Multiple immunoreactive forms of calcitonin in human plasma

Purified rabbit serum haptoglobin was partially characterized, and it was found that the hemoglobin-binding property and subunit structure were similar to those of human type 1-1, swine, canine, and equine haptoglobins. However, rabbit haptoglobin was dissociated into subunits and few intermediates only in the presence of urea or sodium dodecyl sulfate without reduction. Thus the absence of interchain disulfide bonds in rabbit haptoglobin is unique among many animal haptoglobins.     

Haptoglobin polymorphism--not only a genetic marker]

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

Immunochemical characteristics of the ovine polymeric haptoglobin HpC

It was found that haptoglobins of camel, cattle, horse, pig, rat, guinea pig and man form upon immunoelectrophoresis precipitation arcs with antibodies against polymeric sheep haptoglobin C, corresponding to alpha 2-globulins. The immunocrossreactivity of haptoglobins of man and various animal species towards antibody to sheep haptoglobin (100, 88.0, 75.2, 72.1, 56.3, 51.0, 41.3 and 28.0% for haptoglobin of sheep, camel, pig, cattle, man, rat, guinea pig and horse, respectively) was determined. The intensity of crossreactions between sheep haptoglobin and the proteins under study towards antibody to haptoglobin C reflects the similarity of their primary structure and, consequently, the immune homology of their molecules. Using quantitative titration, the antigenic valency values for human (6), sheep (5), cattle (4) and horse (3) haptoglobins were determined.     

Studies on haptoglobin binding to concanavalin A

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.     

Studies on the binding of haemoglobin by haptoglobin using electrofocusing and gradient electrophoresis.

1. Gel electrofocusing followed by gel gradient electrophoresis separated the haptoglobins and their complexes with haemoglobin into characteristic two-dimensional patterns of protein bands. 2. Molecular weights of 107 000, 139 000 and 168 000 were obtained for the three bands seen after a purified preparation of haptoglobin type 1 was partially saturated with haemoglobin. This indicated that free haptoglobin, the intermediate haptoglobin-haemoglobin complex containing one half-haemoglobin and the saturated complex with two half-haemoglobins were present. 3. The three proteins showed considerable microheterogeneity and gave a number of isoelectric points in the pH ranges 4.58-4.77, 5.20-5.40 and 5.74-5.93, free haptoglobin type 1 being the lowest group. These ranges were all 0.15-0.30pH units lower if other values were taken for the isoelectric points of markers used to calibrate the pH gradient. 4. All three proteins were present over a wide range of haemoglobin concentrations, from 0.5% to 92% of that required for saturation. This would be expected if both binding sites have similar affinities for haemoglobin.     

Carbohydrate structures of haptoglobin in sera of healthy people and a patient with congenital disorder of glycosylation

Haptoglobin is one of acute phase glycoproteins often used as markers in glycopathology studies. In this work the oligosaccharide structures of haptoglobin from 'healthy' subjects have been studied in detail, taking into consideration the possible dependence of glycosylation on the phenotype. About 75% of charged haptoglobin glycans were of biantennary complex structure, and some of them lacked one terminal sialic acid molecule. Triantennary structures made up almost 25% of the charged glycans pool, and highly branched tetrasialylated oligosaccharides did not exceed 1%. The main difference between haptoglobin derived from the sample of pooled 44 sera and from the 2-2 phenotype individual concerned the relative content of trisialylated oligosaccharide with one 2-3 linked sialic acid residue. The oligosaccharide profile of haptoglobin derived from serum of a patient suffering from congenital disorder of glycosylation was compared to 'healthy' controls. It was shown, that four main glycans are identical in patient and 'normal' haptoglobins. Some alterations were found in the relative content of mono-, bi-, and trisialylated glycans as well as in the appearance of some tracely abundant oligosaccharides in haptoglobin of the patient with congenital disorder of glycosylation.     

The effect of haptoglobin on prostaglandin H synthase from ram vesicular glands

It was shown that the ability of sheep and horse haptoglobins differing in their immunological properties to inhibit PGH synthetase is about the same. It was found that haptoglobin inhibits the PGH synthetase-catalyzed enzymatic reaction, the inhibiting effect being non-competitive with respect to the electron donor, adrenaline. The degree of PGH synthetase inhibition by haptoglobin depends on the glycoprotein concentration, incubation time and enzyme activity.     

Hereditary polymorphism in the haptoglobin system of schizophrenic patients and healthy subjects]

Hereditary polymorphism of haptoglobin is studied by means of starch gel electrophoresis in schizophrenic patients (200 persons) and in healthy people (154 persons), in habitants of Minsk. It is concluded that Minsk inhabitants do not differ considerably from the rest European population in their frequencies of haptoglobin alleles. No reliable differences in the distribution of haptoglobin genotypes were found between patients and healthy persons. However, hereditary types of haptoglobins turned to be markers of the course and the prognosis of schizophrenia.     

