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PubMed1980

Haemoglobin binding with haptoglobin. Unequivocal demonstration that the beta-chains of human haemoglobin bind to haptoglobin.

Haptoglobin binding to haemoglobin and its isolated alpha- and beta-chains was studied by use of a highly sensitive solid-phase radiometric assay. As expected, adsorbents of haemoglobin bound 125I-labelled haptoglobin more efficiently than did adsorbents of the alpha-chain. However, unexpectedly, adsorbents of the beta-chain were found to be essentially identical with those of the alpha-chain in their ability to bind haptoglobin. These results demonstrate, unequivocally, the ability of beta-chains to bind to haptoglobin, and indicate that this assay is particularly convenient and useful for studying haptoglobin interactions with haemoglobin and its alpha- and beta-chains.

Haptoglobins↗

PubMed1986

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding sites on the beta-chain of human haemoglobin by synthetic overlapping peptides encompassing the entire chain.

A synthetic approach was employed to identify the haptoglobin-binding sites on the beta-chain of human haemoglobin. This approach consists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen synthetic peptides (beta 1-15, beta 11-25 etc.) were examined for their ability to bind human haptoglobin by quantitative solid-phase radiometric titrations of 125I-labelled haptoglobin. Of these 14 peptides only peptides beta 11-25 and beta 131-146 bound haptoglobin significantly; peptide beta 21-35 exhibited a small binding activity as a consequence of the overlap with peptide beta 11-25. On this basis and by examination of the three-dimensional structure of haemoglobin, it was concluded that the beta-chain of haemoglobin has two binding sites for haptoglobin that reside in, but do not necessarily encompass all of, the regions beta 11-25 and beta 131-146.

Binding Sites↗

PubMed1992

Quantitative differentiation of the haptoglobin-related gene product from haptoglobin in human plasma: a possible test for tumor-associated antigen.

The haptoglobin-related gene product (HGRP) having greater than 90% sequence homology with plasma haptoglobin, has recently been implicated as a tumor maker of recurrent breast cancer. Therefore, availability of a monoclonal antibody which is specific to the HGRP and ensuing ELISAs to quantitate the HGRP without cross-reactivity with plasma haptoglobin will allow determination of the clinical value of the HGRP in human plasma under various pathophysiological conditions. A peptide representing the first 34 residues of the N-terminal portion of the HRGP was synthesized and conjugated to keyhole limpet hemocyanin (KLH) to immunize Balb/c mice. Hybrid clones that produced specific antibodies to HRGP were screened with the same peptide but conjugated to bovine serum albumin (HRGP-BSA). From 12 hybridoma clones that recognized HGRP-peptide, we obtained one IgM producing clone (27.5) by limiting dilution technique that selectively reacted with HGRP-peptide with minimal cross-reactivity with haptoglobin. Using a separate monoclonal antibody, clone 21.7, which is directed to the alpha subunit of haptoglobin, we developed an enzyme-linked immunosorbent assay (ELISA) to determine the quantities of the HGRP in human plasma, which will allow assessment of HGRP as a clinical marker of malignancy.

Adolescent↗

PubMed1981

Haemoglobin binding with haptoglobin. Localization of the haptoglobin-binding site on the alpha-chain of human haemoglobin.

A synthetic approach was employed to identify the haptoglobin-binding site on the alpha-chain of human haemoglobin. This approach cosists of the synthesis of a series of consecutive overlapping peptides that, together, systematically represent the entire protein chain. Fourteen peptides were synthesized (alpha 1-15, alpha 11-25, alpha 21-35, alpha 31-45, alpha 41-55, alpha 51-65, alpha 61-75, alpha 71-85, alpha 81-95, alpha 91-105, alpha 101-115, alpha 111-125, alpha 121-135 and alpha 131-141), and their ability bind human haptoglobin was studied, Only peptide alpha 121-135 bound haptoglobin significantly. On this basis we conclude that the haptoglobin-binding site on the alpha-chain of haemoglobin resides within, but does not necessarily encompass all of, the region alpha 121-135.

Binding Sites↗

PubMed1984

Haptoglobin typing, is it clinically necessary for a reliable determination of haptoglobin with the single radial immunodiffusion technique?

Over a period of three years we determined haptoglobin levels by single radial immunodiffusion (RID) and the haptoglobin phenotype in over 1700 samples of patients suspected of a haemolytic disease. As haptoglobin phenotyping is rather laborious and therefore an expensive method, we re-evaluated the diagnostic need for phenotyping. From our reference values for the respective phenotypes of haptoglobin it may be theoretically argued that phenotyping is still desirable when the RID value is in the range 400-1170 mg/l. Limiting ourselves to suspected haemolytic diseases, one can abolish phenotyping beyond this narrow range without withdrawing important clinical information. From the total group it appeared that 480 samples lay in this range. In 72 samples (15% of 480) the evaluation of the RID values was essentially altered by phenotyping. Careful examination of the medical records indicated that in a number of cases laborious phenotyping had indeed contributed to the diagnosis.

