Presence Of Organic Phosphates Research Articles

Adsorption Behavior of Inorganic and Organic Phosphate by Iron Manganese Plaques on Reed Roots in Wetlands

Inorganic and organic phosphate adsorption by iron–manganese (Fe–Mn) plaques extracted from reed roots was investigated. Scanning electron microscopy indicated the roots had rough surfaces and fine particles attached. X-ray photoelectron spectra indicated that Fe and Mn in the Fe–Mn plaques were mainly in the +III and +IV oxidation states, respectively. The contact time, initial phosphate concentration, and temperature effects on inorganic and organic phosphate adsorption were investigated by performing batch tests. Pseudo-second-order model described inorganic and organic phosphate adsorption, indicating the chemisorption was the dominant adsorption process. Langmuir and Freundlich isotherm models were fitted to the equilibrium data, and the Langmuir model fitted best. The maximum inorganic and organic phosphate adsorption capacities at 298 K were 7.69 and 3.66 mg/g, respectively. The inorganic and organic phosphate adsorption processes were spontaneous and exothermic. The inorganic phosphate adsorption capacity was higher than the organic phosphate adsorption capacity, and the presence of organic phosphate did not negatively affect adsorption at inorganic to organic phosphate molar ratios between 1:1 and 3:1. Fourier-transform infrared spectra before and after adsorption showed abundant functional groups on Fe–Mn plaques and that phosphate was probably adsorbed via replacement of hydroxyl groups and inner-sphere surface complexation.

The mechanism of iron phosphide formation during boundary friction in the presence of organic phosphates and phosphites

Thermochemical and mechanochemical reactions of tricresyl phosphate, tributyl phosphate, and tributyl phosphite with an iron surface are studied. A possible mechanism of iron phosphide formation under severe boundary friction conditions is proposed. The antiscoring behavior of phosphine in a vaseline oil solution is studied for the first time. It is found that (a) iron powders modified by tricresyl phosphate or tributyl phosphite do not yield iron phosphide at a temperature of 710°C in vacuum but are reduced to iron phosphide by birch coal; (b) at a temperature of 760°C in a flow reactor, tricresyl phosphate produces a much greater amount of solid phosphorous-containing residue than tributyl phosphite; (c) at a temperature of 760°C in a flow reactor, tributyl phosphite produces white phosphorus; (d) phosphine improves effectively the antiscoring properties of vaseline oil.

Chemical stability study of vitamins thiamine, riboflavin, pyridoxine and ascorbic acid in parenteral nutrition for neonatal use.

BackgroundThe objective of this work was to study the vitamins B1, B2, B6 and C stability in a pediatric formulation containing high amounts of calcium in the presence of organic phosphate, amino acids, glucose, sodium chloride, magnesium sulfate, pediatric vitamins and trace elements under different conditions using developed and validated analytical methods.MethodsThe study was carried out during 72 h with formulations packaged in recommended storage temperature (4°C) and 25°C, with and without photoprotection.ResultsThe results showed that the methodologies used for assessing the chemical stability of vitamins B1, B2, B6 and C in the formulation were selective, linear, precise and accurate. The vitamins could be considered stable in the formulation during the three days of study if stored at 4°C. When stored at 25°C vitamin C presented instability after 48 h.ConclusionThe pediatric formulation containing high amount of calcium in the presence of organic phosphate, amino acids, glucose, sodium chloride, magnesium sulphate, pediatric vitamins and trace elements packaged in bag-type trilaminate presented a shelf life of the 72 h, when maintained under refrigeration, between 2°C and 8°C. This shelf life was measured considering the vitamins studied. Further studies are needed including all the vitamins present in this formulation.

Characteristics of alkaline phosphatase in cyanobacterial strains and in an APasedef mutant of Nostoc muscorum

Repression of alkaline phosphatase (APase) activity in four taxonomically different cyanobacteria Anabaena flosaquae, Calothrix brevissima, Scytonema javanicum and Hapalosiphon intricatus by rifampicin and chloramphenicol led to the conclusion that enzyme synthesis is under transcriptional control. A mutant isolated in Nostoc muscorum after mutagenesis with NTG was found to be defective in APase activity (APasedef). The mutant showed nearly 90% growth compared to wild type strain in the presence of inorganic phosphate (Pi). The wild type Nostoc muscorum showed some growth (33% compared to Pi) in the presence of organic phosphate but the growth of the APasedef mutant was negligible under these conditions since it lacks APase activity. APase activity and phosphate uptake functions were phosphate repressible in the wild type strain. Phosphate uptake in the mutant was comparable to the wild type cyanobacterium. The mutant was also able to accumulate polyphosphate granules like the wild type.

