Ligand binding and conformation change in the dimeric hemoglobin of the clam Scapharca inaequivalvis

The reaction with carbon monoxide of the cooperative dimeric hemoglobin from Scapharca inaequivalvis has been examined by flash photolysis. In the nanosecond time range, geminate rebinding of 5% of dissociated CO occurs with a rate constant of 1.4 x 10(7) s-1. There is a change in absorbance of deoxyhemoglobin following photolysis at a rate of 1.2 x 10(6) s-1, consistent with a shift in the position of the Soret band to longer wavelengths. The amplitude of the change is proportional to the population of deoxydimer. In much of the Soret region this change is greater than the absorbance excursion associated with geminate recombination. There is at least one other slower change associated with the singly liganded species. Geminate rebinding of NO has components of 50, 8, and 0.035 ns-1, accounting for 75%, 25%, and less than 1% of the total reaction observed after a 35-ps photolysis flash. Simulation of diffusion of NO by molecular dynamics shows the ligands moving from the heme pocket to a subsidiary space between the edge of the heme and the surface of the protein.     

Hydroxide rather than histidine is coordinated to the heme in five-coordinate ferric Scapharca inaequivalvis hemoglobin

The ferric form of the homodimeric Scapharca hemoglobin undergoes a pH-dependent spin transition of the heme iron. The transition can also be modulated by the presence of salt. From our earlier studies it was shown that three distinct species are populated in the pH range 6-9. At acidic pH, a low-spin six-coordinate structure predominates. At neutral and at alkaline pHs, in addition to a small population of a hexacoordinate high-spin species, a pentacoordinate species is significantly populated. Isotope difference spectra clearly show that the heme group in the latter species has a hydroxide ligand and thereby is not coordinated by the proximal histidine. The stretching frequency of the Fe-OH moiety is 578 cm-1 and shifts to 553 cm-1 in H218O, as would be expected for a Fe-OH unit. On the other hand, the ferrous form of the protein shows substantial stability over a wide pH range. These observations suggest that Scapharca hemoglobin has a unique heme structure that undergoes substantial redox-dependent rearrangements that stabilize the Fe-proximal histidine bond in the functional deoxy form of the protein but not in the ferric form.     

The apolar distal histidine mutant (His69-->Val) of the homodimeric Scapharca hemoglobin is in an R-like conformation

The effect of the apolar mutation of the distal histidine (His69-->Val) has been studied in the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Absorption, circular dichroism, and resonance Raman spectroscopy point to a more symmetric heme structure of the deoxy derivative, which is indicative of an R-like conformation of the deoxy heme. Resonance Raman spectroscopy also brings out alterations in the geometry and interactions of the bound CO molecule. The iron-carbon stretching frequency is decreased by about 30 cm-1 with respect to the native protein, while the diatomic ligand stretching frequency is increased by about the same degree. Consistent with the structural changes, the ligand binding properties are significantly altered. In the mutant the overall rate and the affinity for CO binding are increased about 100-fold with respect to the native protein, and cooperativity is abolished. In addition, the amplitude and the rate of the geminate rebinding process increase significantly. This finding may be correlated to the longer average residence time of the photolyzed CO molecule within the heme pocket of the H69V mutant, as indicated by molecular dynamics simulations.     

Ligand linked assembly of Scapharca dimeric hemoglobin

The assembly of Scapharca dimeric hemoglobin as a function of ligation has been explored by analytical gel chromatography, sedimentation equilibrium, and oxygen binding experiments to test the proposal that its cooperativity is based on quaternary enhancement. This hypothesis predicts that the liganded form would be assembled more tightly into a dimer than the unliganded form and that dissociation would lead to lower oxygen affinity. Our experiments demonstrate that although the dimeric interface is quite tight in this hemoglobin, dissociation can be clearly detected in the liganded states with monomer to dimer association constants in the range of 10(8) M-1 for the CO-liganded state and lower association constants measured in the oxygenated state. In contrast, the deoxy dimer shows no detectable dissociation by analytical ultracentrifugation. Thus, the more highly hydrated deoxy interface of this dimer is also the more tightly assembled. Equilibrium oxygen binding experiments reveal an increase in oxygen affinity and decrease in cooperativity as the concentration is lowered (in the muM range). These experiments unambiguously refute the hypothesis of quaternary enhancement and indicate that, as in the case of human hemoglobin and other allosteric proteins, quaternary constraint underlies cooperativity in Scapharca dimeric hemoglobin.     

