Calibration and use of a Clark-type oxygen electrode from 5 to 45 degrees C

A calibration procedure for a Clark-type oxygen electrode over a wide range of temperatures is described. The autoxidation of duroquinol (2,3,5,6-tetramethyl-1,4-benzenediol) was used to verify the electrode's ability to accurately sense the total amount of dissolved O2 in an aqueous buffer. Electrode response time was measured by using oxygenated ethanol to deliver a rapid increase in O2 concentration to the reaction medium. An oxygen-producing system (spinach thylakoids) was utilized to test the range of O2-evolution rates able to be sensed. It was concluded that a Clark-type oxygen electrode has the absolute sensitivity, rapidity, and range necessary to accurately track rates of O2 production or consumption from 5 to 45 degrees C.     

A miniature Clark-type oxygen electrode using a polyelectrolyte and its application as a glucose sensor

A miniature Clark-type oxygen electrode was fabricated by anisotropically etching silicon. A two-gold-electrode configuration was used and a double-layered gas-permeable membrane was formed directly on the electrolyte, poly(vinyl-4-ethylpyridinium bromide) in the sensitive area. These materials improved the electrode's stability in long-term storage and sterilization tolerance to a practical level. The 90% response time averaged 80 s and residual current 10%, with a good linear calibration curve. The oxygen electrode was also used to make an integrated sensor for the simultaneous determination of glucose and oxygen. The glucose sensor's response time was 50-110 s, with good linearity in glucose concentrations between 56 microM and 1.1 mM at 37 degrees C, pH 7.0.     

Amperometric responses to acetylene, ethylene, and methane by a Clark-type oxygen electrode

Hydrogen-conditioned Clark electrodes exposed to successive acetylene samples displayed a decreasing curvilinear response. Slight electrode responses are also effected by saturated ethylene solutions. Thus, amperometric measurements of hydrogen or carbon monoxide in the aqueous environment by these electrodes are perturbated by the presence of acetylene and to a much lesser extent by ethylene. Methane-saturated solutions induced no electrode response.     

Compensation for measuring error produced by finite response time in determination of rates of oxygen consumption with a Clark-type polarographic electrode

In the determination of the rates of oxygen consumption with a Clark-type oxygen electrode, and experimental error is caused by finite response time of the oxygen electrode for a rapid oxidation reaction. A theoretical equation between the observed pseudo first-order rate constant (k_obs) and the true rate constant (k)

$\text{1}\text{k}{}_{\mathrm{obs}}\text{=}\text{1}\text{k}\text{+T}$

where T is a time constant for a first-order response of the oxygen electrode, was derived and found to hold up to k = 23 min^?1 in oxidation of hydroquinone at pH 7.60–8.63.     

Steady-state measurements of respiration rate with an oxygen electrode.

An oxygen electrode was developed which measures steady-state respiration rates in a volume of 0.25 ml and at oxygen concentrations as low as 0.1 μm. The steady state was achieved by pumping air-equilibrated buffer into the respirometer at various rates. The method is most suitable for tissue slices.     

Chemical kinetics measurements on the reaction between blood and ozone

The pseudofirst-order ozonization rate constant of whole bovine blood has been measured in comparison to that of free haemin. The free prosthetic group haemin (which has also the central iron atom in the oxidized form) shows k values in the range of 0.20-0.03 s(-1) while the haeme groups inside haemoglobin protein and contained in the whole blood sample show slightly lower k values, just in the range of 0.10-0.02 s(-1). It has been found that ozone even with whole blood reacts specifically with haemoglobin of the red cells because it is adsorbed selectively on the iron atoms of the haeme prosthetic groups of haemoglobin. The absorption implies the oxidation of the central iron atom of the haeme groups with formation of methaemoglobin followed by an oxidative fission of the haeme rings. The other blood components do not exert any significant protection to the reaction between ozone and haemoglobin, which appear extremely specific and selective like the reaction between CO or HCN and haemoglobin. By analogy with the behaviour of these other gases ozone may be classified as a blood poison. The results of this work are discussed in the frame of the risks connected to the ozonotherapy and autohaemotherapy involving the blood ozonization of human or animal subjects and the re-injection of ozonized blood into the bodies.     