Female↗

PubMed1982

Di-, tri-, and tetrapeptide sequences in haptoglobin. Contribution to understanding of haptoglobin structure.

Amino acid sequences of haptoglobin and 11 proteins related and unrelated to haptoglobin were resolved into overlapping di-, tri-, and tetrapeptides. Comparison of the obtained peptides confirmed the existence of homology between haptoglobin and the family of serine proteases. The homology with light chain of immunoglobulins was relatively weak. A surprising similarity with concanavalin A was found. Tetrapeptide beta-turns (chain reversals), characterized by Chou & Fasman (1977, J. Mol. Biol., 115, 135-175) were compared with similar structures in light (alpha) and heavy (beta) subunits of haptoglobin.

Amino Acid Sequence↗

PubMed1993

Quantitation of human haptoglobin by ELISA system based on streptococcal haptoglobin receptors.

Streptococcus pyogenes cells with binding properties for human haptoglobin were used for quantitative determination of the acute phase protein, haptoglobin in various biological fluids. The S. pyogenes cells with protein surface antigen T4 served as solid phase in a microtitre ELISA system. After binding to the bacteria the amount of haptoglobin could be quantified by polyclonal or monoclonal antibodies. The constructed ELISA proved to be sensitive and correlated well with a conventional peroxidase method and with an immunoassay based on hemoglobin binding to haptoglobin.

Enzyme-Linked Immunosorbent Assay↗

PubMed1983

Simultaneous quantitation of free haptoglobin and haemoglobin-haptoglobin (HbHp) complexes by double-decker rocket immunoelectrophoresis.

Existing immunochemical methods for haptoglobin (Hp) determination are incapable of distinguishing between free haptoglobin and haemoglobin-haptoglobin (HbHp) complexes. A one-dimensional double-decker rocket immunoelectrophoretic method (DD-RIE) for simultaneous quantitation of free Hp and Hp bound as HbHp complexes was therefore developed. The DD-RIE can be used for quantitative in vitro studies of the Hb-Hp interactions, for quantitation of Hp's Hb binding capacity and for investigations of haemolytic episodes in patients. The clinical value of the method is illustrated by an investigation of a patient with a gradual accumulation of HbHp complexes in plasma, a steady rise in total Hp and a fall in free Hp during haemolysis. A proposed receptor-mediated clearance of the complexes by the reticuloendothelial system (RES) could not be substantiated, as HbHp receptors were not demonstrable on human blood monocytes.

Haptoglobins↗

PubMed1979

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation.

1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

Animals↗

PubMed1991

DNA polymorphisms in the controlling region of the human haptoglobin genes: a molecular explanation for the haptoglobin 2-1 modified phenotype.

A haptoglobin 2-1 modified (Hp2-1mod) phenotype results when the amount of Hp2 polypeptide synthesized in Hp2/Hp1 heterozygotes is less than that of Hp1 polypeptide. Cloned Hp2 DNA from an individual with the Hp2-1mod phenotype is here shown to have a C in place of the normal A at nucleotide position -61 in one of the interleukin-6 (IL-6) responsive elements of the haptoglobin promoter region. Direct sequencing of the haptoglobin promoter region, amplified by PCR, from DNA from unrelated American blacks showed a C at -61 in all of 10 individuals with the Hp2-1mod phenotype, in two of four with a "possible Hp2-1mod" phenotype, but in none of 15 with the Hp2-1 phenotype. Thus the -61C mutation in the Hp2-61C allele is strongly associated with the Hp2-1mod phenotype. Sequencing results also show that there are three other promoter sequences in the population studied; each can be associated with either Hp2 or Hp1. The variability seen in the Hp2-1mod phenotype, a variability which ranges from close to Hp2-1 to close to Hp1-1, can be explained, in part, by the existence of several Hp2 alleles differing in their promoters--and possibly, in part, by differences in the promoters of the accompanying Hp1 allele. A further part of the variability may be the consequence of differences in the way that the Hp2-61C and the Hp2 alleles respond to the IL-6-dependent factor during an acute-phase response.

Base Sequence↗

PubMed1976

[Haptoglobin in domestic mammals. V. Plasma haptoglobin level in cattle under pathological conditions].

