Kinetics of the reaction between oxygen and haemoglobin bound to haptoglobin.
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ABSTRACT:
SUBMITTER: Chiancone E
PROVIDER: S-EPMC1177685 | biostudies-other | 1973 May
REPOSITORIES: biostudies-other
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Project description:Haptoglobin binds haemoglobin so firmly that there is practically no dissociation. It would be expected that the heat of the reaction would be relatively large. The development of the microcalorimeter by Benzinger offered the opportunity to measure the heat of reaction. The experiments were carried out in the Beckman 190B Microcalorimeter in two ways: (1) a constant amount of haptoglobin (Kabi; 65mg.) with different amounts of haemoglobin, and (2) a constant amount of haemoglobin (32.5mg.) with different amounts of haptoglobin. The proteins, each in 5ml. of 0.15m-phosphate buffer, pH7.4, were placed in equal-volume calorimeter cells. The heat produced/mg. of haemoglobin was calculated from the slope of the curve for a constant amount of haptoglobin and from the maximum heat for a constant amount of haemoglobin. This heat is about 70kcal./mole at 37 degrees . DeltaH varies with temperature, being -70.2 at 37 degrees , -29.7 at 20 degrees and 7.2 at 4 degrees . From the amount of haptoglobin required to attain maximum heat with 32.5mg. of haemoglobin and the amount of haemoglobin required to attain maximum heat with 65mg. of haptoglobin, it appears that at excess of haptoglobin there is competition between the reactions of 2moles of haptoglobin with 1mole of haemoglobin (or 2 alphabeta-chains) and 1mole of haptoglobin with 1mole of haemoglobin.
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Project description:One of the difficulties in creating a blood substitute on the basis of human haemoglobin (Hb) is the toxic nature of Hb when it is outside the safe environment of the red blood cells. The plasma protein haptoglobin (Hp) takes care of the Hb physiologically leaked into the plasma - it binds Hb and makes it much less toxic while retaining the Hb's high oxygen transporting capacity. We used Electron Paramagnetic Resonance (EPR) spectroscopy to show that the protein bound radical induced by H 2O 2 in Hb and Hp-Hb complex is formed on the same tyrosine residue(s), but, in the complex, the radical is found in a more hydrophobic environment and decays slower than in unbound Hb, thus mitigating its oxidative capacity. The data obtained in this study might set new directions in engineering blood substitutes for transfusion that would have the oxygen transporting efficiency typical of Hb, but which would be non-toxic.
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Project description:The haptoglobin-haemoglobin receptor of the African trypanosome species, Trypanosoma brucei, is expressed when the parasite is in the bloodstream of the mammalian host, allowing it to acquire haem through the uptake of haptoglobin-haemoglobin complexes. Here we show that in Trypanosoma congolense this receptor is instead expressed in the epimastigote developmental stage that occurs in the tsetse fly, where it acts as a haemoglobin receptor. We also present the structure of the T. congolense receptor in complex with haemoglobin. This allows us to propose an evolutionary history for this receptor, charting the structural and cellular changes that took place as it adapted from a role in the insect to a new role in the mammalian host.
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Project description: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.
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Project description: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.
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Project description:Haptoglobin is a plasma protein of mammals that plays a crucial role in vascular homeostasis by binding free haemoglobin released from ruptured red blood cells. Trypanosoma brucei can exploit this by internalising haptoglobin-haemoglobin complex to acquire host haem. Here, we investigated the impact of haptoglobin deficiency (Hp-/-) on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in a Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology, similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma between Hp-/- and control Black mice were observed. Similarly, very few quantitative differences were observed between the proteomes of parasites harvested from Hp-/- and Black mice, including both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. Combined, the data support the dispensability of haptoglobin for haemoglobin internalisation by T. b. brucei during infection in mice. Since the trypanosomes knock-outs for their haptoglobin-haemoglobin receptor (HpHbR) internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice, it suggests that T. b. brucei employs also an HpHbR-independent haptoglobin-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.
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Project description:Trypanosoma brucei requires haemoglobin for survival in the mammalian bloodstream. Haptoglobin (Hp), a host protein known to bind haemoglobin, was determined as a key component for haemoglobin acquisition by the parasite. Here, we investigated the impact of haptoglobin deficiency on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in the Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma have been observed between Hp-/- and control Black mice. Similarly, very few quantitative differences have been observed between the proteomes of parasites harvested from Hp-/- and Black mice, entailing both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. This was verified by western blotting, EM immunolabeling of parasites cryosections, and analytical quantification of haem cofactors in the parasites extracts. Combined, the data support the Hp dispensability for haemoglobin internalisation by T. b. brucei during infection in mice. To further explore this phenomenon, we have generated T. b. brucei haptoglobin-haemoglobin receptor (HpHbR) knock-outs and used them to infect Hp-/- and control mice. The HpHbR-knock-out trypanosomes internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice strongly suggesting that T. b. brucei employs an HpHbR-independent Hp-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.
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Project description:The dimeric haemoglobin in the tracheal cells of the Gastrophilus larva was extracted and purified, and the spectral properties of its oxy- and carbon monoxide adducts are recorded. In dilute solutions the kinetic parameters of binding of oxygen and carbon monoxide were determined. In solutions between 0.1 and 50mum for oxygen k(on) is 1x10(7)m(-1).s(-1) and k(off) is 1s(-1); for carbon monoxide l(on) is 6.5x10(5)m(-1).s(-1) and l(off) is 0.14s(-1). These values are in agreement with previous equilibrium results on oxygen binding and carbon monoxide/oxygen partition. These results are discussed and compared with the known values for other monomeric protohaem proteins.
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