Functional and structural properties of the hemoglobin components from Italian sturgeon ( Acipenser naccarii )

The structural and oxygen binding properties of Acipenser naccarii blood have been investigated. The electrophoretic analysis of the hemolysate of this sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. Constituent globin chains were analyzed by urea-Triton acid polyacrylamide gel electrophoresis and isolated by high performance liquid chromatography. N-terminal amino acid sequence analysis revealed the presence of a N-terminal proline in two of the three α-chains present in the globin pattern, and the presence of a histidine residue in 2βposition. Oxygen equilibria reveal a very low sensitivity of the individual hemoglobins to chloride ions and temperature; however, in the presence of organic phosphates the oxygen affinity of the hemoglobin components decreases strongly. In particular, when Guanosine-5-triphosphate (GTP) is added, the reduction of the oxygen affinity, at pH 7.4 and 20 °C, is 60% and 50%, respectively, for HbI (anodic component) and HbII (cathodic component). As the effect of protons is concerned, the small Root effect shown by total hemolysate at physiological conditions, seems to be due mainly to the cathodic component. On the whole, the functional properties shown by sturgeon hemoglobin components seem to be related to the particular physiological needs dictated by the environmental characteristics.

Purification and characterization of the hemoglobin components of Adriatic sturgeon (Acipenser naccarii) blood

The oxygen binding properties of Acipenser naccarii hemoglobins have been investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes and hemolysate in the presence of the physiological cofactors GTP and chloride ions. In particular, the oxygen equilibrium reveal a very low sensitivity of the hemolysate to the chloride ions and temperature while in the presence of organic phosphates their oxygen affinity decreased strongly. The electrophoretic analysis of the hemolysate of the sturgeon showed the presence of two hemoglobin components, each with a considerable globin multiplicity. The partial amino acid sequence of the a and P chain of the single hemoglobins was also established.

A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins.

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the alpha 2 epsilon 2 and zeta 2 epsilon 2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.

The anodic hemoglobin of Anguilla anguilla. Molecular basis for allosteric effects in a root-effect hemoglobin.

The functional and structural basis for the Root effect has been investigated in the anodic hemoglobin of the European eel, Anguilla anguilla. This hemoglobin exhibits a large Bohr effect, which is accounted for by oxygen-linked binding of seven to eight protons in the presence of GTP at pH 7.5. Oxygen equilibrium curves show nonlinear lower asymptote of Hill plots, indicating the occurrence of heme-heme interactions within the T state. Analysis of the curves according to the co-operon model (Brunori, M., Coletta, M., and Di Cera, E. (1986) Biophys. Chem. 23, 215-222) reveals that T state cooperativity is positive at high pH and in the stripped hemoglobin (where the T --> R allosteric transition is operative) and negative at low pH and in the presence of organic phosphate (where the molecule is locked in the low affinity structure), indicating site heterogeneity. The complete amino acid sequence of eel anodic hemoglobin has been established and compared with that of other fish hemoglobins. The presence of the Root effect correlates with a specific configuration of the alpha1beta2 switch interface, which at low pH would stabilize subunit ligation in the T state without changing the quaternary structure. We propose that the major groups involved in the binding of oxygen-linked protons in eel anodic hemoglobin are located on the beta chain and comprise His-HC3 at the C terminus, His-FG4 at the switch interface, and Lys-EF6 and the N terminus at the phosphate-binding site.