The haptoglobine concentration in the blood plasma of cattle tends to rise to more than 10 mg/100 ml in the presence of acute inflammation anywhere in the organism. They were 40 mg/100 ml and above in the presence of traumatic reticuloperitonitis. The absence of haptoglobin multiplication in the plasma is a high-probability indicator to the absence of that inflammatory process. chronic inflammation will only in exceptional cases entail plasma-haptoglobin multiplication. Concentrations will not be pathologically increased in cattle with positive reaction to haematological testing for leucosis.

Actinobacillosis↗

PubMed2000

Reference range of serum haptoglobin is haptoglobin phenotype-dependent in blacks.

Reference values for serum haptoglobin (Hp), were established in a Black Zimbabwean population. The upper limit (2.15 g/l) is comparable to the one in Caucasians, but the lower limit (0.12 g/l) is much lower than the proposed interim international reference limit (0.3 g/l). Subjects that typed as Hp 0-0 by starch gel electrophoresis technique were retyped using high performance gel permeation chromatography. This resulted in a 32% decrease in the frequency of Hp 0-0, but an increase in Hp 2-2 and Hp 2-1M phenotype frequencies. In the Zimbabwean Blacks, the Hp 0-0 frequency was estimated to be 2.9%. Haptoglobin reference values were found to be Hp phenotype-dependent; highest values were found in Hp 1-1 (median 0.88 g/l; range 0.31-1.69 g/l) and in Hp 2-1 (median 0.90 g/l; range 0.31-2.22 g/l) and lower values (median 0.66 g/l; range 0.13-1.79 g/l) in Hp 2-2 subjects. The Hp 2-1M phenotype was characterized by low reference values (0.18-1.25 g/l) (P<0.05). In three cases of the rare variant Hp Johnson, high Hp concentrations were found (median 1. 57 g/l; range 0.98-1.57 g/l).

Adult↗

PubMed1991

Haptoglobin phenotyping from older bloodstains by enzyme immunoassay and haptoglobin phenotypes within a Nebraska Caucasian population.

Enzyme immunoassay and Western blotting (electrophoretic) techniques were used to determine haptoglobin (HP) phenotypes from older bloodstains. Serum was collected from liquid blood and the HP phenotypes were determined. Bloodstains were prepared from these specimens and stored at various temperatures for several months. The stains were extracted and applied to gradient polyacrylamide gels. The Western blotting technique was used to achieve the transfer of HP bands from the gels to the nitrocellulose membranes. Enzyme immunoassay with goat anti-HP antiserum and rabbit anti-goat immunoglobulin peroxidase were used to identify the HP bands from the extracted samples. Enzyme immunoassay was found to be clearly more sensitive than o-dianisidine or o-tolidine in detecting HP bands from diluted serum samples. The haptoglobin frequency in a Caucasian population in Nebraska was calculated. The frequencies of Phenotypes 1, 2-1, and 2 were found to be 15.8, 48.4, and 35.8%, respectively.

Blood Preservation↗

PubMed1977

Influence of modifications of physicochemical and biological properties of haptoglobin. VII. Immunochemical reactivity of haptoglobin with modified tyrosine residues.

Tyrosine residues in human haptoglobin (Hp) type 2-1 were modified by treatment with various amounts of N-acetylimidazole, iodine or tetranitromethane. Rabbit antisera directed against native Hp, Ac-Hp, 1-Hp and NO2-Hp in cross-reactions by immunoprecipitation showed serologic identify of native Hp and most of the modified derivatives, with the exception of derivatives modified by means of a high excess of N-acetylimidazole, which showed partial identity with remaining preparations, irrespective of the antiserum used. The results indicate that tyrosine residues play no essential role in the immunochemical reactivity of haptoglobin.

Acetylation↗

PubMed1996

Haptoglobin phenotyping by newly developed monoclonal antibodies. Demonstration of haptoglobin uptake into peripheral blood neutrophils and monocytes.

We have generated two IgG murine mAbs that recognize native human haptoglobin (Hp). These mAbs, 3A8 and 4B2, efficiently bind to the Hp complex regardless of the serum donor's phenotype. The specificity of mAb 3A8 was confirmed by immunoaffinity purification of 3A8-binding material from human serum and subsequent N-terminal amino acid sequencing of the invariant 40-kDa chain. mAb 3A8 and 4B2 were also reactive with cell-associated Hp when studied by immunocytochemistry. When human peripheral blood leukocytes were tested, 90% of the granulocytes and a lesser (and variable) fraction of monocytes displayed an intense intracytoplasmatic granular staining. This was confirmed by flow cytometric analysis of permeabilized leukocytes and by demonstrating the presence of Hp (of the expected Hp serum phenotype) in extracts of washed granulocytes by immunoblotting. Leukocytes obtained from a Hp phenotype 2-2 donor, incubated in culture medium supplemented with 10% serum from a donor possessing the Hp 1-1 phenotype, contained both Hp phenotypes when analyzed by immunoblotting after a 6-h incubation period. In addition, Hp was actively exocytosed by granulocytes following their exposure to Candida albicans. These observations suggest that exogenous Hp is concentrated within granulocytes and not synthesized de novo and is, in turn, exocytosed following neutrophil activation. Northern blotting analysis is consistent with the lack of haptoglobin gene transcription in granulocytes. These findings together with the earlier observations that Hp modulates granulocyte activity suggest that Hp levels may be enhanced locally at sites of inflammation to modulate granulocyte activity.

