis. We have developed specific monoclonal antibodies (Mab) to human Hb-AN and wish to report evaluation of an immunoassay in rats using an Mab that cross-reacts with rat Hb-AN. A dose response study of LD 0, 10, 50, and 90 in Sprague-Dawley rats was undertaken, with each rat receiving \[2,3-14C]AN at 50 Ci kg-1 sc, and Hb from an aliquot of blood was taken for covalent binding analysis by liquid scintillation spectrometry and fluorescence ELISA. The dose responses of rats at 0.25, 0.5, 1.0, and 2.0 h after AN doses of 20, 50, 80, 115 mg kg-1 were compared by both methods with Hb and globin samples. Regression analysis showed a linear relationship between immunoassay and 14C-AN binding. This indicates that an antigenic form of Hb-AN may be used as a surrogate of Hb-AN adduct. The sensitivity of ELISA was tested in rats exposed for 1 h to sub-toxic doses of AN (10-1.1 mg kg-1). Quantification of Hb-AN by immunoassay was achieved by calibration with a synthetic adduct HbAN4h, a reference adduct prepared by treating rat Hb with excess AN for 4 h. ELISA and GC-MS analysis of N-terminal valine-AN in the Hb-AN adduct were compared and similar detection levels were found. This rat study appears to have validated the new immunoassay method for biomonitoring of AN exposure.     

Immunoassay of haemoglobin-acrylonitrile adduct in rat as a biomarker of exposure

Acrylonitrile (AN) is a rat carcinogen. Human exposure may come from chemical industries and smoking. A haemoglobin adduct of acrylonitrile (Hb-AN) has been used as a biomarker of exposure by means of gas chromatography-mass spectrometry (GC-MS) analysis. We have developed specific monoclonal antibodies (Mab) to human Hb-AN and wish to report evaluation of an immunoassay in rats using an Mab that cross-reacts with rat Hb-AN. A dose response study of LD 0, 10, 50, and 90 in Sprague-Dawley rats was undertaken, with each rat receiving \[2,3-14C]AN at 50 Ci kg-1 sc, and Hb from an aliquot of blood was taken for covalent binding analysis by liquid scintillation spectrometry and fluorescence ELISA. The dose responses of rats at 0.25, 0.5, 1.0, and 2.0 h after AN doses of 20, 50, 80, 115 mg kg-1 were compared by both methods with Hb and globin samples. Regression analysis showed a linear relationship between immunoassay and 14C-AN binding. This indicates that an antigenic form of Hb-AN may be used as a surrogate of Hb-AN adduct. The sensitivity of ELISA was tested in rats exposed for 1 h to sub-toxic doses of AN (10-1.1 mg kg-1). Quantification of Hb-AN by immunoassay was achieved by calibration with a synthetic adduct HbAN4h, a reference adduct prepared by treating rat Hb with excess AN for 4 h. ELISA and GC-MS analysis of N-terminal valine-AN in the Hb-AN adduct were compared and similar detection levels were found. This rat study appears to have validated the new immunoassay method for biomonitoring of AN exposure.     

Study of structural heterogeneity of polymerized hemoglobin using biochemical and immunochemical methods

The structural heterogeneity of crosslinked pyridoxalated hemoglobin (polyHb-PLP) have been investigated by the methods of gel-filtration, isoelectric focusing and immunoelectrophoresis. It was established that the heterogeneity of poly-Hb-PLP is mainly depicted by polydispersion of molecular weight and less by the changes in the isoelectric properties. The qualitative difference has been shown between the immunochemical properties of the native hemoglobin and poly-Hb-PLP, which gives the suggestion of the formation of new antigenic determinants as a result of chemical modification and of more expressed antigenicity of modified hemoglobin compared with native protein. Poly-Hb-PLP has wide antigenic spectrum that corresponds to its structural heterogeneity. The data obtained suggest that the decrease in the antigenic activity of poly-Hb-PLP can be achieved by the lowering of the part of high molecular weight components.     

New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites

A new set of hydrophilicity high-performance liquid chromatography (HPLC) parameters is presented. These parameters were derived from the retention times of 20 model synthetic peptides, Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X was substituted with the 20 amino acids found in proteins. Since hydrophilicity parameters have been used extensively in algorithms to predict which amino acid residues are antigenic, we have compared the profiles generated by our new set of hydrophilic HPLC parameters on the same scale as nine other sets of parameters. Generally, it was found that the HPLC parameters obtained in this study correlated best with antigenicity. In addition, it was shown that a combination of the three best parameters for predicting antigenicity further improved the predictions. These predicted surface sites or, in other words, the hydrophilic, accessible, or mobile regions were then correlated to the known antigenic sites from immunological studies and accessible sites determined by X-ray crystallographic data for several proteins.     