Oxygen binding properties of blood and hemoglobin from the pigeon Columba livia

Oxygen binding curves were measured on pigeon red blood cells (RBCs) suspensions, at the temperatures of 25, 30, 37 and 41°C, from pH 7.0 to 8.2, and on pigeon hemoglobin solutions, in the absence and presence of organic phosphates. Data were analysed on the basis of the MWC model. P 50 for pigeon RBCs is 46.9 mmHg, at pH 7.4 and 41°C. This low O 2 affinity is due, in part, to the existence of a large pH gradient across the membrane of the RBCs. The overall Bohr effect ( Δ logP m Δ pH ) is –0.44 in the 7.0–8.2 extracellular pH range and increases to –0.70 if intracellular pH is considered (pHi range, 6.8–7.5). A significant pH dependence for K T and K R is observed. The maximal Hill coefficient ( n max) for pigeon RBCs never exceeds 3.35 in all the conditions of pH and temperature examined, n max is slightly higher for RBCs than for hemoglobin dilute solutions. This might denote a diminished solubility of hemoglobin molecule in the physiological milieu, and is not observed for the oxygen equilibrium of human RBCs and hemoglobin solutions. Apart from this, the functional properties of pigeon RBCs are close to those of hemoglobin solutions, under comparable concentrations of protons, Cl – and organic phosphates, and for the same temperature. The enthalpy change for overall oxygenation of pigeon RBCs is rather small (about – 7 kcal/mol).

On the presence of organic phosphate in some Camargue sediments: evidence for the importance of phytate

The organic phosphate pool of some Camargue sediments (South of France) was studied, after removal of inorganic phosphate, with Ca-NTA/dithionite (Fe bound phosphate) and Na-EDTA (Ca bound phosphate). The organic phosphate was divided into an acid soluble organic phosphate fraction (ASOP) and a residual organic phosphate fraction (ROP). The extraction of organic matter with 2.0 M NaOH (90 °C) from ROP yielded considerable quantities of Org-P. In this extract the presence of phytate (inositol hexa phosphate) could be demonstrated using phytase to hydrolyse the phytate. Phytate was shown to account for a considerable part of organic phosphate in sediments of freshwater marsh sediments as well as in the sediment of the brackish/salt water lake ‘Etang de Vaccares’. In laboratory experiments phytate was found to precipitate with all poly-valent cations tested. Furthermore, phytate was found to be strongly adsorbed onto Fe(OOH), which may explain its accumulation and its stability in sediments.

Structure-function relationships in the low-affinity mutant haemoglobin aalborg (Gly74 (E18)β → Arg)

Haemoglobin Aalborg (Gly74 (E18)β → Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates: it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)β → Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2·8 Å resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)β protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)β forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18β on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.

Some remarks on the presence of organic phosphates in sediments

This article describes a new method developed to assess the size and nature of the organic phosphate pool. Using sediment suspensions from the Rhone, Garonne and Po rivers, inorganic P compounds, Fe(OOH) and CaCO3 were removed using mild extractants at sediment pH. The residual phosphate was then fractionated into an acid soluble organic phosphate pool and a residual organic phosphate pool by acid hydrolysis (0.5 M H+). Both pools were quantitatively important, accounting for between 16 and 54% and 16 and 51% of total phosphate respectively. Acid hydrolysis was chosen since it yielded a distinct plateau, with high reproducibility, within 30 minutes. This fractionation permits a further study of dynamics and bioavailability of sediment org-P, without interference of Fe(OOH) and CaCO3. In many studies in which changes in the organic pool were examined after extraction of inorganic phosphate, 0.5 M HCl was used to extract apatite bound phosphate. The results presented here show that this is likely to result in a considerable underestimation of the organic phosphate pool.

Respiratory pigments: interactions between oxygen and carbon dioxide transport

Oxygen and carbon dioxide are transported in vertebrates and invertebrates by a wide range of respiratory pigments. These respiratory gases are not transported independently of one another, and this review considers the influence of carbon dioxide on oxygen transport and vice versa. A specific effect of carbon dioxide or bicarbonate, decreasing oxygen affinity, is found in many haemoglobins, but the effect is often reduced in the presence of organic phosphates. Clear experimental data are available for mammalian haemoglobins but in birds and lower vertebrates more data are required to verify the presence and magnitude of the CO2 effect. In erythrocruorins and haemocyanins CO2 increases O2 affinity, whereas in haemerythrins, as in haemoglobin, CO2 again decreases oxygen affinity. Much of our knowledge of invertebrate respiratory pigments is based, however, on data from one or two species. A specific effect of CO2 on O2 affinity has also often been found only at high CO2 partial pressures, which may be outside the physiological range for these species. More in vivo experimental data on CO2 values are required for these species, and further studies on other species may help to explain this discrepancy. The interaction of O2 and CO2 transport is mainly through the Haldane effect, i.e., deoxygenated blood having a greater capacity for CO2 than oxygenated blood. This is due directly to the formation of carbamino groups (carbamate) and also to the fact that deoxygenated blood binds relatively more protons than oxygenated blood. This forms the basis for the linkage between the Bohr and Haldane effects. In some species in which the Bohr coefficient is below −1.0, an akalosis in the tissues may be induced. Large Haldane effects may be particularly effective in promoting CO2 unloading when the partial pressure difference of CO2 between arterial and venous blood is small. Carbamate formation may account for 10–20% of the CO2 transported in mammals, but its role in lower vertebrates and invertebrates has only recently been considered. Carbon dioxide transport is modulated by those factors that influence O2 affinity as these in turn influence the Haldane effect.