The kinetics of the reaction between NO and O2 as studied by a novel approach

The kinetics of the reaction between NO and O2 was determined by measuring the time course of the decrease in the concentration of NO with a quench-flow technique. NO and O2 were mixed rapidly and reacted for periods of time varying from 10 to 50 s. A second rapid mixing with a solution containing an excess of deoxyhemoglobin and sodium hydrosulfite trapped free NO as nitrosylhemoglobin and reduced O2. The spectrum of the mixture of deoxy- and nitrosylhemoglobin was recorded within 30 s from the second mixing, before any appreciable dissociation of NO from the protein, by means of a flow-cell mounted on-line with the quench-flow apparatus. The amount of NO not consumed in the auto-oxidation reaction was calculated from the proportion of nitrosylhemoglobin in the mixture. As NO and O2 bind deoxyhemoglobin at comparable rates and NO is oxidized to nitrate by oxyhemoglobin, the ratio of hemoglobin/(NO + O2) had to be optimized to avoid the interference of this oxidation reaction. The kinetics was first and second order with respect to O2 and NO, respectively and third order overall with a rate constant k = 4 x kaq = 4 x 2.23 (+/- 0.26) x 10(6) M-2 s-1 at 20 degrees C, invariant in the pH range 7-9, in agreement with published values obtained by different methodologies.     

The reaction of aromatic peptides with double helical DNA. Quantitative characterisation of a two step reaction scheme

The binding of LysTrpLys and LysTyrLys to calf thymus DNA has been investigated by the field jump method using fluorescence detection. Two separate relaxation processes, clearly distinguished on the time scale and by opposite ampli- tudes, are observed for the binding of LysTrpLys to DNA with ~ 30000 base pairs. The concentration dependence of the relaxation time constants demonstrates a mechanism with a bimolecular step followed by a slow intramolecular transition with a forward rate of 6.4 X 103 s^?1 and an equilibrium constant of 11. Measurements at various degrees of peptide binding demonstrate that the binding mechanism associated with low binding rates is restricted to a rather low number of binding sites (roughly one site in 15 base pairs). The binding of LysTyrLys to the same DNA is not associated with relaxation pro- cesses of opposite amplitudes; nevertheless two processes could be identified and assigned to a two step mechanism corre- sponding to that observed in the case of LysTrpLys. In the presence of sonicated DNA both peptides show a single relaxa- tion process with characteristics similar to those observed for the slow process in the binding to high molecular DNA. The data indicate that the intramolecular step is faster for low than for high molecular DNA. These results suggest an assignment of the intramolecular step to an insertion of the aromatic residues into the DNA associated with bending of the helix. The increase in the rate of the intramolecular step with decreasing chain length of the DNA may then be explained by a higher flexibility of the double helix at lower chain lengths.     

Electrogenerated chemiluminescence reaction of lucigenin with isatin at a platinum electrode

The electrogenerated chemiluminescence (ECL) reaction of lucigenin with isatin was investigated at a platinum electrode in a neutral aqueous solution. The ECL intensity of lucigenin at ?0.65 V was greatly enhanced by isatin, and the ECL intensity was about 50 times higher than that of lucigenin without isatin. The enhanced ECL was believed to be produced by the chemiluminescence reaction between reduced lucigenin and superoxide anion that was generated by the reaction of electrochemically reduced isatin with dissolved oxygen. The conditions for the determination of isatin were optimized. Under the optimized condition, the enhanced ECL intensity vs. isatin concentration was linear in the range 4.8 × 10^?7?1.9 × 10^?5 g/mL; with a detection limit of 3.3 × 10^?8 g/mL, and the relative standard derivation 1.0 × 10^?6 g/mL isatin was 3.8%. Copyright © 2004 John Wiley & Sons, Ltd.     

D-Fructose-1,6-diphosphatase-AMP binding measurements using a nucleotide selective enzyme electrode.

A newly developed AMP selective enzyme electrode has been used to make direct binding measurements of the allosteric interaction between AMP and D-fructose-1,6-diphosphatase (E.C. No. 3.1.3.11). The proposed technique is based upon the ability of the enzyme electrode to distinguish between free and bound nucleotide. D-fructose-1,6-diphosphatase from rabbit muscle was found to have four binding sites for AMP with an average binding constant of 9 × 10⁴$\text{M}$^?1. The advantages of a direct electrode method for determining nucleotide binding constants are discussed.     

Nitration of angiotensin II by .NO2 radicals and peroxynitrite. .NO protects against .NO2 radical reaction.

