Kinetics of CO and O(2) binding to human serum albumin-heme hybrid

The kinetics of the CO and O(2) binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-?N-(2-methylimidazolyl)?octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [(fast), (slow)] were determined to be 4.9 x 10(6) M(-)(1) s(-)(1) and 6.7 x 10(5) M(-)(1) s(-)(1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O(2) photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG() of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.     

Effect of heme structure on O(2)-binding properties of human serum albumin-heme hybrids: intramolecular histidine coordination provides a stable O(2)-adduct complex

5,10,15,20-Tetrakis[(alpha,alpha,alpha,alpha-o-pivaloylamino)phenyl]porphinatoiron(II) and 5,10,15,20-tetrakis([alpha,alpha,alpha,alpha-o-(1-methylcyclohexanoylamino)]phenyl)porphinatoiron(II) complexes bearing a covalently bound 8-(2-methyl-1-imidazolyl)octanoyloxymethyl or 4-(methyl-L-histidinamido)butanoyloxymethyl side-chain [FeRP(B) series: R = piv or cyc, B = Im or His] have been synthesized. The histidine-bound derivatives [FepivP(His), FecycP(His)] formed five N-coordinated high-spin iron(II) complexes in organic solvents under an N(2) atmosphere and showed large O(2)-binding affinities in comparison to those of the 2-methylimidazole-bound analogues [FepivP(Im), FecycP(Im)] due to the low O(2)-dissociation rate constants. On the contrary, the difference in the fence groups around the O(2)-coordination site (pivaloyl or 1-methylhexanoyl) did not significantly influence to the O(2)-binding parameters. These four porphinatoiron(II)s were efficiently incorporated into recombinant human serum albumin (rHSA), thus providing the synthetic hemoprotein, the albumin-heme hybrid [rHSA-FeRP(B)]. An rHSA host absorbs a maximum of eight FeRP(B) molecules in each case. The obtained rHSA-FeRP(B) can reversibly bind and release O(2) under physiological conditions (in aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. As in organic solutions, the difference in the fence groups did not affect their O(2)-binding parameters, but the axial histidine coordination significantly increased the O(2)-binding affinity, which is again ascribed to the low O(2)-dissociation rates. The most remarkable effect of the heme structure appeared in the half-life (tau(1/2)) of the O(2)-adduct complex. The dioxygenated rHSA-FecycP(His) showed an unusually long lifetime (tau(1/2): 25 h at 37 degrees C) which is ca. 13-fold longer than that of rHSA-FepivP(Im).     

Photoreduction of O(2) Primes and Replaces CO(2) Assimilation

A mass spectrometer with a membrane inlet system was used to monitor directly gaseous components in a suspension of algae. Using labeled oxygen, we observed that during the first 20 seconds of illumination after a dark period, when no net O₂ evolution or CO₂ uptake was observed, O₂ evolution was normal but completely compensated by O₂ uptake. Similarly, when CO₂ uptake was totally or partially inhibited, O₂ evolution proceeded at a high (near maximal) rate. Under all conditions, O₂ uptake balanced that fraction of the O₂ evolution which could not be accounted for by CO₂ uptake.     

Al(III)-binding ability of an octapeptide and its phosphorylated derivative

A synthetic octapeptide, H-GlyGluGlyGluGlySerGlyGly-OH, and its phosphorylated Ser derivative were synthetized and their solution speciation and binding modes in their complexes with Al(III) were measured. One goal of the work was find a lead compound for the design of a selective peptide-based Al(III) chelator. pH-potentiometry was used to characterize the stoichiometry and the stability of the species formed in the interactions of the metal ion and the peptides, while multinuclear NMR was applied to characterize the binding sites of the metal ion in the complexes. CD spectroscopy revealed a difference in the conformational behaviour of the phosphorylated peptide as compared with its non-phosphorylated parent derivative. The Al(III) is presumed to enhance aggregation through the -PO3H(-)-Al(3+)-PO3(2-)-Al(3+)- intermolecular bindings between the peptide chains. The results of molecular dynamics calculations supported the experimentally obtained secondary structures and the binding position of Al(III).     

O2-binding by the blood of the landhopper,Arcitalitrus dorrieni (Hunt) (Crustacea: Amphipoda)

• 1.1. The O₂-binding characteristics of the blood of the euterrestrial amphipod (landhopper) Arcitalitrus dorrieni have been studied.
• 2.2. The blood exhibited a low O₂ affinity, with a p₅₀ (at pH = 7.8) of 21.4 torr (10°C). Affinity decreased with an increase in temperature at constant pH (ΔH = − 79.4kJ/mol) but the Bohr factor (ΔlogP₅₀/Δ pH = −0.67) was unaffected.
• 3.3. The O₂-carrying capacity of the blood was moderate (1.51 ml/100 ml)
• 4.4. The results support the hypothesis that the blood of terrestrial amphipods is characterized by having a low affinity pigment.