Effects of Dexamethasone and Vitamin D3on Cartilage Differentiation in a Clonal Chondrogenic Cell Population

We have investigated the regulation of chondroblast/chondrocyte differentiation using a unique clonal cell population, designated RCJ 3.1C5 (C5), which differentiates into discrete three-dimensional cartilage nodules when grown in the presence of 15% fetal calf serum. Histologically, the nodules resembled hyaline cartilage; they contained large rounded chondrocytes surrounded by a refractile matrix which stained intensely with Alcian blue, exhibited metachromasia after Toluidine blue staining, and stained with an antibody against type II collagen. The cartilage nodules that formed did not mineralize, despite the presence of organic phosphate in the culture medium. The synthetic glucocorticoid dexamethasone (DEX) increased the number of cartilage nodules formed in a dose-dependent manner (ED50, approximately 10(-9) M), with a maximal stimulatory dose of 10(-8) M. DEX had no effect on the population doubling time and saturation density. The effects of DEX on the number of cartilage nodules were similar whether it was added from the beginning of the culture period (starting during exponential growth) or at confluence. In contrast, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited cartilage nodule formation in a dose-dependent manner (IC50, approximately 5 x 10(-10) M), with maximum inhibition at 10(-7) M. In addition, 1,25-(OH)2D3 decreased cell proliferation and saturation density. Equimolar doses of the vitamin D3 metabolites 24,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 had no effect. C5 cells treated with 1,25-(OH)2D3 in the absence of DEX during the exponential growth phase exhibited a reduced capacity to form cartilage nodules upon subsequent exposure to DEX. At confluence, before cartilage nodules had formed, C5 cells responded to PTH and prostaglandin-E2 with increases in intracellular cAMP of about 10- and 95-fold respectively. After cartilage nodules were present, prostaglandin-E2 responsiveness decreased to about 25-fold, whereas there was no significant change in PTH responsiveness. DEX decreased the population alkaline phosphatase levels at all times measured, whereas 1,25-(OH)2D3 had a biphasic effect: an increase at 5 days in culture, followed by a decrease at later times in culture. These data indicate that the clonal cell line RCJ 3.1C5 is a useful model system in which to investigate cartilage differentiation.

Arctic life and adaptation—III. the function of whale ( Balaenoptera acutorostrata) hemoglobin

1. 1. The oxygen binding properties of the hemoglobin from the Lesser Rorqual, Balaenoptera acutorostrata, has been investigated with respect to the possible effects of organic phosphates on gas transport in arctic environments. 2. 2. The intrinsic oxygen affinity of the hemoglobin is high and strongly modulated by the effects of organic phoshates. 3. 3. In the absence of organic phosphates, the temperature sensitivity of oxygen binding expressed by the heat of oxygenation, ΔH is − 16.2 kcal/mol when corrected for the heat of oxygen in solution. 4. 4. In the presence of organic phosphates there is a marked decreased in the temperature sensitivity ΔH ∼ − 5 kcal/mol) . 5. 5. This feature is of great importance for oxygen unloading in the flippers and the tail, where the temperature is lower than the trunk of the whale. 6. 6. Furthermore the organic phoshates strongly increase the Bohr coefficient, Δlog P 50/Δ pH , from less than −0.3 in stripped hemoglobin to about −1.5 when the hemoglobin is saturated with P 6-inositol. 7. 7. This feature may be of great physiological importance by reducing the CO 2 tension and acidosis after a prolonged dive.

Quantitative analysis of linkage in macromolecules when one ligand is present in limited total quantity.