Haptoglobin synergistically potentiates bradykinin and thrombin induced prostaglandin biosynthesis in isolated osteoblasts

Haptoglobin of two different phenotypes (Hp 1-1 and Hp 2-1) dose-dependently (1-4 mg/ml) stimulated the formation of prostaglandin E2 (PGE2) in osteoblast-like cells isolated from neonatal mouse calvarial bones. The degree of stimulation obtained by haptoglobins (4 mg/ml) on PGE2 biosynthesis was in the same range as that caused by bradykinin (1 mumol/l). Pretreatment of osteoblasts with Hp 1-1 or Hp 2-1 (1-4 mg/ml) resulted in a dose-dependent, synergistic potentiation of the stimulatory effect of bradykinin (1 mumol/l) on PGE2 formation. Thrombin (7 U/ml) stimulated PGE2 formation in the osteoblast-like cells by a mechanism that was also synergistically potentiated by haptoglobin (2 mg/ml). These data show that haptoglobin per se stimulates PGE2 biosynthesis in isolated osteoblasts and, in addition, synergistically potentiates the effect of bradykinin and thrombin. Consequently, the enhanced production of haptoglobin seen in different inflammatory processes may contribute to the destruction of bone by inducing the formation of prostanoids capable of stimulating bone resorption.     

A population genetic survey of the haptoglobin polymorphism in Melanesians by DNA analysis.

We have determined the haptoglobin (Hp) genotypes of 831 Melanesians from Vanuatu, Papua New Guinea, and New Caledonia by Southern blot analysis of DNA extracted from umbilical cord and peripheral blood samples. There was complete agreement between these genotypes and the protein phenotype in cases where both were determined, and genotyping was possible in cases where no serum haptoglobins were measurable. Subtyping of Hp1 alleles using restriction enzymes showed that Melanesians, like Mongoloids and Australian Aboriginals, have only the Hp1S allele. Three cases of Hp Johnson were found in Vanuatu, and further restriction mapping supported a partial gene triplication model for the structure of this variant. We also report a new common BclI restriction enzyme polymorphism upstream of the Hp1 gene. The advantages of using DNA for haptoglobin typing are discussed.     

Haptoglobin patterns in essential hypertension and associated conditions--increased risk for Hp 2-2

Blood samples from 257 hypertensive patients and 180 normotensive controls were analysed for their association with haptoglobin levels and phenotypes. Compared to controls, patients with Hp 2-2 phenotype showed a significantly increased risk for essential hypertension (p less than 0.001) and hypertension associated with ischaemic heart disease (p less than 0.05). There was a significant decrease in the mean levels of serum haptoglobins in hypertension as compared to controls, suggesting the possibility for intravascular haemolysis due to vascular damage leading to further complications.     

Serum proteins among Dusads of Bihar

Horizontal starch gel electrophoresis has been employed for the detection of haptoglobin, transferrin and albumin phenotypes among 88 Dusads of Bihar. No variant of the haptoglobins or transferrins has been found in this sample, whereas one individual showed bisalbuminemia.     

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

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

The antioxidant activity of haptoglobin towards haemoglobin-stimulated lipid peroxidation

Haemoglobin stimulates the peroxidation of lipids in two discernable phases. The first phase is inhibited by binding haemoglobin to the protein haptoglobin. The second phase is stimulated by complexable iron released from the haemoglobin molecule during the process of lipid peroxidation. This latter peroxidation is inhibitable by transferrin and the iron chelator desferrioxamine. Heat-denatured haemoglobin and haemin both stimulated lipid peroxidation but this is not inhibitable by haptoglobin. It is suggested that the haptoglobins play an important antioxidant role in vivo by preventing iron-stimulated formation of oxygen radicals.     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Mass spectrometric protein structure characterization reveals cause of migration differences of haptoglobin alpha chains in two-dimensional gel electrophoresis

Haptoglobin belongs to the major constituents of plasma and acts as hemoglobin-binding and acute-phase protein. Due to the occurrence of three major allelic variants and further structural modifications, the alpha chains of haptoglobin form varying spot patterns in two-dimensional gel electrophoresis (2-DE) gels, which is generally observed in differential proteome analyses using plasma or related body fluids of humans. In the present study plasma samples from 10 donors of initially unknown haptoglobin phenotype were separated by 2-DE and tryptic digests of excised haptoglobin alpha chain spots were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and MALDI-quadrupole ion trap TOF-MS. Haptoglobin alpha1S, alpha1F, as well as alpha2 chains were found to occur each with at least three structurally differing protein species: (i) the unmodified form, which corresponds to the sequence database entries; (ii) derivatives, in which asparagine at position five is deamidated to aspartic acid; and (iii) derivatives with an additional C-terminal arginine residue. These structural variants account for the most commonly observed spot patterns of haptoglobin alpha chains in Coomassie-stained gels. Additionally, a minor derivative of the haptoglobin alpha2 chain carrying both modifications, deamidation at position five and the C-terminal arginine residue, was identified. Theoretical pI values of the characterized structural variants are, consistent with their observed migration in the 2-DE gels.     

