Elimination from rat circulation of goat and sheep haptoglobin and their complexes with rat haemoglobin

The rate of elimination from rat circulation of 125I-labelled human, goat and sheep haptoglobin, and their complexes with rat haemoglobin was studied. The clearance of human haptoglobin-rat haemoglobin complex from rat circulation was about 5 times faster than that of free haptoglobin. For sheep and goat haptoglobin and their complexes with rat haemoglobin the rate of elimination was identical, the half-life time t 1/2 ranging from 8 to 9 h.     

Elimination of goat haemoglobin and its complexes with goat haptoglobin from goat and rat circulation

The rate of elimination from rat and goat circulation of 125I-labelled goat haemoglobin and its complexes with human and goat haptoglobin in goat circulation, was studied. The half-life time t1/2 of haemoglobin in rat circulation was 1.5h, whereas in goat circulation -- 4 h, and was 10 times shorter than t1/2 for its complexes.     

Haptoglobin biosynthesis in rats Immunological identification of polysomes synthesizing haptoglobin and quantitation of haptoglobin in the cytoplasm of liver cells

A quantitative enzyme-linked immunosorbent assay was developed and utilized to study the stimulation of haptoglobin biosynthesis during an acute inflammatory challenge. A 10-fold increase in intracellular haptoglobin was measured at the peak of the inflammatory response. The increase in serum haptoglobin levels was concomitant with the intracellular levels, demonstrating the secretory output is also elevated during the inflammatory period. A monospecific antihaptoglobin was produced and used to detect the specific polysomes involved in haptoglobin synthesis. The amount of radioactively labeled antibody bound to the nascent haptoglobin chain was increased approx. 3-fold during the inflammatory response, indicating that new haptoglobin was being synthesized and suggesting an increase in functional haptoglobin mRNA resulting from the inflammatory signal.     

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.     

Immunochemical comparison of human and goat haptoglobin and alpha 2-macroglobulin.

1. A non-haptoglobin alpha 2-globulin was observed in the plasma of goats (Capra hircus) after inflammatory stresses. 2. This stress reaction protein exhibited an electrophoretic mobility and molecular weight similar to goat haptoglobin. 3. Complete immunological cross-reactivity was demonstrated between the goat protein and human alpha 2-macroglobulin. 4. The functional and evolutionary significance of these observations are discussed.     

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.     

Canine haptoglobin: a unique haptoglobin subunit arrangement.

1. Isolated canine haptoglobin behaved identically to the alpha 2 beta 2 structure typical of human haptoglobin type 1-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptoglobin dissociated into alpha beta subunits that separated into alpha and beta chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine alpha chain when compared to human alpha 1 chain. 4. These results provide evidence that there is no inter alpha chain disulphide in canine haptoglobin comparable to the alpha 1 20-alpha 1 20 disulphide in human haptoglobin that links the two alpha beta subunits.     

Fluorescent probe studies of haptoglobin type 2-1.

Haptoglobin is an alpha2 serum protein that forms an irreversible complex with hemoglobin. The combination between these two macromolecules resembles the binding of an antigen to its antibody except that the complex remains soluble. This investigation was undertaken to determine the nature of the hydrophobic sites on haptoglobin type 2-1. The interaction of 1-anilinonphthalene-8-sulfonate (ANS) with haptoglobin type 2-1 is characterized by a flourescence intensity in solutions containing ANS and haptoglobin as the pH is decreased from 9 to 4. The dissociation constant for the ANS interaction with haptoglobin 2-1 is 5.8 x 10--5 M at pH 7.0, 5.2 X 10--5 M at pH 5.0 AND 30.3 X 10--5 M at pH 4.0. Fmax shows no change in the pH range 6-9 but does show an increase at pH 4.0 when compared to the neutral region.     

Mass spectrometric assay for analysis of haptoglobin fucosylation in pancreatic cancer

A mass spectrometric method was developed to elucidate the N-glycan structures of serum glycoproteins and utilize fucosylated glycans as potential markers for pancreatic cancer. This assay was applied to haptoglobin in human serum where N-glycans derived from the serum of 16 pancreatic cancer patients were compared with those from 15 individuals with benign conditions (5 normals, 5 chronic pancreatitis, and 5 type II diabetes). This assay used only 10 μL of serum where haptoglobin was extracted using a monoclonal antibody and quantitative permethylation was performed on desialylated N-glycans followed by MALDI-QIT-TOF MS analysis. Eight desialylated N-glycan structures of haptoglobin were identified where a bifucosylated triantennary structure was reported for the first time in pancreatic cancer samples. Both core and antennary fucosylation were elevated in pancreatic cancer samples compared to samples from benign conditions. Fucosylation degree indices were calculated and show a significant difference between pancreatic cancer patients of all stages and the benign conditions analyzed. This study demonstrates that a serum assay based on haptoglobin fucosylation patterns using mass spectrometric analysis may serve as a novel method for the diagnosis of pancreatic cancer.     