Hemoglobin Kariya [alpha 40 (C5) Lys leads to Glu]: a new hemoglobin variant with an increased oxygen affinity

A new abnormal hemoglobin, Hb Kariya [alpha 40 (C5) Lys leads to Glu], with an amino acid substitution at the alpha 1 beta 2 contact was discovered in a young Japanese man. This variant migrated to the anode faster than Hb A, being nearly the same as Hb I in electrophoretic mobility. It amounted to about 6% of the total hemoglobin of the hemolysate. This hemoglobin showed an increased oxygen affinity, decreased heme-heme interaction and a lowered 2,3-DPG effect.     

Oxygen binding and other physical properties of human hemoglobin made in yeast.

Wagenbach et al. (1991, BioTechnology, 9, 57-61) have recently developed a system for producing soluble recombinant tetrameric hemoglobin in yeast: hemoglobin begins to appear 4-5 h after induction with galactose, alpha- and beta-globin chains fold in vivo and endogeneously produced heme is incorporated into hemoglobin tetramers. We have further characterized the oxygen-binding properties, as well as the tetramer stability, of recombinant human Hb A made in yeast. After purification by ion-exchange chromatography, a single band at the same position as normal human Hb A was obtained using cellulose acetate electrophoresis. Although the oxy and deoxy forms of purified recombinant Hb A made in yeast were spectrophotometrically identical to native human Hb A, the oxygen-binding curve was shifted slightly left of that for native human Hb A. Further purification of recombinant hemoglobin by FPLC revealed two fractions: one (fraction B) with low cooperativity and high oxygen affinity, and the other (fraction A) with almost identical cooperativity and oxygen affinity compared with native human Hb A. The Bohr effect of fraction A was also identical to native human Hb A. Hemoglobin in fraction B with lowered cooperativity precipitated approximately 1.5 times faster than normal human Hb A during mechanical agitation, while hemoglobin in fraction A with normal cooperativity precipitated with kinetics identical to native human Hb A. These results suggest that some of the recombinant molecules made in yeast fold improperly, and that these molecules may exhibit decreased cooperativity for oxygen binding and decreased stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosylated minor components of human adult hemoglobin. Purification, identification, and partial structural analysis

Human hemolysate contains several minor components designated Hb A1a, Hb A1b, Hb A1c, which are post-translational modifications of the major hemoglobin component A0. Individuals with diabetes mellitus have elevated levels of Hb A1c, a hemoglobin modified with a glucose moiety at the NH2 terminus of each beta chain. A new chromatographic technique using Bio-Rex 70 is described which not only allows complete separation of Hb A1a from Hb A1b but also resolution of Hb A1a into two components, designated Hb A1a1 and Hb A1a2. Carbohydrate determinations with the thiobarbituric acid procedure revealed that Hb A1a1, Hb A1a2, and Hb A1b as well as Hb A1c were glycosylated. Total phosphate analysis revealed 2.06 and 1.01 mol of phosphorus/alphabeta dimer for Hb A1a1 and Hb A1a2 respectively; Hb A1b and Hb A1c contained no detectable phosphate. Hemoglobin incubated with D-[14C]glucose-6-P co-chromatographs precisely with Hb A1a2, strongly suggesting that Hb A1a2 is glucose-6-P hemoglobin. Levels of Hb A1a1 and Hb A1a2 are normal in individuals with diabetes mellitus. Furthermore, diabetic red cells contain normal levels of glucose-6-P. Therefore, glucose-6-P hemoglobin does not serve as a significant precursor to Hb A1c. Instead Hb A1c is formed by the direct reaction of hemoglobin with glucose. This suggests that hemoglobin can serve as a model system for nonenzymatic glycosylation of protein.     

