Phillip C. Chan

SOME PROPERTIES OF THE ASCORBATE FREE RADICAL

A study of the reactivity of the ascorbate radical with biological materials was performed using dopamine, cytochrome C, O/sub 2/, methanol, lactate, pyruvate, fumarate, and L-..cap alpha..-ketoglutarate. Interferring conditions were controlled by use of a modified Durrum fast kinetics spectrophotometer on line with a Van de Graaf generator. The reaction conditions and rate constants are presented in tabular form. (DDA)

Ascorbic acid as a scavenger of singlet oxygen

The role of the reactive forms of oxygen in physiologic and pathologic processes is a topic under intense investigation at the present time. Similarly, the role of biological anti-oxidants such as ascorbic acid and tocopherol as inhibitors of and therapy for pathologic processes is a subject of considerable experimental and clinical interest. The reactive forms of oxygen include O;-, OH’, HZOz , ‘02 and metal ion-oxygen complexes. Nishikimi [l] has shown that ascorbate can scavenge O;with a second-order rate constant of 2.7 X 10’ M” .s.-l . Bielski et al. [2] have demonstrated ascorbate to be an effective scavenger for OH’ at neutral pH. Singh and Vadasz [3] demonstrated that ascorbate inhibited the methylene blue-catalyzed photoinactivation of Escherichia coli ribosomes by 60% and suggested that the inhibition was mediated via the scavenging of ‘02 by ascorbate. This communication presents further experimental evidence indicating that ascorbate is a ‘02 scavenger.

Copper(II)-catalyzed lipid peroxidation in liposomes and erythrocyte membranes

Cu++ was uniquely capable of catalyzing the peroxidation of rat erythrocyte membrane lipid in the presence of 10 mM H2O2, whereas several other transition metal ions were without significant effect. In contrast, peroxidation of soybean phospholipid liposomes could be catalyzed with decreasing efficiency by Co++, Cu++, Pb++, or Cr+++ also in the presence of H2O2. The effect of imidazole on Cu++-catalyzed lipid peroxidation was stimulatory in liposomes and inhibitory in membrane preparations, whereas EDTA, histidine, citrate and alanine inhibited peroxidation in both systems. EDTA could stop the peroxidation after initiation, but catalase could not, indicating that Cu++ alone was necessary for the propagation of the chain reaction. Competitive inhibition studies with various scavengers of hydroxyl radicals or singlet oxygen and the absence of significant reaction enhancement by D2O indicated that neither of these reactive oxygen species was a major mediator in the Cu++-H2O2 oxidative system. A copper-oxygen complex may be directly involved in the initiation of peroxidation. Normal erythrocyte membranes and phospholipid liposomes also differ in their sensitivities toward external oxidative stress. In the absence of H2O2, Cu++ (0.2 mM) was capable of catalyzing lipid peroxidation in liposomes, aged erythrocyte membranes and membranes from vitamin-E-deficient rats; however, freshly prepared membranes from control rats and liposomes containing α-tocopherol required H2O2 greater than 2 mM for the catalytic effect of Cu++ to be observed.

Enzyme-catalyzed free radical reactions with nicotinamide-adenine nucleotides: I. Lactate dehydrogenase-catalyzed chain oxidation of bound NADH by superoxide radicals

The radiation-induced oxidation of NADH by Superoxide radicals proceeds in the presence of lactate dehydrogenase by a chain mechanism. The reaction is initiated by O2− radicals, and propagated by O2. The chain length is a function of [NADH]/[lactate dehydrogenase], the concentration of O2, the dose rate, and pH. The chain reaction can be inhibited by addition of ascorbic acid.

Singlet oxygen in copper-catalyzed lipid peroxidation in erythrocyte membranes

Lipid hydroperoxide was generated in human erythrocyte membranes by irradiation with near ultraviolet (UV) light in the presence of a photosensitizer, hematoporphyrin, but no production of 2-thiobarbituric acid-reactive materials (malonaldehyde and its precursors) was detected. Incubation of the irradiated membranes with CuSO4 led to increased levels of hydroperoxide and formation of malonaldehyde. Hydroperoxides were essential for initiating the Cu(II)-catalyzed peroxidation as no significant activity was observed with nonirradiated membranes and Cu(II) unless an organic peroxide, either t-butyl hydroperoxide or cumene hydroperoxide, was added. Catalytic activity was also found with Fe(II), but not with other metal ions tested. The peroxidation catalyzed with Cu(II) was partially inhibited by several singlet oxygen quenchers but was not affected by superoxide dismutase, catalase or OH• radical scavengers. The possible involvement of singlet oxygen in the Cu(II)-catalyzed peroxidation reaction was further supported by a 3-fold enhancement of malonaldehyde production in D2O.

Reversible effect of sodium dodecyl sulfate on human erythrocyte membrane adenosine triphosphatase

Abstract A study was made on the effect of sodium dodecyl sulfate on human erythrocyte membrane ATPase. The ATPase was activated by low concentrations of sodium dodecyl sulfate and was inhibited by the detergent above 0.4 mM. At optimum concentration of sodium dodecyl sulfate the membrane ATPase was stimulated about two-fold by either Na + or K + alone. This stimulation was not affected by ouabain. The effect of sodium dodecyl sulfate was abolished by a preincubation at 50° for 5 min. Tracer studies indicated that within the range tested less than 1% of sodium dodecyl sulfate in the medium was bound to the membrane fragments. Heating at 50° did not dissociate the bound sodium dodecyl sulfate. The evidence indicated that the reversible alteration of membrane ATPase characteristics by sodium dodecyl sulfate was not simply due to the binding and unbinding of the detergent molecules to the enzyme system.

The influence of a low-potassium diet on rat-erythrocyte-membrane adenosine triphosphatase.

Abstract Wistar rats were provided a low-potassium dietary regimen for varying time intervals up to 10 weeks. Within 2 weeks plasma potassium concentration was reduced; from the fifth to the tenth week it remained at about half the value for normal plasma. Starting at the fifth week of the low-potassium dietary regimen, the (Na + K)-dependent ATPase in the erythrocyte membrane of the potassium-depleted rats showed a steady increase in specific activity; by the tenth week, it was 50–100% higher than that of control rats. On the other hand, there were no detectable changes in another stromal enzyme, acetylcholine esterase, in the membrane preparations of potassium-depleted rats. In subsequent experiments low-potassium rats (depleted for 10 weeks) were returned to a complete diet for varying intervals. Plasma potassium concentration became normal within 1 week. But (Na + K)-dependent ATPase in the erythrocyte membrane decreased slowly and reached a normal level only after about 5 weeks on the complete diet. The results suggest that the decrease in the plasma potassium concentration results in an induction of the erythrocyte membrane (Na + K)-dependent ATPase.

Study of peroxidase mechanisms by pulse radiolysis. II. Reaction of horseradish peroxidase compound I with O2

Abstract 1. 1. Sodium formate has no effect upon the absorption spectra and the rates of reaction of horseradish peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7) and its derivative Compound I. It is a good radiation protector for horseradish peroxidase in aerated aqueous solutions. 2. 2. The rate of formation of Compound I has been determined in a fast kinetics spectrophotometer at 335 nm. During the conversion of horseradish peroxidase to Compound I, no other intermediates are detectable in the range from 300 to 450 nm. 3. 3. Radiation generated superoxide radicals (O2−), reduce Compound I to Compound II with a rate constant of 1.60·10 6 ± 0.08·10 6 M −1 ·s −1 . 4. 4. A three-dimensional computer presentation (absorbance vs wavelength vs time) shows spectral changes observed after pulse irradiation of an air-saturated formate solution containing horseradish peroxidase. 5. 5. Studies with both pulse radiolysis and γ-ray irradiation indicate that Compound II does not react with O2−.

Studies on the lipoic acid free radical

Abstract 1. 1. When dihydrolipoic acid is irradiated by 60Co γ rays at pH 8.0 and in the presence of N2O, it is converted to lipoic acid with a G value of 3.3 ± 0.3, where G is the number of molecules formed per 100 eV energy absorbed. No other products were detectable. The radiation product was identified by its absorption spectrum and thin-layer chromatography. 2. 2. The lipoic acid free radical was studied with pulse radiolysis. Its absorption spectrum has a maximum at 410 nm with a molar extinction coefficient of 7760 M−1 cm−1. The radicals derived from either oxidation of dihydrolipoic acid or reduction of lipoic acid are the same. At pH 8 the radical decays by second order, k = (7.45 ± 0.22) s 10 7 M −1 s s −1 . Ionic strength studies indicate that the radical decay is influenced by a charge of one. 3. 3. The formation of the lipoic acid radical by pulse radiolysis and the rate of its decay are independent of the concentration of dihydrolipoic acid in the solution, which excludes the possibility of an interaction between the radical and its patent compound to form a complex. 4. 4. The lipoic acid radical reduces FAD to form FADHṡ at a rate of k = (2.40 ± 0.18) × 10 8 M −1 s s −1 .

Degradation of articular cartilage by copper and hydrogen peroxide

When porcine articular cartilage particles were incubated in the presence of Cu2+ and H2O2 at pH 7.4, solubilization of collagen and proteoglycan was observed. Both agents were necessary and the rate of solubilization was concentration dependent. Other transition metal ions showed much lower catalytic activity. The solubilized polypeptides were polydispersed in size and the hydroxyproline content of the larger fragments was 13% by weight. Further incubation of the released material with Cu2+ and H2O2 resulted in further degradation and partial destruction of hydroxyproline residues. Competitive studies with scavengers of OH· and1O2 as well as the effect of D2O excluded these two species as major mediators in this system.