Effect of modification on physicochemical and biological properties of haptoglobin. VI. Reaction with azlactone of p-nitrobenzoyl-valine.

The azlactone of p-nitrobenzoyl-valine (Nbz-Val) has been used for modification of xi-amino groups of lysine in haptoglobin type 1-1, in hemoglobin, and in the haptoglobin-hemoglobin complex. By the use of this reagent 95% of amino groups in haptoglobin and 90% in hemoglobin have been blocked without any changes in peroxidase activity of the formed complexes: Nbz-Val.haptoglobin with hemoglobin, Nbz-Val. hemoglobin with haptoglobin, and Nbz-Val.(haptoglonin-hemoglobin). After reduction and reoxidation, Nbz-Val.haptoglobin was found to retain 90% of peroxidase activity when complexed with hemoglobin. Beta chains separated either from haptoglobin or Nbz-Val.haptoglobin showed 15% of peroxidase activity in the complex with hemoglobin, alpha chains of the same origin were completely inactive. Whereas recombination of haptoglobin from alpha and beta chains resulted in 42% hemoglobin-binding capacity, renaturation of Nbz-Val.haptoglobin from separated subunits was found to proceed with almost 100% yield. In immunodiffusion with rabbit anti-haptoglobin or anti-Nbz-Val.haptoglobin sera, preparations of haptoglobin and Nbz-Val.haptoglobin after reduction and reoxidation or after recombination from separated subunits gave similar precipitation arcs showing the reaction of immunological identity.     

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.     

Structure of haptoglobin and the haptoglobin-hemoglobin complex by electron microscopy

The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 Mr. In the complex, each H chain binds an alpha beta dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124(+/- 8) A and the interhead distance is 87 (+/- 7) A. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal . Thus, the alpha beta dimers are binding to the H chains, but off the long axis of the barbell by 127 degrees in a trans configuration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5(+/- 6.3) A or 30 A greater than for haptoglobin. The N terminus of the beta chain was located on the trans off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the alpha beta dimers in the region of the beta N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the alpha beta dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the alpha beta dimer to the H chain of 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.     

Reaction of haptoglobin with hemoglobin covalently cross-linked between the alpha beta dimers.

Hemoglobin tetramers which cannot split into alphabeta dimers, because they are covalently cross-linked between the beta chains across the polyphosphate binding site, form complexes with haptoglobin. The reaction is biphasic as measured by fluorescence quenching and peroxidase activity. A complex in which one of the alpha beta dimers of the cross-linked hemoglobin is bound to one of the sites in the divalent haptoglobin molecule, is formed reversibly during the initial fast phase. In the subsequent slower step, this product then either polymerizes, adds another cross-linked hemoglobin molecule or, in the presence of excess haptoglobin, combines with a second haptoglobin molecule. This latter complex, in which two haptoglobin molecules are bridged by a cross-linked hemoglobin tetramer, can still combine with normal alpha beta dimers at the vacant haptoglobin combining sites. In spite of the very low oxygen affinity of the cross-linked hemoglobin, combination with haptoglobin shifts if oxygen affinity to the very high value of the normal hemoglobin-haptoglobin complex.     

Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix.

The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.     

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.     

Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria

The abilities of Staphylococcus aureus and Streptococcus pyogenes to remove iron from mouse 59Fe hemoglobin that was either in free form or complexed with human haptoglobin, were evaluated. 59Fe hemoglobin from the amphibian Taricha granulosa was also used in free form or complexed with the amphibian's hemoglobin-binding proteins. Contrary to what was reported from a study using pathogenic Escherichia coli, haptoglobin failed to exhibit a bacteriostatic influence when complexed with hemoglobin. In our study, more 59Fe was removed by the bacteria from the haptoglobin-hemoglobin complex than from free mouse hemoglobin. The hemoglobin and hemoglobin-plasma protein complexes of Taricha were stripped of 59Fe at similar rates and extents by both bacterial species.     