The transition from HK to LK phenotype in the red cells of newborn genetically LK lambs

Red cells from newborn lambs were separated into different age populations by centrifugation, and cells with fetal hemoglobin (Hb) were distinguished from those with adult Hb by an acid elution technique. Changes were followed during development in rates of K+ transport (active and passive), numbers of Na+/K+ pump sites per cell, cell volumes, and numbers of Lp and L1 antigen sites per cell. These changes were correlated with the percentage of cells with adult hemoglobin. (The Lp and L1 antigens are associated with K+ transport in that specific alloantibody against Lp, anti-Lp, stimulates active transport, and anti-L1 inhibits passive transport.) Active K+ transport decreased during development because of a decline in number of Na+/K+ pumps (from measurements of ouabain binding) and because of an alteration in the affinity of the pumps for intracellular K+ (from kinetic studies in which the intracellular K+ concentration was varied). Cells with fetal Hb had fewer Lp sites and were larger than cells with adult Hb. As transport properties changed, the number of Lp sites increased and continued to increase after all the cells had adult Hb Cells with fetal Hb had as many L1 sites as lamb cells with adult Hb, but the number of L1 sites was less than those found previously for adult sheep. A population of small cells with intermediate K+ concentration and intermediate numbers of Lp sites appeared soon after birth. The various points of evidence suggested that the developmental process leading to cells with adult transport properties was a gradual one and did not coincide precisely with the switch from fetal to adult Hb.     

The effect of crosslinking by bis(3,5-dibromosalicyl) fumarate on the autoxidation of hemoglobin

Bis(3,5-dibromosalicyl) fumarate was used to crosslink hemoglobin both in the oxy and deoxy states. This double headed diaspirin was known to crosslink oxy Hb A selectively between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxy Hb A between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R. Y., et al. (1986) J. Biol. Chem. 261, 9929). The autoxidation at 37 degrees C of oxy alpha 99 crosslinked hemoglobin was found to be 1.8 times as fast as that of Hb A while that of the oxy beta 82 crosslinked hemoglobin was only 1.2 times as fast. After 5 hours the formation of methemoglobin in the alpha crosslinked Hb A is 21.3% compared to 10.8% in beta crosslinked Hb A and 6.4% in Hb A. These results may effect the proposed use of alpha 99 crosslinked hemoglobin as a blood substitute by demonstrating the need for protection from autoxidation during storage.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.     

Strong conformational propensities enhance T cell antigenicity

The ability to predict T cell antigenic peptides would have important implications for the development of artificial vaccines. As a first step towards prediction, this report uses a new statistical technique to discover and evaluate peptide properties correlating with T cell antigenicity. This technique employs Monte Carlo computer experiments and is applicable to many problems involving protein or DNA. The technique is used to evaluate the contribution of various peptide properties to helper T cell antigenicity. The properties investigated include amphipathicities (alpha and beta), conformational propensities (alpha, beta, turn and coil), and the correlates of alpha-helices, such as the absence of helix-breakers and the positioning of the residues which stabilize alpha-helical dipoles. We also investigate segmental amphipathicity. (A peptide has this property when it contains at least two disjoint subpeptides, one hydrophobic, one hydrophilic.) Statistical correlations and stratifications assessed independent contributions to T cell antigenicity. The findings presented here have important implications for the manufacture of peptide vaccines. These implications are as follows: if possible, peptide vaccines should probably be those protein segments which have a propensity to form amphipathic alpha-helices, which do not have regions with a propensity to coil conformations, and which have a lysine at their COOH-terminus. The last two observations are of particular use in manufacturing peptides vaccines: they indicate where the synthetic peptides should be terminated. These implications are supported by the findings given below. The significances (p values) support the following statistical generalites about antigenic conformations: most helper T cell antigenic sites are amphipathic alpha-helices; alpha-helical amphipathicity and propensity to an alpha-helical conformation contribute independently to T cell antigenicity; there is evidence that some T cell antigenic sites are beta conformations instead of alpha-helices; T cell antigenic sites avoid random coiled conformations; and T cell antigenic sites are usually not segmentally amphipathic. alpha-Helical amphipathicity was significant, but segmental amphipathicity was not. This has implications for the dimensions of the structure interacting with the hydrophobic portion of an amphipathic T cell antigenic site. Lysines are unusually frequent at the COOH-terminal of T cell antigenic sites, even after accounting for tryptic digests. These lysines can stabilize alpha-helical peptides by a favorable interaction with alpha-helical dipoles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Improved production of human hemoglobin in yeast by engineering hemoglobin degradation