trypsin inhibition by sheep haptoglobin

It was found that sheep haptoglobin causes non-competitive inhibition of trypsin. The enzyme inactivation is due to its interaction with haptoglobin. MetHb and thionine do not influence the efficiency of haptoglobin as a trypsin inhibitor. It is concluded that the haptoglobin molecule has a trypsin-binding site which differs from the sites responsible for the interaction with MetHb and thionine.     

Hemoglobin binding to deglycosylated haptoglobin

The carbohydrate portion of polymeric haptoglobin was gradually removed by exoglycosidases in order to investigate its role in complex formation between haptoglobin and hemoglobin. Total removal of sialic acid diminished the haptoglobin-hemoglobin complex formation 15%. Removal of about 25% of the galactose residues from asialohaptoglobin, i.e., about 40% of the total weight of the carbohydrate moiety, totally inhibited the ability of haptoglobin to form complex with hemoglobin and react with haptoglobin-specific antibodies. Liberation of further galactose residues resulted in slow precipitation of the protein. Removal of a similar part of the carbohydrate moiety from haptoglobin-hemoglobin complex did not liberate hemoglobin from it, and the complex reacted with haptoglobin antibodies. The combined data indicate that the carbohydrate portion is essential for the functionally active form of polymeric haptoglobin to complex with hemoglobin, but it hardly has any direct role in the binding event, and other factors are responsible for the stability of the complex.     

Regulation of mouse haptoglobin synthesis

A cloned line of mouse hepatoma cells (Hepa-1) responded to treatment with dexamethasone by a 30-80-fold increase in synthesis and secretion of functional haptoglobin. Under the same conditions, the production of albumin was only slightly elevated whereas that of alpha 1-fetoprotein was reduced by 50%. The hormone concentration for half-maximal stimulation of haptoglobin synthesis was between 1 and 2 X 10(-8) M. The time course of induction is characteristic for a glucocorticoid- regulated protein. Cell-free translation of RNA indicated an increase in the amount of functional haptoglobin mRNA that can account for the change in the protein production. To correlate our findings on Hepa-1 cells with those on nontransformed liver cells, we tested the hormonal response of isolated hepatocytes in tissue culture. Haptoglobin was first synthesized and secreted by hepatocytes from 17-19-d-old fetuses. But neither prenatal nor adult hepatocytes showed a dexamethasone- dependent increase in haptoglobin synthesis. However, when several independent clones of hybrid cells formed from adult mouse hepatocytes and rat hepatoma cells were treated with dexamethasone, the synthesis of mouse haptoglobin was in all cases elevated. It appears that haptoglobin expression in mouse liver cells is potentially sensitive to glucocorticoids, but this modulation is manifested only in transformed cells and their derivatives.     

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.     

Human haptoglobin binds to human myoglobin

Human myoglobin, obtained from human heart, was purified to homogeneity by salt precipitation, crystallization and ion-exchange chromatography. Trace contamination by haemoglobin, if any, was removed by repeated adsorption on an immunoadsorbent of anti-haemoglobin antibodies. The interaction between human haptoglobin and human myoglobin was investigated by a solid-phase radioimmunoassay. Myoglobin adsorbents bound 125I-labelled haptoglobin in a specific manner. Linear Scatchard plots of the data indicate that human myoglobin has only one binding site for haptoglobin in terms of the binding affinity (Ka = 8.5 X 10(6) M-1). These results suggest that haptoglobin not only binds haemoglobin but also binds human myoglobin, although with an affinity that is much lower than that of haemoglobin. The physiological significance of this interaction is discussed.     

Serum haptoglobin type and liver cirrhosis.

Haptoglobin phenotypes were studied by a polyacrylamide gel electrophoresis on 200 blood donors and 105 patients with liver cirrhosis, of which 79% belonged to non-alcoholic etiology. Though no difference of haptoglobin types could be found between blood donors with positive and negative hepatitis B antigen, the cirrhois patients had an excess haptoglobin gene 1. The patients with haptoglobin gene 1 were associated with severe liver dysfunction. Since the family pedigrees of the patients with type 1--1 excluded individuals with type 2--2, the phenotypes seemed to be stable in the cirrhotic process. The possibility that the haptoglobin 2 gene offered resistence to the non-alcoholic cirrhosis was discussed.     