Haptoglobin-hemoglobin binding involves the heavy chain of haptoglobin and the α and β chains of hemoglobin

Previous studies from this laboratory have demonstrated unambiguously that the isolated β chain of human adult hemoglobin binds human haptoglobin (Hp). In the present work, the ability of the isolated subunits of haptoglobin and hemoglobin to form complexes is investigated. In quantitative radiometric adsorbent titrations, the H chain of haptoglobin bound to hemoglobin whereas the L chain had no binding activity. Also, the H chain of haptoglobin bound to the isolated α and β subunits of hemoglobin, but its binding to the α or β chain was less than the binding it exhibits to hemoglobin. The isolated L chain was able to reassociate with the H chain to form a complex that binds to hemoglobin or its subunits. Although the L chains had no binding activity, its association with the H chain increased the binding of the latter to Hb or its isolated α and β subunits suggesting a more indirect role for the L chain in haptoglobin-hemoglobin interactions.     

17 Beta-estradiol interaction with haptoglobin and its complex with hemoglobin

The estradiol-binding capacity of sheep haptoglobin and its complex with hemoglobin was investigated. It was shown that in the presence of H2O2 the haptoglobin-hemoglobin complex tightly binds 17 beta-estradiol. No dissociation of the estradiol-protein complex occurs after its precipitation with trichloroacetic acid and steroid extraction with the ester. It is supposed that the 17 beta-estradiol is bound to the protein by covalent bonds; this binding is characteristic only of the haptoglobin-hemoglobin complex but not of haptoglobin alone.     

Studies on camel hemoglobin. 1. Physico-chemical properties and some structural aspects of camel hemoglobin (Camelus dromedarius).

Hemoglobin from an adult camel (Camelus dromedarius) was prepared from the red cell lysate by CM- and DEAE-cellulose chromatography. The purified hemoglobin showed a lesser mobility on starch gel electrophoresis at pH 8.5 than that of human hemoglobin C. Native camel hemoglobin contains 95-99% alkali-resistant hemoglobin and in soluble in 2.94 M K2HPO4/KH2PO4 buffer. Different forms of camel hemoglobin show similar ammonium sulfate precipitation curves. Indirect evidence for the stability of camel hemoglobin solutions was obtained from several sources. Spontaneous met-hemoglobin formation is extremely slow and minimal quantities of degradation products appear on starch gel electrophoresis and on chromatographic separation. The alpha and beta chains of camel hemoglobin A were separated on a CM-23 column by the use of a pyridine formate gradient. Large peptide fragments were obtained by tryptic digestion of maleylated alpha and beta chains. The N-terminal structure of the alpha and beta chains and of tryptic maleylated peptides derived from alpha and beta chains are presented. Between adult camel hemoglobin and adult human hemoglobin six amino acid differences in the N-terminal 20 amino acid residues of the alpha chain, at residues: 4, 5, 12, 14, 17, and 19; eight amino acid substitutions were found in the beta chain at positions: 4, 5, 6, 9, 12, 13, 16, and 19. Substitutions at alpha5 Ala leads to Lys, and beta19 Asn leads to Lys, increase the net positive charge of camel hemoglobin by two, while other substitutions result in no charge differences. The molecular basis of the stability of camel adult hemoglobin is discussed.     

Identification of alpha 2-macroglobulin as a cytokine binding plasma protein. Binding of interleukin-1 beta to "F" alpha 2-macroglobulin