We present a general framework for analysis of two closely related problems in biochemical studies: (1) The first is analysis of binding data obtained under conditions in which a second, linked ligand is present in limited total quantity. In such conditions the free activity of the second ligand varies throughout the primary ligand binding curve, and the resultant behavior can be quite complex. Analysis of such curves enables one to quantitatively extract detailed information regarding the linkage of the two ligands at intermediate stages of ligation. The treatment is applied in an accompanying paper to oxygen binding in human hemoglobin in the presence of organic phosphates [Robert, C.H., Fall, L., & Gill, S. J. (1988) Biochemistry (following paper in this issue)]. (2) The second treatment we outline regards the analogous problem of analyzing differential scanning calorimetry (DSC) data obtained for a macromolecule binding a ligand present in limited quantity. A simple model is presented that accounts for dual transitions like those already seen in DSC data for human serum albumin in the presence of nonsaturating amounts of fatty acids [Ross, P., & Shrake, A. (1987) Abstracts of the 42nd Calorimetry Conference, University of Colorado, Boulder, CO].

Is there a root effect in Xenopus hemoglobin?

The reaction of Xenopus hemoglobin with oxygen and carbon monoxide has been reinvestigated over the pH range 8.5–6.0, in the absence and presence of organic phosphates (2,3-diphosphoglycerate or inositol hexakisphosphate), to establish if the tetramer can be stabilized in a T-quaternary state by protons and polyphosphate; the equilibrium and kinetic data indicate that Xenopus hemoglobin does exhibit a Root effect. These new results are discussed with reference to those reported by Bridges et al. [(1985) Resp. Physiol. 61, 125-136] on Xenopus blood and, more generally, to the molecular definition and the structural basis of the Root effect as an extreme form of the Bohr effect.

Intrinsic fluorescence of carp hemoglobin: a study of the R → T transition

The intrinsic fluorescence of hemoglobins is known to respond to ligand-induced changes in the quaternary structure of the protein. Carp hemoglobin is an interesting model to study the quaternary transition since its R ↔ T equilibrium is pH-dependent and at low pH, in the presence of organic phosphate, it remains in the T or ‘deoxy’ quaternary structure, even when saturated with ligand. In this study, using front-face fluorometry, we show that the intrinsic fluorescence intensity exhibited by carp carboxyhemoglobin increases as the pH is lowered below 6.5 in the presence of inositol hexaphosphate. At low pH, carp methemoglobin is less affected by the addition of inositol hexaphosphate than is the CO derivative, while little or no change is observed in the met-azide derivative. We conclude: (1) the exact nature of the R to T state transition induced by inositol hexaphosphate differs for carp carboxy-, met- and met-azide hemoglobin derivatives; (2) the chromophores responsible for the changes observed with absorption spectroscopy may not be the same as those chromophores responsible for the fluorescence differences; and (3) α46-Trp is tentatively assigned as one source of fluorescence emission. Furthermore, fluorescence properties of carp hemoglobin are compared to those of human hemoglobin.

Mineralized bone nodules formed in vitro from enzymatically released rat calvaria cell populations.

Single-cell suspensions obtained from sequential enzymatic digestions of fetal rat calvaria were grown in long-term culture in the presence of ascorbic acid, Na beta-glycerophosphate, and dexamethasone to determine the capacity of these populations to form mineralized bone. In cultures of osteoblastlike cells grown in the presence of ascorbic acid and beta-glycerophosphate or ascorbic acid alone, three-dimensional nodules (approximately 75 micron thick) covered by polygonal cells resembling osteoblasts could be detected 3 days after confluency. The nodules became macroscopic (up to 3 mm in diameter) after a further 3-4 days. Only in the presence of organic phosphate did they mineralize. Nodules did not develop without ascorbic acid in the medium. Dexamethasone caused a significant increase in the number of nodules. Histologically, nodules resembled woven bone and the cells covering the nodules stained strongly for alkaline phosphatase. Immunolabeling with specific antibodies demonstrated intense staining for type I collagen that was mineral-associated, a weaker staining for type III collagen and osteonectin, and undetectable staining for type II collagen. Nodules did not develop from population I and the number of nodules formed by populations II-V bore a linear relationship to the number of cells plated (r = .99). The results indicate that enzymatically released calvaria cells can form mineralized bone nodules in vitro in the presence of ascorbic acid and organic phosphate.

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p 50(CO) of 100 mmHg at 15°C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10 3 M −1 · s −1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.