Hydrazinoacridine staining of proteins and glycoproteins in polyacrylamide gels

The histochemical method for staining polyaldehydes in tissue sections with p-hydrazinoacridine has been adapted for use in polyacrylamide gels. While staining of histological preparations was reported to be specific for polyaldehydes and independent of bisulfite, both glycoproteins (β chain of haptoglobin) and nonglycoproteins (lysozyme and α chain of haptoglobin) were stained following periodate oxidation, and satisfactory results were highly dependent on the presence of bisulfite. Hydrazinoacridine staining of periodate-treated gels produced an extremely sensitive fluorescent labeling of the haptoglobin β chain and also stained haptoglobin α chain and lysozyme. The proteins could be visualized under visible light as yellow bands which were scanned spectrophotometrically at 440 nm. The β chain of haptoglobin could be subjectively distinguished from the nonglycoproteins both by differential intensity of staining with hydrazinoacridine and Coomassie brilliant blue and by the yellow nature of the fluorescence. The sensitivity of hydrazinoacridine staining of the β chain of haptoglobin compared favorably to that of the commonly used periodic acid-Schiff staining procedures and provided the advantage that nonglycoproteins in complex mixtures could be localized in the gels.     

Hemoglobin-binding site on haptoglobin probed by selective proteolysis

Selective proteolysis has been used to delineate the hemoglobin-binding site on haptoglobin heavy chain. Haptoglobin was cleaved specifically by plasmin, trypsin, chymotrypsin, staphylococcal protease, and thermolysin. Haptoglobin-hemoglobin complex was treated with these enzymes to determine which sites were protected from cleavage by the hemoglobin. The modified haptoglobins were tested for changes in their hemoglobin and hemoglobin alpha chain-binding properties. The sites of proteolytic cleavage were identified from the newly generated NH2 termini by automated Edman degradation amino acid-sequencing techniques. The results suggest that residues 128 through 131, 136 and 137, as well as 9 and 10 of the heavy chain may be involved in the binding of hemoglobin. On the other hand, residues 159 and 160, which lie in the 17-residue additional loop that is unique to haptoglobin among its homologous serine protease family, and residues 73 and 74, which lie close to the carbohydrate-binding residues, appear to be remote from the hemoglobin-binding site.     

Studies on several genetic hematological traits of the Mexican population. XVI. Hemoglobin, S and glucose-6-phosphate dehydrogenase deficiency in the east coast

The distribution of abnormal hemoglobins, glucose-6-phosphate dehydrogenase deficiency, blood group antigens A, B, M, N, C, c, D, E, e, V, Duffy and Diego, serum haptoglobins, transferrins and pseudocholinesterase types, were investigated in five villages of the East Coast of Mexico, where vivax malaria was endemic until recently. Hemoglobin S and G-6-PD deficiency were present in variable degrees in all five locations. All patients with the enzyme deficiency had the A band by electrophoresis, which coupled with the presence of hemoglobin C and haptoglobin 2-1 M in a few individuals strongly suggested Negro admixture. The high frequency of antigen V and the relatively low frequency of Fy (a +) were congruent with the above hypothesis and the conclusion was drawn that the presence of hemoglobin S and G-6-PD deficiency in this area is due to Negro admixture. Unusually high frequencies of the Diego antigen were observed in three of the five villages studied.     

Pig MAP/ITIH4 and haptoglobin are interleukin-6-dependent acute-phase plasma proteins in porcine primary cultured hepatocytes.

The acute-phase expression of pig MAP (major acute-phase protein)/ITIH4 (inter-alpha-trypsin inhibitor heavy chain 4) and haptoglobin were analysed in primary cultures of isolated pig hepatocytes in response to recombinant human (rh) cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), as well as to bacterial lipopolysaccharide (LPS). Analysis of pig MAP/ITIH4 and haptoglobin mRNAs was carried out by RT-PCR amplification. Secreted proteins from the cytokine-treated hepatocytes were quantified by immunochemical techniques. Time-course and dose-response experiments show that pig MAP/ITIH4 and haptoglobin belong to the type II acute-phase proteins, as they are specifically induced by rhIL-6 and not by rhTNF-alpha or rhIL-1. Stimulation of cultured pig hepatocytes with rhIL-6 for 48 h at doses of 1000 U.mL-1 showed a fourfold to fivefold increase in pig MAP/ITIH4 concentration in the medium, while the concentration of haptoglobin only increased twofold. A similar increase in the concentration of pig MAP/ITIH4 was also observed in media of LPS-treated hepatocytes with the simultaneous generation of IL-6 by the Kupffer cells present in the cultures. Albumin secretion decreased after stimulation with doses of 100 or 1000 U.mL-1 rhTNF-alpha, rhIL-1 or rhIL-6. Therefore, it can be concluded that pig MAP/ITIH4 behaves as a major acute-phase protein produced by porcine hepatocytes under the effect of inflammatory cytokines.