Determination of hemoglobin affinity for oxygen by a polarographic method

The method of determination of hemoglobin affinity for oxygen based on polarographic analysis and the method of half-saturation values P50 calculation are described. A special design of a polarographic cell is proposed for measurements in a wide temperature range. The results of temperature and pH dependences of the hemoglobin affinity for oxygen and erythrocytes of the White-Sea cod are presented.     

Determination of effective diffusion coefficient of substrate in gel particles with immobilized biocatalyst

A method of determining of the effective diffusion coefficient of substrate in a particle, where the diffusion and consumption of substrate by biocatalytic reaction are present simultaneously, was designed and experimentally verified. The method is based on measuring the overall rate of heterogeneous biocatalytic reaction in particles of varying diameter. The effective diffusion coefficient, D_e, was determined by fitting the measured reaction rates with the solution of the reaction-diffusion equation. The method is tailored for cases where the enzyme reaction is governed by the Michaelis-Menten kinetics. The value of K_m required for the solution of the mathematical model was adopted from the measurement of the kinetics of free cells, whereas the rate parameter, k₂, was optimized together with D_e. As an experimental model, the sucrose hydrolysis catalyzed by Ca-alginate-entrapped yeast cells was examined. The particle diameter varied in the range of 1.2–3.9 mm and the initial reaction rates were measured in a batch-stirred reactor at a sucrose concentration of 100 m. The D_e of sucrose at 30°C was found to be 2.9 · 10⁻¹⁰ m²s⁻¹.     

Dynamic response of a polarographic oxygen probe

The dynamic characteristic of dissolved oxygen probes is usually modeled as being equivalent to a single diffusion layer. Other workers have shown that in response to a downstep in oxygen tension a polarographic probe initially follows single diffusion layer dynamics but that during the last 10% of response the probe deviates significantly from this behavior. Probe response to a series of downsteps of various magnitudes after exposure to calibration gases for 1, 2, and 3 min was recorded. When the probe membrane was new the response behavior was found to be largely independent of the step size as well as the exposure duration. The deviation fro the single diffusion layer model was explained in terms of lateral diffusion of oxygen from the anodic compartment to the cathode. By use of a model incorporating the lateral diffusion, probe response to a general oxygen tension-time function was calculated.     

Denitrification and oxygen respiration in biofilms studied with a microsensor for nitrous oxide and oxygen

Depth distributions of O₂ respiration and denitrification activity were studied in 1- to 2-mm thick biofilms from nutrient-rich Danish streams. Acetylene was added to block the reduction of N₂O, and micro-profiles of O₂ and N₂O in the biofilm were measured simultaneously with a polarographic microsensor. The specific activities of the two respiratory processes were calculated from the microprofiles using a one-dimensional diffusion-reaction model. Denitrification only occurred in layers where O₂ was absent or present at low concentrations (of a fewM). Introduction of O₂ into deeper layers inhibited denitrification, but the process started immediately after anoxic conditions were reestablished. Denitrification activity was present at greater depth in the biofilm when the NO₃ ^– concentration in the overlying water was elevated, and the deepest occurrence of denitrification was apparently determined by the depth penetration of NO₃ ^–. The denitrification rate within each specific layer was not affected by an increase in NO₃ ^– concentration, and the half-saturation concentration (K_m) for NO₃ ^– therefore considered to be low (<25M). Addition of 0.2% yeast extract stimulated denitrification only in the uppermost 0.2 mm of the denitrification zone indicating a very efficient utilization of the dissolved organic matter within the upper layers of the biofilm.     

Continuous measurement of oxygen concentration in citrus soil by means of a waterproof zirconia oxygen sensor

A limiting-current type of zirconia oxygen sensor has been developed and its efficiency was tested for the determination of oxygen in soil air. This oxygen sensor is waterproof and has a very short response time. By the use of the sensor, changes in soil oxygen concentration, due to undecomposed organic material and waterlogging, were observed. The sensor was found to be highly stable in the soil and suitable for continuous oxygen monitoring in the field.     

Limitation of oxygenic photosynthesis and oxygen consumption by phosphate and organic nitrogen in a hypersaline microbial mat: a microsensor study

Microbial mats are characterized by high primary production but low growth rates, pointing to a limitation of growth by the lack of nutrients or substrates. We identified compounds that instantaneously stimulated photosynthesis rates and oxygen consumption rates in a hypersaline microbial mat by following the short-term response (c. 6 h) of these processes to addition of nutrients, organic and inorganic carbon compounds, using microsensors. Net photosynthesis rates were not stimulated by compound additions. However, both gross photosynthesis and oxygen consumption were substantially stimulated (by a minimum of 25%) by alanine (1 mM) and glutamate (3.5 mM) as well as by phosphate (0.1 mM). A low concentration of ammonium (0.1 mM) did not affect photosynthesis and oxygen consumption, whereas a higher concentration (3.5 mM) decreased both process rates. High concentrations of glycolate (5 mM) and phosphate (1 mM) inhibited gross photosynthesis but not oxygen consumption, leading to a decrease of net photosynthesis. Photosynthesis was not stimulated by addition of inorganic carbon, nor was oxygen consumption stimulated by organic compounds like glycolate (5 mM) or glucose (5 mM), indicating that carbon was efficiently cycled within the mat. Photosynthesis and oxygen consumption were apparently tightly coupled, because stimulations always affected both processes to the same extent, which resulted in unchanged net photosynthesis rates. These findings illustrate that microsensor techniques, due to their ability to quantify all three processes, can clarify community responses to nutrient enrichment studies much better than techniques that solely monitor net fluxes.     

Determination of oxygen profiles in agar-based gelled in vitro plant tissue culture media

Keeping account of the limited knowledge concerning the relevance of the oxygen status of the medium during in vitro culture, a technique was elaborated to systematically study the oxygen concentration in gelled media. In a first series of experiments, the Oxygen Diffusion Rate (ODR) technique was used to investigate the dissolved oxygen concentration as a function of time at different depths. The results obtained demonstrated that the oxygen concentration in agar media was reduced by 80% during the heating steps included in the preparation procedure. It took about one week to reach an oxygen concentration equal to 90% of the equilibrium value at a depth of 1 cm, irrespective of the brand of agar used. As the recovery of the oxygen concentration at various depths could be nicely modelled by Fick's law, it follows that this process is diffusion limited. In this respect, fluorescence recovery after photobleaching (FRAP) measurements revealed that the diffusion coefficient of oxygen in gelled media was only affected to a very small extent by the presence of up to 2% (w/v) agar. In a final experiment, explants of Ficus benjamina were cultured on a rooting medium. As the oxygen concentration in the gelled medium was not significantly affected by the presence of the biological material, it was concluded that the oxygen uptake by explants from gelled media is negligibly small and hence cannot be considered as being a growth-limiting factor during in vitro micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Interpretation of mixed sediment profiles by means of a sediment-mixing model and radioactive fallout

Chernobyl fallout provided a pulsed release to the environment and an opportunity to study sediment mixing processes in lakes. The paper presents the application of a mathematical modelling technique using a radionuclide as tracer. The model simulates the radionuclide profiles in sediments and computes the concentrations by solving numerically a one-dimensional mass-balance equation. The mixing-advection-decay model has been designed to include time-variable input capabilities and a depth-dependent mixing coefficient.     

Simultaneous measurements of oxygen diffusion coefficients and solubilities in fermentation media with polarographic oxygen electrodes

A membrane-covered oxygen electrode was used to measure oxygen diffusion coefficients and solubilities in aqueous glucose solutions and various fermentation media following a newly developed methodology. The fermentation media studied were tryptic soy broth and those for fermentations of Penicillium chrysogenum, Saccharomyces cerevisiae, and Micrococcus glutamicus. The experimental results of oxygen diffusion coefficients and solubilities in glucose solutions were in good accord with the literature data. As for the fermentation media, both oxygen diffusion coefficients and solubilities were found to decrease with an increased fractional composition of these media, and log-additive behaviors of the oxygen diffusion coefficients and solubilities in fermentation media were observed.     

High-resolution profiles of temperature and dissolved oxygen in a river

Longitudinal profiles of water quality along a well-mixed tidal river are, ideally, based on simultaneous measurements at fixed stations distributed along the river. The resolution of the profiles is limited by the density of the stations. However, for a given number of stations the resolution is greatly increased if water quality date can be extrapolated upstream and downstream of the stations, making use of velocity data; the resolution is then determined by the density of the extrapolated data points, which may be an order of magnitude higher than the density of the stations. A 15-km length of river was investigated using 5 current meters equipped to measure depth, temperature, conductivity and dissolved oxygen. Data were recorded simultaneously every 10 minutes. When the average cross-sectional speed was 0.25 ms⁻¹ (typical of tidal conditions), the extrapolated data points were 150 m apart, so the resolution of the resulting profiles (7 per kilometre) was much higher than that of the stations alone (0.3 per kilometre). The extrapolation process required a means of deducing the average cross-sectional speed from the speed measured at a given station. The key to this was provided by temperature data recorded during the onset of a spate, when tidal flow was suspended and the average cross-sectional speed was uniformly about 0.75 ms ⁻¹ at four of the stations. Profiles of temperature and dissolved oxygen were generated by this method; the resolution was about 2 data points km⁻¹ during the onset of the spate, and 6 points km⁻¹ during tidal flow.     

New solution method for equations of reaction and mass transfer in biocatalyst particles

Summary For numerical solution of the reaction-mass transfer equations for immobilised biocatalysts it may be better to start integration at the particle surface and proceed inwards: calculations are targetted on the region to which practically interesting changes are often confined (because concentrations are effectively zero in the interior); and during iterative solution wrong initial estimates may be rejected after detecting anomalies early in the integration.Symbols C_b substrate concentration in bulk (mol m^–3) - c dimensionless substrate concentration (C/C_b) (-) - D_e effective diffusion coefficient (m²s^–1) - Da Damkohler number (V.r_o ²/D_e.K_s) (-) - K_s substrate concentration kinetic coefficient (mol m^–3) - k_e external mass transfer coefficient (ms^–1) - r_o bead radius (m) - Sh Sherwood number (k_e.r_o/D_e) (-) - V maximum rate per unit volume in beads (mol m^–3s^–1) - x dimensionless distance from bead centre (r/r_o) (-) - dimensionless kinetic coefficient (K_s/C_b) (-) - _o effectiveness factor (-)     

Intracellular singlet oxygen generation by phagocytosing neutrophils in response to particles coated with a chemical trap.

To determine if singlet oxygen (O2(1 delta g)) is produced by neutrophils (PMNs) during the process of phagocytosis, glass beads were coated with a specific chemical trap for O2(1 delta g), 9,10-diphenylanthracene (DPA). Singlet oxygen, but not other reactive oxygen species, reacts rapidly with DPA at a rate of kr = 1.3 x 10(6) M-1 s-1 to form a stable product, DPA-endoperoxide (Corey, E. J., and Taylor, W. C. (1964) J. Am. Chem. Soc. 86, 3881-3882; Wasserman, H. H., Scheffer, J. R., and Cooper, J. L. (1972) J. Am. Chem. Soc. 94, 4991-4996; Turro, N. J., Chow, M.-F., and Rigaudy, J. (1981) J. Am. Chem. Soc. 103, 7218-7224). The production of DPA-endoperoxide was determined by ultraviolet spectroscopy as a decrease in DPA absorbance at 355 nm. The absorbance of DPA was normalized to the absorbance of perylene, which was included in the coating on the beads as a nonreactive, internal standard. In the present study, DPA- and perylene-coated beads were initially allowed to adhere to fibronectin-coated coverslips. PMNs were then added to the bead-coated coverslips and allowed to adhere and phagocytose the beads for 1 h at 37 degrees C. In some experiments, 4B-phorbol-12-myristate-13-acetate (PMA) (1 ng/2.5 x 10(7) cells/ml), a known activator of the PMN NADPH-oxidase, was added as a co-stimulant. The amount of O2(1 delta g) produced by phagocytically stimulated PMNs was calculated to be 11.3 +/- 4.9 nmol of O2(1 delta g)/1.25 x 10(6) cells. Low dose PMA co-stimulation increased the production of O2(1 delta g) to 14.1 +/- 4.1 nmol/1.25 x 10(6) cells. Averaged together these amounts represent approximately 19 +/- 5.0% of the total oxygen consumed by PMNs in response to DPA- and perylene-coated beads. The specificity of the DPA reaction with O2(1 delta g) was confirmed by warming to 120 degrees C, which releases O2(1 delta g) from the DPA-endoperoxide, regenerating the parent DPA compound (Wasserman et al., 1972; Turro et al., 1981) and the absorbance at 355 nm. In addition, beta-carotene, an avid quencher of O2(1 delta g), was included in the coating of some bead preparations; assays in which these beads were used showed no change in the absorbance at 355 nm. Singlet oxygen production by myeloperoxidase was also measured using the coated bead assay and the results suggest that this is a major pathway by which singlet oxygen is generated in phagocytically stimulated PMNs.(ABSTRACT TRUNCATED AT 400 WORDS)