     

Human serum albumin hybrid incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer as an O2-binding site

We have found that recombinant human serum albumin (HSA) incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer can reversibly bind and release O2 under physiological conditions (pH 7.3, 37 degrees C) like hemoglobin and myoglobin. beta-2-Methylimidazolyl-tailed porphyrinatoirons (6a, 6b) are synthesized via four steps from the atropisomers of tetrakis(o-aminophenyl)porphyrin. The stereochemistry of the alpha,alpha,alpha,beta-conformer has been determined by NMR spectroscopy. 6a and 6b form stable O2-adduct complexes in toluene solution at room temperature. The association rate constants of O2 are 3.1- and 1.9-fold lower than those of the corresponding alpha,alpha,alpha,alpha-conformers (1a, 1b), indicating that the three substituents (cyclohexanamide or pivalamide groups) are close to each other on the porphyrin platform and construct a narrow encumbrance around the O2-coordination site. Although 6a and 6b are incorporated into the hydrophobic domains of HSA to produce the albumin-heme hybrid, only HSA-6a can bind O2 in aqueous medium. The cyclohexanamide fences are necessary for the tailed porphyrinatoiron to form a stable O2-adduct complex under physiological conditions. The O2-binding affinity (P(1/2)) of HSA-6a is 45 Torr (37 degrees C), and the O2 transporting efficiency between lungs and muscle tissues in the human body is estimated to be identical to that of human red blood cells. The HSA-6a solution will become one of the most promising materials for red blood cell substitutes, which can be manufactured on an industrial scale.     

Low-affinity Ca(2+)-binding sites versus Zn(2+)-binding sites in histidine-rich Ca(2+)-binding protein of skeletal muscle sarcoplasmic reticulum.

Histidine-rich Ca(2+)-binding protein (HRC) is a 170 kDa protein that can be identified in the isolated sarcoplasmic reticulum from rabbit skeletal muscle by its ability to bind [125I]low-density lipoprotein on blots after SDS-PAGE and that appears to be bound to the junctional membrane through calcium bridges. Molecular cDNA cloning of this protein predicts the existence of a Ca(2+)-binding domain and of a distinct heavy-metal binding domain at the cystein-rich COOH-terminus. Here we demonstrate, using radioactive ligand blot techniques, that HRC protein binds 45Ca at low affinity, as well as being able to bind 65Zn, but at different sites, that are largely inhibitable by prior reductive alkylation of the protein. In contrast to Ca(2+)-binding protein calsequestrin not having detectable 65Zn-binding sites, HRC protein bound selectively to immobilized Zn2+ on IDA-agarose affinity columns. Our results also indicate that rabbit and human 140 kDa HRC protein have common properties.     

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO₂ fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q_a, the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O₂ photoreduction. Hence, O₂ could serve as an electron acceptor to allow oxidation of Q_a even in the absence of CO₂ fixation.     

O(2) affinity of cross-linked hemoglobins modifies O(2) metabolism in proximal tubules.

Previous experiments using cross-linked tetrameric hemoglobins (XLHb) to perfuse isolated rat kidneys showed that high-O2-affinity XLHb improved proximal tubule function more effectively than low-O2-affinity XLHb. To determine how function was improved, proximal tubule fragments were incubated with albumin, Hb34 [half-saturation point (P50) 34 Torr], or Hb13 (P50 13 Torr) with Po2 values ranging from 22 to 147 Torr. ATP content reflected O2 delivery to mitochondria. Both XLHb increased ATP, Hb34 with Po2 >or= 47 Torr and Hb13 with Po2 相似文献   

Reduction of methemoglobin via electron transfer from photoreduced flavin: restoration of O2-binding of concentrated hemoglobin solution coencapsulated in phospholipid vesicles