Mutation of residue Phe97 to Leu disrupts the central allosteric pathway in Scapharca dimeric hemoglobin

Residue Phe97, which is thought to play a central role in the cooperative functioning of Scapharca dimeric hemoglobin, has been mutated to leucine to test its proposed role in mediating cooperative oxygen binding. This results in an 8-fold increase in oxygen affinity and a marked decrease in cooperativity. Kinetic measurements of ligand binding to the Leu97 mutant suggest an altered unliganded (deoxy) state, which has been confirmed by high resolution crystal structures in the unliganded and carbon monoxide-liganded states. Analysis of the structures at allosteric end points reveals them to be remarkably similar to the corresponding wild-type structures, with differences confined to the disposition of residue 97 side chain, F-helix geometry, and the interface water structure. Increased oxygen affinity results from the absence of the Phe97 side chain, whose tight packing in the heme pocket of the deoxy state normally restricts the heme from assuming a high affinity conformation. The absence of the Phe97 side chain is also associated with diminished cooperativity, since Leu97 packs in the heme pocket in both states. Residual cooperativity appears to be coupled with observed structural transitions and suggests that parallel pathways for communication exist in Scapharca dimeric hemoglobin.     

Azide, cyanide, fluoride, imidazole and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative: a comparative study

Azide, cyanide, fluoride, imidazole, and pyridine binding to ferric and ferrous native horse heart cytochrome c and to its carboxymethylated derivative has been investigated, from the thermodynamic viewpoint, at pH 7.5 and 25.0 degrees C. Ligand affinity for ferric and ferrous carboxymethylated cytochrome c is higher by about 30- and 400-fold, respectively, than that observed for the native protein. The results here reported: (i) allow the estimation, for the first time, of the ligand-independent free energy associated with the heme-iron sixth coordination bond in ferric and ferrous native cytochrome c, which turns out to be +8.4 kJ mol-1 and +14.6 kJ mol-1, at 25.0 degrees C, respectively, and (ii) suggest an interplay between redox, structural, ligand binding, and recognition properties of cytochrome c.     

Calorimetric studies of carbon monoxide and inositol hexaphosphate binding to hemoglobin A

Heats of CO and IHP binding to hemoglobin A have been determined under a variety of buffer and pH conditions. From these data heats of ion binding linked to hemoglobin oxygenation have been estimated. For IHP binding to deoxyhemoglobin the buffer-corrected enthalpies are surprisingly large, reaching -25 kcal/mol of IHP at pH 7.4. These values correspond to approximately -11 kcal/mol of proton absorbed upon IHP binding and may rise largely from the protonation of hitidine and NH2-terminal groups in the binding site (Arnone, A., and Perutz, M.F. (1974) Nature 249, 34-36). The decreased magnitude of delta HIHP observed at low pH parallels the decreased proton uptake at low pH. In 0.1 M chloride (pH 7.4) the reaction Hb(aq) + IHP leads to Hb x IHP(aq) has a standard free energy change (Edalji, R., Benesch, R.E., and Benesch, R. (1976) J. Biol. Chem. 251, 7720-7721) of -10 kcal and an enthalpy change of -25 kcal. Therefore, enthalpic forces provide the dominant driving force of this process. The origin of these large negative enthalpy changes is attributed to the exothermic protonation of protein basic groups induced by the proximity of phosphate negative charges. The importance of protonation in the binding of organic phosphates to hemoglobin may well extend to the specific binding of other phosphate substrates to enzyme reaction sites.     