With the increasing demand for blood transfusions, the production of human hemoglobin (Hb) from sustainable sources is increasingly studied. Microbial production is an attractive option, as it may provide a cheap, safe, and reliable source of this protein. To increase the production of human hemoglobin by the yeast Saccharomyces cerevisiae, the degradation of Hb was reduced through several approaches. The deletion of the genes HMX1 (encoding heme oxygenase), VPS10 (encoding receptor for vacuolar proteases), PEP4 (encoding vacuolar proteinase A), ROX1 (encoding heme-dependent repressor of hypoxic genes) and the overexpression of the HEM3 (encoding porphobilinogen deaminase) and the AHSP (encoding human alpha-hemoglobin-stabilizing protein) genes — these changes reduced heme and Hb degradation and improved heme and Hb production. The reduced hemoglobin degradation was validated by a bilirubin biosensor. During glucose fermentation, the engineered strains produced 18% of intracellular Hb relative to the total yeast protein, which is the highest production of human hemoglobin reported in yeast. This increased hemoglobin production was accompanied with an increased oxygen consumption rate and an increased glycerol yield, which (we speculate) is the yeast's response to rebalance its NADH levels under conditions of oxygen limitation and increased protein-production.     

Fetal hemoglobin distributions among adult baboon and macaque species

Heritable variation in fetal hemoglobin (Hb F) in erythrocytes of the adult human has been shown to occur at more than one genetic locus. Heritable variation has also been reported in adult baboons. Nonhuman primates thus may serve as useful models for understanding how Hb F is regulated in the human. In the study reported here we identified Hb F in hemolysates from 27 of 32 rhesus macaques, from 32 of 32 baboons, and from none of 35 cynomoglus macaques. Hb F as a percentage of total hemoglobin occurred as a normally distributed variable among rhesus macaques but among baboons the distribution was both skewed and kurtotic. Such difference could be either a consequence of nonrandom sampling of the gene pool in one of the species, or a consequence of species evolution. A technique of single cell hemoglobin electrophoresis was applied to erythrocytes from three adult pig-tailed macaques. This demonstrated that erythrocytes which contain Hb F (F-cells) also customarily contain Hb A and that the proportions of these two hemoglobins varies substantially among the F-cells, as we previously noted for human F-cells. We conclude that the macaques could serve as useful models for understanding Hb F regulation in the human.     

Preparation and in vivo evaluation of two bovine hemoglobin-based plasma expanders

A hemoglobin (Hb)-based oxygen carrier was successfully transfused into rats. An ultrapure lipid-free bovine Hb was prepared by hypotonic dialysis and ultrafiltration. The Hb was polymerized with glutaraldehyde and the P50 was 24.3 mm Hg. On the basis of immunological analysis, immuno-dot blot, the Hb preparations were not antigenic. A second transfusion produced no adverse immunological side effects. A right shift in P50 was obtained by further treatment of polymerized Hb with inositol hexaphosphate; however, this Hb preparation was unsuitable for transfusion as all animals died within a few minutes. A 30% exchange transfusion in rats with the polymerized bovine Hb resulted in a 100% survival of all animals. P50 values of treated animals were reduced by about 2 mm Hg for 14 days. The Hb product circulated for 14 days as determined by 51Cr labeling. Ultrapure bovine Hb has the potential to circulate and carry oxygen in rats and causes no immunological side effects.     

Identifying antigenicity-associated sites in highly pathogenic H5N1 influenza virus hemagglutinin by using sparse learning

Since the isolation of A/goose/Guangdong/1/1996 (H5N1) in farmed geese in southern China, highly pathogenic H5N1 avian influenza viruses have posed a continuous threat to both public and animal health. The non-synonymous mutation of the H5 hemagglutinin (HA) gene has resulted in antigenic drift, leading to difficulties in both clinical diagnosis and vaccine strain selection. Characterizing H5N1's antigenic profiles would help resolve these problems. In this study, a novel sparse learning method was developed to identify antigenicity-associated sites in influenza A viruses on the basis of immunologic data sets (i.e., from hemagglutination inhibition and microneutralization assays) and HA protein sequences. Twenty-one potential antigenicity-associated sites were identified. A total of 17 H5N1 mutants were used to validate the effects of 11 of these predicted sites on H5N1's antigenicity, including 7 newly identified sites not located in reported antibody binding sites. The experimental data confirmed that mutations of these tested sites lead to changes in viral antigenicity, validating our method.     