Ferric methemoglobin is reduced to its ferrous form by photoirradiation either by direct photoexcitation of the heme portion to induce electron transfer from the surrounding media (Sakai at al. (2000) Biochemistry 39, 14595-14602) or by an indirect electron transfer from a photochemically reduced electron mediator such as flavin. In this research, we studied the mechanism and optimal condition that facilitates photoreduction of flavin mononucleotide (FMN) to FMNH(2) by irradiation of visible light, and the succeeding reduction of concentrated metHb in phospholipid vesicles to restore its O(2) binding ability. Visible light irradiation (435 nm) of a metHb solution containing FMN and an electron donor such as EDTA showed a significantly fast reduction to ferrous Hb with a quantum yield (Phi) of 0.17, that is higher than the method of direct photoexcitation of heme (Phi = 0.006). Electron transfer from a donor molecule to metHb via FMN was completed within 30 ns. Native-PAGE and IEF electrophoresis indicated no chemical modification of the surface of the reduced Hb. Coencapsulation of concentrated Hb solution (35 g/dL) and the FMN/EDTA system in vesicles covered with a phospholipid bilayer membrane (Hb-vesicles, HbV, diameter: 250 nm) facilitated the metHb photoreduction even under aerobic conditions, and the reduced HbV restored the reversible O(2) binding property. A concentrated HbV suspension ([Hb] = 8 g/dL) was sandwiched with two glass plates to form a liquid layer with the thickness of about 10 microm (close to capillary diameter in tissue, 5 microm), and visible light irradiation (221 mW/cm(2)) completed 100% metHb photoreduction within 20 s. The photoreduced FMNH(2) reacted with O(2) to produce H(2)O(2), which was detected by the fluorescence measurement of the reaction of H(2)O(2) and p-nitrophenylacetic acid. However, the amount of H(2)O(2) generated during the photoreduction of HbV was significantly reduced in comparison with the homogeneous Hb solution, indicating that the photoreduced FMNH(2) was effectively consumed during the metHb reduction in a highly concentrated condition inside the HbV nanoparticles.     

Quantitative separation of bacteria in saline solution using lanthanide Er(III) and a magnetic field.

A trivalent lanthanide ion, erbium (Er3+), has been used in combination with a magnetic separation technique to isolate seven bacterial species from suspensions in 0.9% saline. Erbium has an exceptionally high atomic magnetic moment of 9.3 Bohr magnetons, and following addition as ErCl3 (final concentration 5 mM) to bacterial suspensions, it imparts the magnetic moment to the bacterial cells by ionic binding to the cell surface. Strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Staphylococcus saprophyticus and Enterococcus faecalis were obtained from the Quality Control Depository of The Cleveland Clinic Foundation, Cleveland, Ohio, USA as suspensions in 0.9% NaCl, in concentrations ranging from 10(2) to 10(8) c.f.u. ml-1. Bacteria were separated from solution inside a capillary flow cell exposed to a highly non-homogeneous magnetic field (maximum field intensity was 0.4 T) and quantified by a light scattering method. The quantity of cellular deposition in the magnetic field was correlated with the initial concentration of cells in the suspension, expressed in c.f.u. ml-1, and sample volume (1.5 and 3.0 ml), sample pH (prior to ErCl3 addition), affinity to Gram stain (negative vs positive) and species.(ABSTRACT TRUNCATED AT 250 WORDS)     

O2-binding albumin thin films: solid membranes of poly(ethylene glycol)-conjugated human serum albumin incorporating iron porphyrin

Poly(ethylene glycol) (PEG)-conjugated human serum albumin (HSA) incorporating the tetrakis(alpha,alpha,alpha,alpha-o-amidophenyl)porphinatoiron(II) derivative (FeP) [PEG(HSA-FeP)] is a unique plasma protein-based O2 carrier as a red blood cell substitute. The aqueous solution of PEG(HSA-FeP) [mw of PEG: 2-kDa (PEG2) or 5-kDa (PEG5)] was evaporated on a glass surface to produce a red-colored solid membrane. Scanning electron microscopy observations revealed that the PEG2(HSA-FeP) membrane consisted of two parts: (i) a surface layer made of a fibrous component (10 microm thickness), and (ii) a bottom layer of an amorphous phase (5 microm thickness). The condensed solution provided a thick membrane (70 microm), which also has the amorphous bottom layer. On the other hand, the PEG5(HSA-FeP) produced homogeneous membrane made of the fibrous component. The FeP active sites in the solid membrane formed very stable O2-adduct complexes at 37 degrees C with a half-lifetime of 40 h. The O2-binding affinity of the PEG2(HSA-FeP) membrane (P1/2 = 40 Torr, 25 degrees C) was 4-fold lower than that in aqueous solution, which is kinetically due to the low association rate constant. The membrane was soluble again in water and organic solvents (ethanol and chloroform) without deformation of the secondary structure of the protein. The addition of hyaluronic acid gave a free-standing flexible thin film, and it can also bind and release O2 as well. These O2-carrying albumin membranes with a micrometer-thickness would be of significant medical importance for a variety of clinical treatments.     

Photoreduction of 18O2 and H218O2 with Concomitant Evolution of 16O2 in Intact Spinach Chloroplasts: Evidence for Scavenging of Hydrogen Peroxide by Peroxidase

Illuminated intact spinach chloroplasts decomposed one moleculeof H₂¹⁸O₂ which resulted in the evolution of a half moleculeof ¹⁶O₂, but little ¹⁸O₂. The chloroplasts showed the same rateof photoreduction of ¹⁸C₂ as that of the evolution of ¹⁶O₂ withoutaccumulation of H₂¹⁸O₂. These reactions were suppressed by DCMU,and also by several inhibitors of ascorbate peroxidase and dehydroascorbateand monodehydroascorbate reductases in chloroplasts. These observationsindicate that the hydrogen peroxide produced in chloroplastsis reduced to water by a peroxidase using a photoreductant asthe electron donor. The hydrogen peroxide scavenging systemof chloroplasts was inactivated if hydrogen peroxide was addedin the dark, but not if added during the light. (Received May 4, 1984; Accepted July 10, 1984)     

Acute vs. chronic effects of elevated hemoglobin O(2) affinity on O(2) transport in maximal exercise.