Amino Acid Sequence↗

PubMed1988

Forensic aspects of haptoglobin: electrophoretic patterns of haptoglobin allotype products and an evaluation of typing procedure.

A procedure used for haptoglobin (Hp) typing in paternity cases has been evaluated. All serum samples have been subtyped with a one-dimensional isoelectric focusing/immunoblotting method, and samples with rare or questionable patterns have been further examined by two-dimensional electrophoresis with isoelectric focusing in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The electrophoretic Hp-patterns of common and rare alpha- and beta-chain variants are shown, including allotype patterns of two new beta-chain variants and three new alpha-chain variants. Retyping of nearly 2000 individuals at intervals between 1 to 12 months revealed a typing error frequency of about 0.3%, which is considered acceptable, provided new blood samples are required in every case of paternity exclusion. Comparison of typing results obtained with the present procedure and with routine starch gel electrophoresis in more than 5000 serum samples gave conflicting results in 6 samples. The sensitivity of the described one-dimensional subtyping method was slightly better than that of starch gel electrophoresis. In 4110 unrelated individuals, involved in cases of disputed paternity the Hp 2SS 0.038, Hp 2FF 0.004, and Hp 3 (Johnson) 0.0005. These allele frequencies give a theoretical paternity exclusion rate of 32.5%, which is in accordance with the observed rate in 2200 paternity cases with more than 600 non-fathers. It is concluded that the present procedure represents a definite improvement for Hp subtyping in practical paternity diagnostics. Preliminary results with retyping of weak Hp patterns using a staining technique involving the biotin/avidin complex indicate that the sensitivity of the one-dimensional subtyping method may be substantially increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Gel, Two-Dimensional↗

PubMed1990

Haptoglobin subtyping with anti-haptoglobin alpha chain antibodies.

Specific antibodies against the human haptoglobin (HP) alpha chain were raised and used for immunoblotting after isoelectric focusing to determine Hp alpha subtypes in a reproducible and simplified manner. By eliminating the staining of the HP beta chain, HP alpha subtypes were visualized more precisely and simply than by previously reported methods. Subtypes of a total of 1211 sera, obtained from two different populations in Japan, were examined by the new method. Four HP alpha subtypes (HP 1S-1S,2FS-1S, 2FS-2FS and 2FS-2SS) and five subtypes combined with a new variant (HP 2FS-1V1, 2V1-1S, 2FS-2V1, 2FS-2V2 and 2FS-2V3) were observed. HP 1V1 belonged to HP alpha 1, and HP 2V1, 2V2 and 2V3 belonged to HP alpha 2, and their alleles were designated HP A*1V1, HP A*2V1, HP A*2V2 and HP A*2V3, respectively. The allele frequencies were calculated to be as follows: HP A*1S, 0.2597; HP A*1V1, 0.0004; HP A*2FS, 0.7333; HP A*2SS, 0.0008; HP A*2V1, 0.0045; HP A*2V2, 0.0008; and HP A*2V3, 0.0004. The allele frequency of HP A*2SS, which is common in the European population, is less frequent than HP A*2V1 in the Japanese population.

Alleles↗

PubMed1985

Haptoglobin subtypes in Berlin, GDR. A simple procedure for haptoglobin purification and subtyping.

Sera were obtained from 1,275 blood donors in Berlin, probands involved in paternity tests, and from 119 families with 235 children; the sera were subtyped by isoelectric focusing, following preparation and reductive molecular cleavage of haptoglobin. In this paper, an uninvolved preparation technique is described for routine testing. Allelic frequencies are: Hp *1F = 0.1471; *1S = 0.2502; *2FF = 0.0020; *2FS = 0.5753; *2SS = 0.0251. Only one deviation from autosomal codominant inheritance was recorded in the family examinations, with illegitimacy considered possible. In the region of Berlin, the changes of ruling out uninvolved individuals in paternity suits have gone up from 18% (conventional technique recording two frequent alleles) to 33% (subtyping).

Adult↗