Treatment of normal human plasma with methylamine resulted in the discovery of an interleukin-1 beta(IL-1 beta) binding protein. The protein was labeled with 125I-IL-1 beta and the relative molecular mass (Mr) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein-IL-1 beta complex had a Mr of approximately 400,000 in non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis but became dissociated when exposed to beta-mercaptoethanol. The 125I-IL-1 beta labeled protein complex could be immunoprecipitated from plasma by using an anti-alpha 2-macroglobulin (alpha 2M) antiserum. Similarly, a monoclonal antibody (mAb) specific for electrophoretically fast ("F")alpha 2M was able to adsorb the 125I-IL-1 beta labeled complex from plasma. The mAb was also capable of adsorbing "F" alpha 2M-125I-IL-1 beta complexes from binary reaction mixtures, but failed to adsorb free 125I-IL-1 beta. Experiments carried out with purified plasma alpha 2M established that IL-1 beta became bound to alpha 2M only upon reaction with trypsin or methylamine, which results in the appearance of free thiol groups in alpha 2M ("F" alpha 2M). There was no binding of IL-1 beta to the native form of alpha 2M (electrophoretically slow or "S" alpha 2M), which lacks free thiol groups. Pretreatment of "F" alpha 2M with N-ethylmaleimide or [ethylenebis(oxyethylenenitrilo)] tetraacetic acid prevented complex formation between "F" alpha 2M and IL-1 beta. In contrast, the yield of "F" alpha 2M IL-1 beta complex formation was increased severalfold in the presence of 2.5 mM Zn2+. These findings indicate that "F" alpha 2M interacts with IL-1 beta through a thiol-disulfide exchange reaction. Zn2+ may play a major role in bringing together the reactive domains of the adjoining peptide backbones into proper orientation. The ready complex formation between "F" alpha 2M and the pleiotropic cytokine IL-1 beta suggests a novel biological role for "F" alpha 2M, since "F" alpha 2M-IL-1 beta complexes, but not "F" alpha 2M alone, retained IL-1-like activity in the thymocyte costimulator bioassay.     

Reduction of disulphide bonds in human haptoglobin 2-1

Gradual reduction of disulphide bonds in human haptoglobin, type 2-1, was carried out either by the use of sodium borohydride or 2-mercaptoethanol, newly exposed sulphydryl groups as determined by the Ellman's reagent and by the incorporation of [(14) C] acetamide, respectively. Cleavage of disulphide bonds resulted in the formation of a number of intermediates, separated in polyacrylamide gel electrophoresis, with sulphydryl groups blocked by the radioactive label. On the basis of molecular mass determinations, subunit composition of intermediates, was postulated. The ability of haptoglobin to form an active peroxidase-like complex with hemoglobin depended to a considerable extent on the presence of intact disulphide bonds. On the contrary,throughout the course of reduction of inter- and intrachain disulphides, antigenic reactivity was found to remain unchanged.     

A haptoglobin radioassay based on binding to solid-phase hemoglobin.

A specific and sensitive assay for haptoglobin based on binding to an easily prepared Sepharose-bound hemoglobin reagent is described. The assay is suitable for directly determining radiolabeled amino acid incorporation into haptoglobin in several liver cell systems in vitro and can be adapted to measure unlabeled free haptoglobin in plasma samples regardless of the presence of the haptoglobin-hemoglobin complex.     

Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Cycloclavine from the Culture Broth of Aspergillus japonicus SAITO

Changes of protein components (LA, LB, LC, and X) in mouse serum after administration of an antitumor agent, LC 9018 (lyophilized preparation of Lactobacillus casei, YIT 9018), were investigated. The intraperitoneal injection of LC 9018 caused an increase in these proteins one or two days after administration. The LC component was purified from mouse serum and was identified as haptoglobin. In addition, LA was identified as an intermediate of a haptoglobin-hemoglobin complex [Hp-Hb(α₁β₁)]and X as a haptoglobin-hemoglobin complex [Hp-Hb(α₂β₂)] because addition of increasing amount of hemoglobin to purified haptoglobin formed X via LA and all of these components were immunoprecipitated with anti-haptoglobin IgG.     

Properties of the interspecies hybrids between haptoglobin alpha and beta subunits

1. Subunits alpha isolated from human haptoglobin were recombined with beta subunits of equine haptoglobin, and vice versa. Both hybrid proteins were separated on electrophoresis in polyacrylamide gel into four bands with mobilities corresponding to tetramers 2alpha.2beta, trimers 2alpha.beta, and dimers alpha.beta, in addition to free subunits beta. 2. The binding ability of haemoglobin and the antigenic specificity of tetramers depended on the origin of beta subunit. 3. Reduction of native and hybrid proteins with 2-mercaptoethanol led to gradual formation of alpha.beta, alpha, and beta; the components 2alpha.beta and 2alpha appeared in trace amounts.