To react with peptides, nitric oxide.NO has to be activated by oxidation, or by coupling with superoxide (O.-2) thereby producing peroxynitrite. In the course of.NO oxidation,.NO2 free radicals and N2O3 may be formed. Using gamma-irradiation methods, we characterized the products formed by these nitrogen oxides with angiotensin II. Angiotensin II is specifically nitrated at its tyrosinyl residue by.NO2 or peroxynitrite. Equimolecular amounts of each reagent in K+/Pi solutions at pH 7.4 led to 56% and 5% nitration yields, respectively. Nitrogen oxides produced by autoxidation of.NO, as well as.NO2 under.NO, reacted only with the arginine residue, giving a mixture of peptides containing citrulline, a N-(hydroxylamino-cyanamido-) instead of guanido group, and a conjugated diene derived from an arginine side-chain. However, nitrosation reactions by N2O3 occurred only when the initial concentration of.NO2 was 10 times that able to react with angiotensin II. Thus, in this case.NO appears to protect against.NO2 action.     

Quantitative detection of a DNA ligase reaction on a quartz-crystal microbalance.

We report here kinetic analyses of DNA ligation by using a DNA-immobilized quartz-crystal microbalance (QCM), which enables in situ real-time monitoring of both the binding of ligase and ligation reaction on DNA strands, as mass changes.     

The kinetics of S-transnitrosation--a reversible second-order reaction.

Transnitrosation between thiols and S-nitrosothiols has been suggested to be a mechanism of signal transduction. This kinetics of such reactions fit well to a reversible second-order model. Parameters derived from this model give both the forward and reverse rate constants and the equilibrium position at physiological temperature and pH. In addition, methods are shown for calculating the equilibrium distribution of the nitrosyl function group in mixtures of up to three thiols.     

Equilibrium and kinetics of the reaction of Aplysia myoglobin with azide.

The present paper reports a study on the equilibria and kinetics of the acid-alkaline transition and the azide binding reaction by ferric Aplysia myoglobin. A single completely reversible spectrophotometric titration curve is found over the pH range from similar to 5 to similar to 9, with an apparent pK equals to 7.5 for the acid-alkaline transition. The kinetics of the process, followed by the temperature-jump method, gives, at pH values close to the pK of the transition, one single, well-resolved, relaxation independent of protein concentration and of type of buffer used. The pattern accords to a simple pH dependent reaction, in buffered medium, between the two forms of the protein. The results of the azide binding reaction show that the process conforms to simple equilibrium as expected for a single site protein. The méasured association constant is reported as a function of pH. The kinetics of the reaction of Aplysia metMb with N3- minus shows, on the other hand, a complex behavior. The relaxation pattern is found to strongly depend on pH and ligand concentration in such a way to suggest a linkage between ligand binding and acid-alkaline transition. The system is discussed on the basis of two simplifying conditions, i.e., at low and higher pH with respect to the pK of the acid-alkaline transition. At acid pH the reaction corresponds to a single bimolecular process as expected for a simple binding reaction; at alkaline pH, the dependence of relaxation time on ligand concentration implies the existence of a rate-limiting monomolecular step. On the basis of a reaction scheme implying that binding of the ligand can only occur through the acid (aquomet) form of the protein via the displacement of the water molecule, the experimental data are quantitatively accounted for.     

Catalase activity measured with a micro oxygen electrode in a pressurized reaction vessel

The assembly and the use of a simple airtight pressurized reaction vessel are described for the measurement of catalase activity with a micro oxygen electrode in an optically heterogenous medium. The oxygen concentration is expressed as the ratio of observed current to the current in an air-saturated solution. Thus, an individual standard can be obtained for each measurement and the calibration is less affected by changes in the amplification factor. Different procedures for calibration of the oxygen electrode were compared. Specific activities of crystalline catalase, of red blood cells from humans and from normal or acatalasemic mice, and of liver homogenates from normal or amino-triazole-pretreated rats were determined. The specific catalase activity of human erythrocytes, as found by this method, agrees with that obtained photometrically.     

Photodegradation mechanism and reaction kinetics of recombinant human interferon-alpha2a.

The photodegradation mechanism of recombinant human interferon-alpha2a (IFNalpha2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNalpha2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNalpha2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNalpha2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNalpha2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.     

The kinetics of chemical reaction of activated nitrosamine with organic substrates.

The chemical reaction of tosylated N-nitrosobis (2-hydroxylpropyl) amine with aniline, 1-naphthol, pyridine, and hydroxylamine follows kinetics which can be described in a mixed order rate equation. This nitrosamine, considered an activated nitrosamine with a potential for alkylating a substrate, was seen to alkylate aniline, pyridine, 1-naphthol, and hydroxylamine. The presence of salts in the solution as well as an increase in solution pH increased the rate of reaction of the nitrosamine with substrates. The nitrosamine reacts quickly with water but will not react with solvents such as acetonitrile, dimethylsulfoxide, and methylene chloride.