There is, I think, something ethereal about i —the square root of minus one. I remember first hearing about it at school. It seemed an odd beast at that time—an intruder hovering on the edge of reality.

Usually familiarity dulls this sense of the bizarre, but in the case of i it was the reverse: over the years the sense of its surreal nature intensified. It seemed that it was impossible to write mathematics that described the real world in …

I and i

Publisher Summary Catalase exerts a dual function: (1) decomposition of H 2 O 2 to give H 2 O and O 2 (catalytic activity) and (2) oxidation of H donors, for example, methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxide activity) The kinetics of catalase does not obey the normal pattern Measurements of enzyme activity at substrate saturation or determination of the K s is therefore impossible In contrast to reactions proceeding at substrate saturation, the enzymic decomposition of H 2 O 2 is a first-order reaction, the rate of which is always proportional to the peroxide concentration present Consequently, to avoid a rapid decrease in the initial rate of the reaction, the assay must be carried out with relatively low concentrations of H 2 O 2 (about 001 M) This chapter discusses the catalytic activity of catalase The method of choice for biological material, however, is ultraviolet (UV) spectrophotometry Titrimetric methods are suitable for comparative studies For large series of measurements, there are either simple screening tests, which give a quick indication of the approximative catalase activity, or automated methods

Catalase in vitro

The Carbon Monoxide-binding Pigment of Liver Microsomes I. EVIDENCE FOR ITS HEMOPROTEIN NATURE

At high concentrations, free radicals and radical-derived, nonradical reactive species are hazardous for living organisms and damage all major cellular constituents. At moderate concentrations, how...

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Free Radicals in the Physiological Control of Cell Function

Living in an oxygenated environment has required the evolution of effective cellular strategies to detect and detoxify metabolites of molecular oxygen known as reactive oxygen species. Here we review evidence that the appropriate and inappropriate production of oxidants, together with the ability of organisms to respond to oxidative stress, is intricately connected to ageing and life span.

Oxidants, oxidative stress and the biology of ageing.

Hydroperoxide metabolism in mammalian organs.

Publisher Summary This chapter discusses the assay of catalases and peroxidases are: (1) catalase assay by disappearance of peroxide; (2) method for crude cell extracts; (3) direct spectrophotometric assay of catalase and peroxidase in cells and tissues; and (4) peroxidase assay by spectrophotometric measurements of the disappearance of hydrogen donor or the appearance of their colored oxidation products. Two methods are described for the catalase assay by disappearance of peroxide are: ultraviolet spectrophotometry and permanganate titration. Ultraviolet spectrophotometryis a method devised, on the basis of the absorption curves for peroxide solutions, for determining the activity of catalase by direct measurements of the decrease of light absorption in the region 230 to 250 mμ caused by the decomposition of hydrogen peroxide by catalase. In the case of method for crude cell extracts, oxygen evolution caused by the decomposition of hydrogen peroxide is measured with the conventional manometric technique. Peroxidase assay by spectrophotometric measurements of the disappearance of hydrogen donor or the appearance of their colored oxidation products includes the guaiacol test and the pyrogallol test.

The assay of catalases and peroxidases.

1. Pigeon heart mitochondria produce H(2)O(2) at a maximal rate of about 20nmol/min per mg of protein. 2. Succinate-glutamate and malate-glutamate are substrates which are able to support maximal H(2)O(2) production rates. With malate-glutamate, H(2)O(2) formation is sensitive to rotenone. Endogenous substrate, octanoate, stearoyl-CoA and palmitoyl-carnitine are by far less efficient substrates. 3. Antimycin A exerts a very pronounced effect in enhancing H(2)O(2) production in pigeon heart mitochondria; 0.26nmol of antimycin A/mg of protein and the addition of an uncoupler are required for maximal H(2)O(2) formation. 4. In the presence of endogenous substrate and of antimycin A, ATP decreases and uncoupler restores the rates of H(2)O(2) formation. 5. Reincorporation of ubiquinone-10 and ubiquinone-3 to ubiquinone-depleted pigeon heart mitochondria gives a system in which H(2)O(2) production is linearly related to the incorporated ubiquinone. 6. The generation of H(2)O(2) by pigeon heart mitochondria in the presence of succinate-glutamate and in metabolic state 4 has an optimum pH value of 7.5. In states 1 and 3u, and in the presence of antimycin A and uncoupler, the optimum pH value is shifted towards more alkaline values. 7. With increase of the partial pressure of O(2) to the hyperbaric region the formation of H(2)O(2) is markedly increased in pigeon heart mitochondria and in rat liver mitochondria. With rat liver mitochondria and succinate as substrate in state 4, an increase in the pO(2) up to 1.97MPa (19.5atm) increases H(2)O(2) formation 10-15-fold. Similar pO(2) profiles were observed when rat liver mitochondria were supplemented either with antimycin A or with antimycin A and uncoupler. No saturation of the system with O(2) was observed up to 1.97MPa (19.5atm). By increasing the pO(2) to 1.97MPa (19.5atm), H(2)O(2) formation in pigeon heart mitochondria with succinate as substrate increased fourfold in metabolic state 4, with antimycin A added the increase was threefold and with antimycin A and uncoupler it was 2.5-fold. In the last two saturation of the system with oxygen was observed, with an apparent K(m) of about 71kPa (0.7-0.8atm) and a V(max.) of 12 and 20nmol of H(2)O(2)/min per mg of protein. 8. It is postulated that in addition to the well-known flavin reaction, formation of H(2)O(2) may be due to interaction with an energy-dependent component of the respiratory chain at the cytochrome b level.

The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen.

When a picosecond light pulse is incident on biological tissue, the temporal characteristics of the light backscattered from, or transmitted through, the sample carry information about the optical absorption and scattering coefficients of the tissue. We develop a simple model, based on the diffusion approximation to radiative transfer theory, which yields analytic expressions for the pulse shape in terms of the interaction coefficients of a homogeneous slab. The model predictions are in good agreement with the results of preliminary in vivo experiments and Monte Carlo simulations.

Time resolved reflectance and transmittance for the non-invasive measurement of tissue optical properties.

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H 2 O 2 indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H 2 O 2 generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H 2 O 2 . Succinate, the most effective substrate, yields H 2 O 2 at a rate of 0.5nmol/min per mg of protein in state 4. H 2 O 2 generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H 2 O 2 modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H 2 O 2 at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H 2 O 2 rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H 2 O 2 (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H 2 O 2 /mol of NADP + during the oxidation of NADPH. H 2 O 2 is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H 2 O 2 /mol of NAD + ). 8. Estimation of the rates of production of H 2 O 2 under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H 2 O 2 /min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H 2 O 2 on cellular metabolism.

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Britton Chance

Papers

Hydroperoxide metabolism in mammalian organs.

The assay of catalases and peroxidases.

The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen.

Time resolved reflectance and transmittance for the non-invasive measurement of tissue optical properties.

The cellular production of hydrogen peroxide