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E Bothe

Publications and source records attributed to E Bothe.

41 records · Page 3Linked to original sources
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Release of K+ and H+ from poly U in aqueous solution upon gamma and electron irradiation. Rate of strand break formation in poly U.

Conductivity changes were found which followed the reaction of radiolytically generated OH radicals with the potassium salt of polyuridylic acid (poly U) in aqueous solution. After 60Co-gamma-irradiation the observed increase of conductivity at pH = 6.8 was shown to consist of the liberation of K+ ions from the stock of K+ ions electrostatically bound to the polyanion. The initial G(K+) is 36 and hence 6 times higher than the G value of OH radicals in N2O saturated solutions. At a poly U concentration of 60 mg l-1 half of the ion release occurred at 12 J kg-1 and nearly all ions are released at 40 J kg-1. The liberation of K+ is explained to be a consequence of the formation of chain breaks leading to an increase of the degree of dissociation. The rate of the ion release was studied under pulse radiolysis conditions. Because of the high G-value of counterion liberation and the use of conductivity as analytical quantity the method is very sensitive. With 6 mg l-1 poly U the rate could be measured even at a dose per pulse of 0.25 J kg-1. The kinetics of the ion release can be described in terms of two parallel first order reactions of comparable contribution with an average rate constant of 0.8 s-1 at 20 degrees C, 60 mg l-1 poly U and pH = 6.8 with a small contribution of slower components. In more acidic solutions, besides K+ ions H+ are also liberated since at low pH values bound K+ is replaced by H+. The rate of the ion release was found to increase with increasing replacement of K+ by H+ (kobs = 100 s-1 at pH = 3.4, 60 mg l-1 poly U and T = 18 degrees C). With potassium ion free polyuridylic acid the corresponding rate constant amounted to 220 s-1, nearly independent of pH. From the temperature dependence activation parameters for the ion release were derived (Ea = 57 kJ mol-1, A = 1.0 X 10(10) s-1 at pH = 6.8). Addition of p-benzoquinone at pH = 3.7 and dithiothreitol (DTT) at pH 6.8 were found to decrease the size of the conductivity changes and to increase the rate. The results show that p-benzoquinone and DTT react with poly U radicals and that these reactions prevent chain breaks and ion liberation. It is concluded that the rate determining step of the conductivity increase is the formation of strand breaks by a cleavage of poly U radicals and that this reaction is pH dependent. The pH dependence and the observed value for the activation energy was found to be in agreement with the behaviour of a model system for the earlier postulated C-4' mechanism for strand break formation of polynucleotides and DNA.

DNA↗
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Rate of OH radical induced strand break formation in single stranded DNA under anoxic conditions. An investigation in aqueous solutions using conductivity methods.

Single stranded DNA (ssDNA) in aqueous N2O-saturated solution was pulse-irradiated with electrons or irradiated under steady-state conditions with 60Co-gamma rays. The conductivity increase after irradiation was measured as a function of concentration, pH, temperature, metal cation content and additives. The conductivity increase could be shown to be due to the release of associated counterions (Na+ or H+) as a result of the formation of chain breaks. At 28 mg 1(-1) DNA approximately 8 sodium ions are liberated per 100 eV absorbed energy (G (Na+) = 8.3). On the basis of G value for single-strand breaks (ssb) of G (ssb) = 0.55, it is calculated that 8.3/0.55 = 15 sodium ions per strand break are set free. The release of Na+ monitored by 23Na-NMR at pH 7 as a function of dose corresponds to that of the conductivity increase. The rate of the conductivity increase does not depend on dose/pulse (range 2-20 J kg-1) and shows at least two components. The rate constant of the fast and dominant component is constant above pH 8.5 (k = 38 s-1, 20 degrees C) and increases linearly with proton concentration below pH 7. Values of 13 kcal/mol for the activation energy and 5 X 10(11) s-1 for the frequency factor were obtained at pH 7.3. Addition of p-benzoquinone (pBQ) increases the rate constant of the fast component proportionally to the concentration of pBQ. From these results the rate-determining step of the fast conductivity increase is concluded to be the splitting of DNA radicals. The pH dependence and the magnitude of the activation energy agree with results from low molecular weight model compounds for the C-4' mechanism; this mechanism involves a heterolytic splitting of the phosphoric acid ester bond starting from the 4' radical of DNA.

Animals↗
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Rate and rate-determining step of hydrogen-atom-induced strand breakage in poly(U) in aqueous solution under anoxic conditions.

The rate constant for strand breakage in poly(U) after reaction with hydrogen atoms in deoxygenated aqueous solution has been determined to be k = 1.5 s-1 at pH = 4-5 and 24 degrees C. Dithiothreitol has been found to prevent strand break formation by reacting with H-adduct radicals of poly(U) with a rate constant of 5 X 10(6) M-1 s-1. It is concluded that the rate-determining step in H atom-induced strand breakage in poly(U) at pH less than or equal to 6 is the decay of uracil moiety H-adduct radicals via H-abstraction from the ribose moiety.

Chemical Phenomena↗
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The kinetics of hydroxyl-radical-induced strand breakage of hyaluronic acid. A pulse radiolysis study using conductometry and laser-light-scattering.

Hydroxyl radicals were generated radiolytically in N2O- and N2O/O2(4:1)-saturated aqueous solutions of hyaluronic acid. The hydroxyl radicals react rapidly with hyaluronic acid mainly by abstracting carbon-bound H atoms. As a consequence of subsequent free-radical reactions, chain breakage occurs the kinetics of which has been followed using the pulse radiolysis technique. In the absence of oxygen, strand breakage was followed by the change in conductivity induced by the release of cationic counterions condensed at the surface of hyaluronic acid which is a polyanion consisting of subunits of glucuronic acid alternating with N-acetyl-glucosamine. It appears that strand breakage is not due to one single first-order process, however, the contributions of the different components cannot be adequately resolved. At pH 7 the overall half-life is 1.4 ms, in both acid and basic solutions the rate of free-radical induced strand breakage is accelerated (at pH 4.8, t1/2 = 0.6 ms; at pH 10, t1/2 = 0.18 ms). In the absence of oxygen there is no effect of dose rate on the kinetics of strand breakage. In the presence of oxygen in addition to conductometric detection, strand breakage was also followed by changes in low-angle laser light-scattering. These two techniques are complementary in that in this system the conductometry requires high doses per pulse while the light-scattering technique is best operated in the low-dose range. In the presence of oxygen a pronounced dose-rate effect is observed, e.g. at pH 9.7 after a dose of 9.4 Gy the overall half-time is approx. 0.5 s, while after a dose of 6.6 Gy the half-time is approx. 0.23 s. Both the yield and the rate of strand breakage increase with increasing pH, e.g. at pH 7 G(strand breaks) = 0.7 x 10(-7) mol J-1 and at pH 10.4, 4.8 x 10(-7) mol J-7. The radiolytic yields of CO2, H2O2, organic hydroperoxides, O2.- and oxygen consumption have been determined in gamma-irradiated N2O/O2(4:1)-saturated solutions of both hyaluronic acid and beta-cyclodextrin.

Carbohydrate Sequence↗