These studies were conducted to compare the effects on systemic O(2) transport of chronically vs. acutely increased Hb O(2) affinity. O(2) transport during maximal normoxic and hypoxic [inspired PO(2) (PI(O(2))) = 70 and 55 Torr, respectively] exercise was studied in rats with Hb O(2) affinity that was increased chronically by sodium cyanate (group 1) or acutely by transfusion with blood obtained from cyanate-treated rats (group 2). Group 3 consisted of normal rats. Hb O(2) half-saturation pressure (P(50); Torr) during maximal exercise was approximately 26 in groups 1 and 2 and approximately 46 in group 3. In normoxia, maximal blood O(2) convection (TO(2 max) = cardiac output x arterial blood O(2) content) was similar in all groups, whereas in hypoxia TO(2 max) was significantly higher in groups 1 and 2 than in group 3. Tissue O(2) extraction (arteriovenous O(2) content/arterial O(2) content) was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05) at all exercise PI(O(2)) values. In normoxia, maximal O(2) utilization (VO(2 max)) paralleled O(2) extraction ratio and was lowest in group 1, intermediate in group 2, and highest in group 3 (P < 0.05). In hypoxia, the lower O(2) extraction ratio values of groups 1 and 2 were offset by their higher TO(2 max); accordingly, their differences in VO(2 max) from group 3 were attenuated or reversed. Tissue O(2) transfer capacity (VO(2 max)/mixed venous PO(2)) was lowest in group 1 and comparable in groups 2 and 3. We conclude that lowering Hb P(50) has opposing effects on TO(2 max) and O(2) extraction ratio, with the relative magnitude of these changes, which varies with PI(O(2)), determining VO(2 max). Although the lower O(2) extraction ratio of groups 2 vs. 3 suggests a decrease in tissue PO(2) diffusion gradient secondary to the low P(50), the lower O(2) extraction ratio of groups 1 vs. 2 suggests additional negative effects of sodium cyanate and/or chronically low Hb P(50) on tissue O(2) transfer.     

Perfusion of intact skin by a saline solution of bis(glycinato) copper(II)

The perfusion of intact cat skin by a saline solution of bis(glycinato) copper(II), labelled with Cu⁶⁴, has been studied in a diffusion cell. It has been shown by counting the activity that the copper(II) complex perfuses the skin and over a period of 6-7 hr reaches the isotonic saline solution underneath. After this inaugural delay period a steady rate of penetration was maintained and after 24 hr about 1 mg of the complex perfused the skin. Skin samples, taken before and after, were fixed in buffered formalin and embedded in paraffin. Five micron sections were taken, stained with rubeanic acid and counterstained with 1% Eosin Y solution. Histological examination showed that copper was present in all layers of the perfused skin. This work is relevant to the solubility of metallic copper in human sweat and to the possible therapeutic value of the “copper bracelet.”     

O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation

Leaves of C₃ plants which exhibit a normal O₂ inhibition of CO₂ fixation at less than saturating light intensity were found to exhibit O₂-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O₂-insensitive photosynthesis has been reported in nine other C₃ species and usually occurred when the intercellular CO₂ pressure was about double the normal pressure. A lack of O₂ inhibition of photosynthesis was always accompanied by a failure of increased CO₂ pressure to stimulate photosynthesis to the expected degree. O₂-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O₂ and CO₂ insensitive at physiological CO₂ pressures. Postillumination CO₂ exchange kinetics showed that O₂ and CO₂ insensitivity was not the result of elimination of photorespiration.

It is proposed that O₂ and CO₂ insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO₂ assimilation.

     

Direct electrochemistry of hemoglobin in egg-phosphatidylcholine films and its catalysis to H(2)O(2)

Direct electrochemistry of hemoglobin was observed in stable thin film composed of a natural lipid (egg-phosphatidylcholine) and hemoglobin on pyrolytic graphite (PG) electrode. Hemoglobin in lipid films shows thin layer electrochemistry behavior. The formal potential E degrees ' of hemoglobin in the lipid film was linearly varied with pH in the range from 3.5 to 7.0 with a slope of -46.4 mV pH(-1). Hemoglobin in the lipid film exhibited elegant catalytic activity for electrochemical reduction of H(2)O(2), based which a unmediated biosensor for H(2)O(2) was developed.