Carbon monoxide binding properties of hemoglobin M Iwate

The kinetic and equilibrium CO binding properties of hemoglobin (Hb) M Iwate (alpha2 87 His leads to Tyr beta2) have been investigated. The results show that the alpha(Met) beta2 (CO) tetramer of this protein has a low affinity for CO, as indicated by the stopped-flow and flash-photolysis kinetic, as well as the CO binding equilibrium, measurements. However, it has been found that the phosphate-free alpha2(Met)beta2(CO) tetramer does tend to dimerize extensively (K4.2 = 55 muM). The high-affinity forms seen in earlier kinetic measurements may be explained by this fact. When dimers are accounted for in the functional studies, the results show that the tetramer binds CO noncooperatively either with or without the allosteric cofactor, inositol hexaphosphate (IHP). IHP appears to influence the functional properties of a solution of Hb M Iwate by stabilizing the tetrameric state of aggregation, thereby greatly reducing the population of high-affinity dimers. When the CO "off" rate with IHP present (0.23 s-1) and the CO "on" rate to the tetramer either with or without IHP (1.9 X 10(5) M-1 s-1) at 25 degrees C are sued to calculate the equilibrium constant, the value obtained (8.3 X 10(5) M-1 s-1) is similar to that in equilibrium binding measurements on the phosphate-free tetramer (9.5 X 10(5) M-1) estimated from the observed P 1/2 value at 0.48 mM total heme concentration. By showing that dimers account for the high-affinity component seen in earlier kinetic experiments with Hg M Iwate, we can now more strongly suggest that cooperative CO binding to this tetramer is minimal or absent, with both of the active beta-hemes presenting a very low affinity.     

Mutational destabilization of the critical interface water cluster in Scapharca dimeric hemoglobin: structural basis for altered allosteric activity

We determined the proportion of survival variability explained by the usual prognostic factors in childhood acute lymphoblastic leukaemia (ALL) during a prognostic study of 1552 patients enrolled in three consecutive Fralle group protocols (Fralle 83, Fralle 87 and Fralle 89). The event-free survival rates at 5 years were 54.8% (SD 1.9), 43.1%) (SD 2.7) and 55.6% (SD 2.2), respectively. In the univariate analysis the following variables were predictive of poor outcome: male gender, elevated leucocytosis (> 50 x 10(9)/l), circulating blastosis. haemoglobin >12 g/dl, platelet count <100 x 10(9)/l, age under 1 year or over 9 years, enlarged mediastinum, nodes, spleen and liver, T phenotype, absence of CD10+ cells; testicular and meningeal involvement, poor response to induction therapy (CCSG M3), and LDH >400 U/l. Among the cytogenetic features, hyperdiploidy had a protective effect, whereas hypodiploidy, translocation and other structural abnormalities had a negative influence, particularly in cases of t(9;22) or t(4;11). Multivariate analysis summarized the prognostic information in terms of four variables: age, gender, leucocytosis and cytogenetic features. Missing data had little influence on the results. However, despite their significance in the multivariate analysis, these four variables each had very low predictive power (1.1% for gender, 2.0% for age, 3.5% for leucocytosis, and 1.6% for cytogenetic features). Thus, the most significant prognostic factors in childhood ALL each explain no more than 4% of the variability in prognosis. This may explain the disappointing practical value of these factors and underlines the need for prognostic tools in childhood ALL.     

The binding and utilization of hemoglobin by Prevotella intermedia

Personalism in ethics denotes any system based upon the value of the person. Several versions of personalist morals have been developed over the past 50 years. Some have had particular interest in the field of medical ethics. Here the question is being studied about one such system, the so-called Leuven personalist morals and its usefulness in today's world of bioethics. In order to test the usefulness of this system the case of artificial insemination is examined both in the early 1970s in the context of the Leuven clinics and, subsequently, in the 1990s in a US policy document. The investigation reveals strengths and weaknesses of this personalism. Regarding AID it reveals unresolved oppositions. The conclusion seems to be that this personalism had, no doubt, a profound impact upon medical ethics within its own circle but, as regards the universal usefulness of the system, serious doubts remain.     

Rapid multicomponent analysis of hemoglobin derivatives for controlled antidotal use of methemoglobin-forming agents in cyanide poisoning

When cyanide poisoning is treated with a methemoglobin-forming agent, oxidative metabolism is protected at the expense of the oxygen capacity of the blood. The affinity of methemoglobin for CN- is high enough to compete with cytochrome oxidase, which protects the latter from becoming blocked, but all hemoglobin used for this purpose is lost for the transport of oxygen. Therefore, the fractions of the various hemoglobin derivatives present in the blood should be carefully monitored during this kind of treatment. After we had developed a multiwavelength spectrophotometric method for this purpose, we studied the feasibility of using a modified commercial six-wavelength hemoglobin photometer (Radiometer OSM3) for easy and rapid analysis of methemoglobin and methemoglobin cyanide in small samples of blood. All conditions appeared to be fulfilled for the construction of a practical multiwavelength photometer for reliably monitoring methemoglobin therapy in patients with cyanide poisoning, even in the presence of carboxyhemoglobin, as often occurs in fire victims.     

NMR study of relative oxygen binding to the alpha and beta subunits of human adult hemoglobin

NMR spectra of the downfield region of normal adult hemoglobin are reported as a function of oxygenation and temperature. Spectra were run in D2O at pD 7.4. A specially made NMR tube insert allowed precise measurement of the degree of oxygenation and of methemoglobin formation before and after taking the NMR spectrum. Plots of the estimated intensity of the most downfield prominent NMR peak, identified as arising from a deoxy-beta subunit by Davis et al. ((1971) J. Mol. Biol. 60, 101-111), versus the average degree of oxygenation y, measured optically, yield a nearly straight line within experimental error, for samples stripped of organic phosphates and for samples containing 2,3-diphosphoglycerate or inositol hexaphosphate. Intensities of peaks further upfield than this peak, previously attributed to deoxy-alpha subunits, are difficult to measure directly especially for samples containing inositol hexaphosphate. The latter samples show broadening in these alpha peaks as the degree of oxygenation increases. This extra broadening appears to increase with temperature. Linearity of the beta peak intensity with oxygenation is expected if there is no large oxygen affinity difference between alpha and beta subunits. However, the cooperativity of binding, and inaccuracy of the data, make it impossible to make accurate estimates of affinity differences.     

Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase

BACKGROUND: Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear, non-heme Fe2+ site in this biodegradative enzyme. RESULTS: The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate-enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogenous ligand and the substrate. CONCLUSIONS: We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe2+ site, even in the presence of the enzyme's [2Fe-2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.     

Prooxidant effects of ferrous iron, hemoglobin, and ferritin in oil emulsion and cooked-meat homogenates are different from those in raw-meat homogenates

Oil emulsion and raw and cooked tissue homogenates were used to determine the mechanisms of various iron forms on the catalysis of lipid peroxidation. Flax oil (0.25 g) was blended with 160 mL maleate buffer (0.1 M, pH 6.5) to prepare an oil emulsion. Raw or cooked turkey leg meat was used to prepare meat homogenates. Samples were prepared by adding iron from each of the various sources, reactive oxygen species, or enzyme (xanthine oxidase and superoxide dismutase) systems into the oil emulsion or meat homogenates. In oil emulsion and cooked-meat homogenates, ferrous iron and hemoglobin had strong prooxidant effects, but ferritin became prooxidant only when ascorbate was present. Hemoglobin and ferritin had no prooxidant effect in raw-meat homogenates. The status of heme iron and the released iron from hemoglobin had little effect on the prooxidant effect of hemoglobin in oil emulsion and cooked meat homogenate systems. The prooxidant effect of ferrous iron in oil emulsion and cooked-meat homogenates disappeared in the presence of superoxide (.O2-), H2O2, or xanthine oxidase systems. In raw-meat homogenates, however, ferrous had strong prooxidant effects even in the presence of .O2-, or H2O2. The status of free iron was the most important factor in the oxidation of oil emulsion and cooked-meat homogenates but the impact in raw-meat homogenates was small.     

从选金厂废液中回收氰化物的一种新方法：Vitrokele 912氰化物回收工艺

介绍了一种从选金厂废液中回收氰化物和除去有毒金属的Vitrokele新工艺。这种Vitrokele金属整合剂还可替代活性炭用于吸附回收金。     

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阐述了钢铁材料的性能可以通过细晶强化得到改善,对细晶材料的制备工艺的进展特别是对强烈塑性变形制备块状超细晶材料的工艺方法加以重点阐述。