Interaction of sheep haptoglobin with trypsin inhibitors

It was found that polymeric sheep haptoglobin C interacts with duck egg ovomucoid and with maize trypsin inhibitor. These inhibitors do not block the region in haptoglobin C molecule which is responsible for the formation of its complex with hemoglobin. The binding of the natural protein inhibitors is suggestive of homology of the haptoglobin site involved in the interaction with the substrate-specific site of trypsin. It is assumed that the regions in these protein molecules adjacent to the active and specific sites also possess a high degree of homology.     

Effect of dystocia on serum haptoglobin in Awassi ewes

Serum haptoglobin concentration was determined in 102 Iraqi Awassi ewes. Blood samples were collected from 82 ewes before the correction of dystocia, 10 ewes with eutocia 2 to 4 h after parturition and 10 nonpregnant ewes during the seasonal anestrus phase. The mean serum haptoglobin concentration was significantly higher (P < 0.01) in ewes with dystocia than in ewes with normal births and in the nonpregnant ewes. No significant difference was found between serum haptoglobin concentrations of ewes with ringwomb and ewes with dystocia due to other causes. There was a significant elevation (P < 0.01) of serum haptoglobin in cases treated 24 h after labor compared with those treated during the first 24 h. Significant (P < 0.05) differences were found between serum haptoglobin concentrations in ewes treated surgically and those treated manually.     

Rate of nitric oxide scavenging by hemoglobin bound to haptoglobin

Cell-free hemoglobin, released from the red cell, may play a major role in regulating the bioavailability of nitric oxide. The abundant serum protein haptoglobin, rapidly binds to free hemoglobin forming a stable complex accelerating its clearance. The haptoglobin gene is polymorphic with two classes of alleles denoted 1 and 2. We have previously demonstrated that the haptoglobin 1 protein–hemoglobin complex is cleared twice as fast as the haptoglobin 2 protein–hemoglobin complex. In this report, we explored whether haptoglobin binding to hemoglobin reduces the rate of nitric oxide scavenging using time-resolved absorption spectroscopy. We found that both the haptoglobin 1 and haptoglobin 2 protein complexes react with nitric oxide at the same rate as unbound cell-free hemoglobin. To confirm these results we developed a novel assay where free hemoglobin and hemoglobin bound to haptoglobin competed in the reaction with NO. The relative rate of the NO reaction was then determined by examining the amount of reacted species using analytical ultracentrifugation. Since complexation of hemoglobin with haptoglobin does not reduce NO scavenging, we propose that the haptoglobin genotype may influence nitric oxide bioavailability by determining the clearance rate of the haptoglobin–hemoglobin complex. We provide computer simulations showing that a twofold difference in the rate of uptake of the haptoglobin–hemoglobin complex by macrophages significantly affects nitric oxide bioavailability thereby providing a plausible explanation for why there is more vasospasm after subarachnoid hemorrhage in individuals and transgenic mice homozygous for the Hp 2 allele.     

Binding of alpha 2-macroglobulin and haptoglobin to Actinomyces pyogenes

All 25 cultures of Actinomyces pyogenes tested in the present study bound 125I-labelled human alpha 2-macroglobulin with a mean binding of 65.6%. Thirteen cultures also bound 125I-labelled human haptoglobin with a mean of 51.5%. None interacted with fibrinogen, fibronectin, immunoglobulin G, or albumin. Twenty-eight cultures representing other species of actinomycetaceae did not show any interaction with alpha 2-macroglobulin, haptoglobin, and other plasma proteins tested. The binding of alpha 2-macroglobulin and haptoglobin to A. pyogenes was saturable and could be completely inhibited by the respective unlabelled plasma proteins. The binding of alpha 2-macroglobulin could not be inhibited by unlabelled haptoglobin. On the other hand, alpha 2-macroglobulin blocked the binding of haptoglobin, possibly by steric hindrance. Treatment of the bacteria with trypsin reduced their binding activities for alpha 2-macroglobulin and haptoglobin indicating the protein nature of the binding sites. Exposure to heat (1 h, 80 degrees C) significantly diminished the binding activity for haptoglobin, but not that for alpha 2-macroglobulin. The binding of alpha 2-macroglobulin and haptoglobin could be an important feature in the classification of A. pyogenes among the members of actinomycetaceae.     

Quantitative determination of haptoglobin glycoform variants in psoriasis

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity.