Oxygen electrode uptake

A different and simpler approach to the measurement of P/O ratios came from the introduction of an oxygen electrode suitable for biochemical studies. Chance and Williams (1955) established conditions under which mitochondrial respiration, in the presence of excess substrate, was totally dependent on the amount of ADP available, i.e., the mitochondria were exhibiting respiratory control. From the change in potential when a known amount of ADP was admitted into the electrode vessel, the oxygen uptake and thus the P/O ratio could be determined, completely confirming the earlier results. 

Oxygen uptake during substrate oxidation was measured with a Clark oxygen electrode (Rank Brothers, Cambridge, U.K.) at room temperature with 1 mM substrate in 0.25 mM sodium tartrate buffer, pH 3.0 (3 mL). Rates are expressed relative to veratryl alcohol oxidation. 

Second in popularity only to the pH electrode is the oxygen electrode. This device is a polarographic electrode system that can be used to measure oxygen concentration in a liquid sample or to monitor oxygen uptake or evolution by a chemical or biological system. 

BASIC PROTOCOL 1 POLAROGRAPHIC MEASUREMENT OF OXYGEN UPTAKE Oxygen electrode equipment is used to determine oxygen uptake as a function of time. After preparing the enzyme (see Support Protocol) and calibrating the instrument, substrate and enzyme are added sequentially to a buffered solution, and the rate of oxygen uptake is monitored by a recorder. 

Figure C4.2.2 LOX activity measurements by both oxygen electrode (right) and UV absorbance (left). The graph shows a hypothetical reaction using identical conditions of equal substrate and LOX concentrations and temperature set at 25°C. Over the range generally used for measurement, the UV method gives a 3.24-fold greater response compared to the oxygen electrode that is, the full-scale of oxygen uptake (0 to 100) represents 0.242 pmol 02/ml of solution and the full-scale of UV-absorbance (0 to 2.0) represents 0.0746 pmol product/ml of solution. Also shown is the lag phase followed by a rapid linear rate used for activity calculation.

The respiratory activity of the brain tissue was determined by measuring the rate of oxygen uptake with a Clark oxygen electrode (7). The sample of tissue (a brain half) was treated exactly as if used in a calcium efflux experiment except no radioactivity or RF power was used. Following this procedure which required about 55 minutes, the tissue was placed in the oxygen electrode cell containing 1.6 ml of the standard medium (pH 7.8) at 37°C and the rate of oxygen uptake was recorded. 

The reaction can be followed by O2 uptake (manometrically or oxygen electrode), by determination of ammonia, or by determination of hydrogen peroxide. A fluorimetric assay procedure for determining the hydrogen peroxide produced permits determinations of n-amino acids in the 1-100 /xg/ml range (53). 

Subsequently, Ulmaim et al. have established that relatively stable (i.e., persistent several hours after the electrode illumination had been stopped), surface-bonded species are photogenerated at Ti02 in alkaline solutions, both at open circuit and under anodic bias. The main scope of the latter work was to characterize electrochemically the species accumulating on the surface of titanium dioxide exposed to the near-UV illumination (k > 335 nm), in a sodium hydroxide solution, under conditions simulating a) photochemical cleavage of water b) oxygen photo-uptake and c) photoassisted anodic (under external bias) oxidation of water. 

The issue of whether tunicate blood cells are involved in oxygen uptake and transport could, of course, be settled by experimentation. This challenge was met for tunicate specimens collected from both Atlantic " ] and Pacific ocean waters, " ] using an oxygen electrode to measure dissolved dioxygen concentrations. It was found that the oxygenbinding capacity of tunicate blood cells was indistinguishable from that of sea water. 

Analysis of the reaction rates is achieved in terms of oxygen uptake measured with an oxygen electrode, or by product separation and quantification. DPBF absorbs intensely at 415 nm and reacts rapidly with singlet oxygen to form a colorless intermediate endoperoxide. The DPBF reaction can be used as a benchmark against which the effect of an added quencher is compared. A note of caution must be applied the use of inhibitors and quenchers alone is not unambiguous in its outcome and should be strictly supplemented with flash photolysis experiments. Thus, if a photosensitized reaction is quenched by millimolar concentrations of azide ion, it should also be established that azide does not quench the triplet state of the sensitizer directly because that would also affect the reaction rate. 

The respiration activity of the immobilized mycelium was examined by using an oxygen electrode. The rate of oxygen uptake by the immobilized mycelium was about 30 % of that of the washed mycelium. Furthermore, penicillin production by the immobilized mycelium was performed under air and nitrogen atmospheres and the penicillin produced was determined. Only a small amount of penicillin was produced by the immobilized mycelium under anaerobic conditions. Therefore, oxygen was required for the system synthesizing penicillin in mycelium. 

Figure 1 Oxygen uptake rates with PSI incubated 5 min at various temperature with (- -) and without (-0-) addition of 10 mM MgCl2. The samples were transfered to a Clark oxygen electrode cell and equilibrated at 22°C for 2 min before measurements. The reaction medium contained 11 pg Chl/ml, 0.2 mM DCIP, 1 mM sodium ascorbate, 0.5 mM methyl viologen, 1 mM NaN3, 20 mM Tricine-NaOH (pH 7.8)...

Determination of specific analytes The rapid response of bacterial cells to the appearance of respiratory substrates in their enviromnent was the first feature exploited in microbial biosensors. Monitoring oxygen uptake of a heterotrophic species by means of the Clark oxygen electrode, it is possible to determine accurately the substrate concentration, over a limited range, from the respiratory response of the biocatalyst. Respiratory activity can also be monitored using carbon dioxide probes, mediated amperometry or calorimetry. 

Lipoxygenase assay. Oxygen uptake by lipoxygenase reaction was measured at 25°C with a Clark type oxygen electrode, on assumption that there was 0.24 mM dissolved oxygen In an air-saturated solution at 25 C. (13) The reaction mixture contained 0.33 mM substrate (0.1 ml of substrate dispersed In 0.2%... 

Another important field where PAS has been successfully employed is green plant photosynthesis. Although there are many techniques available to study photosynthesis, not many are applicable to the comprehensive investigation of photosynthesis of intact plants. Even among the techniques that measure the intact leaf photosynthesis, their applications are limited to specific photosynthetic parameters. For example, an infrared gas analyzer can be used only to measure the carbon dioxide uptake during photosynthesis. Similarly, the oxygen electrode technique can... 

Subsequently one plots InNo vs tHe and extrapolates to tHe=0. This plot provides the 02 desorption kinetics at the chosen temperature T. The intersect with the N0 axis gives the desired catalyst surface area NG (Fig. 4.8) from which AG can also be computed. More precisely NG is the maximum reactive oxygen uptake of the catalyst-electrode but this value is sufficient for catalyst-electrode characterization. 

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