Interactions between amyloid-β and hemoglobin: implications for amyloid plaque formation in Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides in the brain is one of the central pathogenic events in Alzheimer's disease (AD). However, why and how Aβ aggregates within the brain of AD patients remains elusive. Previously, we demonstrated hemoglobin (Hb) binds to Aβ and co-localizes with the plaque and vascular amyloid deposits in post-mortem AD brains. In this study, we further characterize the interactions between Hb and Aβ in vitro and in vivo and report the following observations: 1) the binding of Hb to Aβ required iron-containing heme; 2) other heme-containing proteins, such as myoglobin and cytochrome C, also bound to Aβ; 3) hemin-induced cytotoxicity was reduced in neuroblastoma cells by low levels of Aβ; 4) Hb was detected in neurons and glial cells of post-mortem AD brains and was up-regulated in aging and APP/PS1 transgenic mice; 5) microinjection of human Hb into the dorsal hippocampi of the APP/PS1 transgenic mice induced the formation of an envelope-like structure composed of Aβ surrounding the Hb droplets. Our results reveal an enhanced endogenous expression of Hb in aging brain cells, probably serving as a compensatory mechanism against hypoxia. In addition, Aβ binds to Hb and other hemoproteins via the iron-containing heme moiety, thereby reducing Hb/heme/iron-induced cytotoxicity. As some of the brain Hb could be derived from the peripheral circulation due to a compromised blood-brain barrier frequently observed in aged and AD brains, our work also suggests the genesis of some plaques may be a consequence of sustained amyloid accretion at sites of vascular injury.     

Static accessibility model of protein antigenicity: the case of scorpion neurotoxin

Scorpion neurotoxins are a family of homologous, 64 to 65 residue containing proteins with four invariant disulfide bridges. Previous experimental work established four antigenic epitopes in the Androctonus australis neurotoxin and localized them in the amino acid sequence. Using crystallographic coordinates of the Centruroides sculpturatus neurotoxin and computing its large sphere (radius 1 nm or 10 A) accessibility profiles, we identified six antigenic sites clustered into four surface regions. Three of four computed sites coincided with the epitopes identified and localized experimentally in the A. australis neurotoxin, while two of the computed sites partially overlapped the fourth epitope. To investigate the relationship between antigenicity and segmental flexibility, 8-ps molecular dynamics simulations were performed on the C. sculpturatus structure, average backbone temperature factors computed from the simulation, and results compared with the X-ray-derived B values. Most of the neurotoxin structure and, in particular, three of the four antigenic sites were found inflexible, as judged by the computed and/or crystallographic temperature factors. The remaining epitope was associated with only marginal above-average maxima of backbone B values, corresponding to root mean square atomic displacements of 0.5 A (50 pm). We conclude that neurotoxin antigenicity is determined by an exceptional surface exposure of relatively short loop segments and that segmental flexibility is not an essential component of antigenicity.     

Stopped-flow front-face fluorometer: a prototype design to measure hemoglobin R----T transition kinetics

Stopped-flow techniques are successfully used to study the kinetics of the R----T transition of hemoglobin (Hb). We have previously used front-face fluorometry to demonstrate that (i) the intrinsic fluorescence of Hb primarily originates from beta 37 Trp; (ii) the intrinsic fluorescence is sensitive to the R----T transition; and (iii) the emission of the fluorescent probes bound to specific sites on the Hb molecule (beta 93 Cys) is sensitive to the R----T transition. These findings suggested that a stopped-flow front-face fluorometer could probe R----T transitions at specific sites, such as the aromatic amino acids and sites selectively binding extrinsic fluorophores. We have developed a prototype instrument using as the core a Gibson-Durrum stopped-flow apparatus on line with a digital data analysis system using a modified Marquardt algorithm. Excitation (470 nm) and emission light (520 nm) were selected by narrow band pass filters. To study the R----T transition, a solution of purified oxy Hb A covalently bound to the fluorescent probe 5-iodoacetamidofluorescein (Hb A-AF) (1.0 g%) was mixed rapidly with deoxygenated buffer (pH 7.35, 0.05 M potassium phosphate) containing 2 mg/ml of sodium dithionite. The hemoglobin, at a final concentration of 0.5 g% after mixing, is essentially completely tetrameric. A first-order reaction was observed with a rate constant near 8 s-1, similar to the oxygen dissociation rate reported for oxy Hb A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis.

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.