On the dynamics of DNA in aqueous solution. Pulse radiolysis studies using the light scattering detection method

Summary The kinetics of DNA chain breakage in solution induced by 2 µs pulses of 15 MeV electrons were investigated by light scattering. On irradiating native calf thymus DNA at room temperature the decrease of light scattering intensity (LSI) - due to double strand ruptures - shows a fast decay with a half life _1/2 of about 30 ms as well as a slow decay with _1/2 of about 10 s. With increasing temperature (20–40° C) both the total degree of degradation and the fraction of the fast decay increase due to the facilitated melting of segments between two single strand breaks on alternate strands forming a double strand break. Above 40° C a third mode of LSI decay with _1/2 of 5–10 s arises, indicating detachment of relatively long segments.The total relative decrease of LSI after irradiation A, which can be taken as a measure of the degree of degradation, follows the square of the absorbed dose in the case of native DNA, whereas on irradiating denatured DNA A rises linearly with dose. The decay of LSI due to the degradation of denatured DNA is much faster than that of native DNA with _1/2 down to 150 µs, depending on the absorbed dose. The half lives are interpreted in terms of the separation of fragments by diffusion and of the melting of double strand segments between two single strand breaks.     

Pulse radiolysis of lactate dehydrogenase.

Pulse radiolysis has been used to investigate the rates and transient spectra for the reactions of free radicals with beef heart lactate dehydrogenase at pH 7. Analysis of the results leads to second-order rate-constants for eaq-, .OH, .I, .Br2-, .I2- and .(CNS)2- which are, respectively, 24, 21, 10, 0.55, 0.43 and 0.15 in units of 10(10) M-1 s-1 with uncertainties of +/- 20 per cent. Those for .I and .I2- are similar to the corresponding rate-constants for the related enzyme alcohol dehydrogenase. The spectra of the transient species produced by .OH, .Br2- and .(CNS)2- all showed evidence for reactions with tyrosine and tryptophan residues, and in general terms the magnitudes of the rate-constants appeared to increase with the oxidizing abilities of the radicals. The implication of the results for understanding the mechanism of deactivation by free radicals is discussed.     

Pulse radiolysis studies on superoxide reductase from Treponema pallidum

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.     

Formation of super-reduced Chromatium high-potential iron--sulphur protein in aqueous solution by pulse radiolysis.

Both the oxidized and reduced forms of Hipip (high-potential iron--sulphur protein) are reduced (approx. 30% yields) by eaq.- in a single-stage process, rate constants 1.7 x 10(10) and 1.8 x 10(10) M-1 . s-1 respectively, at 25 degrees C, pH 7.0 (5 mM-phosphate). Super-reduced Hipip, which is formed in the latter case, has a spectrum which closely resembles that of reduced ferredoxin, i.e. Fe4S4 (SR)4(3-) clusters. The spectrum is stable over 2 s periods investigated. Super-reduced Hipip is reoxidized with O2, rate constant 4.8 x 10(6) M-1 . s-1 at 25 degrees C.     

Chromatographic studies on the radiolysis of DNA in aqueous solution

Radiation-induced degradation of double-stranded DNA from calf thymus in aqueous solution with sodium phosphate was studied by conventional gel chromatography and by high-performance liquid-gel permeation chromatography. Comparison of the data after radiolysis of aqueous solutions of DNA under anaerobic and aerobic conditions indicates that double-strand breakage is not enhanced by oxygen. An increase of ionic strength impedes the break-down of the DNA molecules, so that loss of DNA can only be observed at doses above 100 Gy. Only reactions of OH-radicals contribute to the fragmentation of DNA, while the presence of hydrated electrons, H.-or formate radicals does not lead to a loss of highly polymerized DNA up to doses of 1500 Gy. High-performance liquid-chromatography proved to be an excellent method of studying the degradation of macromolecules as a function of dose.     

Pulse radiolysis studies of some aza analogues of nucleic acid components

The technique of pulse radiolysis has been utilized to study the reactions of some aza analogues of nucleic acid components with hydrated electrons and OH radicals. The absorption spectra of the transient free radical adducts which result from these reactions and their decay kinetics were determined. The 5-aza analogues gave similar results to those of pyrimidine bases. The 6-aza analogues also showed similar kinetics, however, transient spectra were different. The presence of the sugar moiety in these aza analogues changed the rate law of the OH adduct transient decay from second order to first order kinetics. This finding may have implications for the understanding of the radiation chemistry of DNA.     

Formation of porphyrin pi cation radical in zinc-substituted horseradish peroxidase

Zinc-substituted horseradish peroxidase is oxidized by K2IrCl6 to a characteristic state which retains one oxidizing equivalent more than the zinc peroxidase. The oxidized enzyme gives an optical absorption spectrum similar to that of compound I of peroxidase and catalase, and a g = 2 electron paramagnetic resonance signal which has an intensity corresponding to the porphyrin content. It is reduced back to the zinc peroxidase by a stoichiometric amount of ferrocyanide or by a large excess of K3IrCl6. From the equilibrium data, the value of E0' for the zinc peroxidase couple is estimated to be 0.74 V at pH 6. The oxidized zinc peroxidase is also formed by the addition of H2O2 or upon illumination with white light. The rate constants for the oxidation by K2IrCl6 and H2O2 at pH 8.0 are 8 x 10(5) and 8 x 10(2) M-1 s-1, respectively. No essential spectral change can be observed when K2IrCl6 is added to the metal-free peroxidase (protoporphyrin--apoperoxidase complex) or to zinc-substituted sperm whale myoglobin.     

Pulse radiolysis study of the reactivity of Trolox C phenoxyl radical with superoxide anion

The reaction between the phenoxyl radical of Trolox C, a water-soluble vitamin E analogue, and superoxide anion radical was examined by using the pulse radiolysis technique. The results indicate that the Trolox C phenoxyl radical may undergo a rapid one-electron transfer from superoxide radical [k = (4.5 +/- 0.5) x 10(8) M-1.S-1] to its reduced form. This finding indicates that superoxide radical might play a role in the repair of vitamin e phenoxyl radical.     

Formation of a steady state in the radiolysis of ferrimyoglobin in aqueous solution

The interaction of radiation-generated · OH radicals with ferrimyoglobin in deaerated aqueous solution at neutral pH has been quantitatively studied. Changes in the visible absorption spectrum have been analyzed on the basis of composition changes of the ferri, deoxy, and ferriperoxide forms of the metalloprotein. A postirradiation thermal process must be considered in order to evaluate the radical-induced composition changes. Initially, ·OH induces reduction of ferrimyoglobin to the deoxy form with a G value (molecular yield/100 eV of absorbed energy) in the zero-dose limit of 1.4 (±0.2). Radiation-generated H₂O₂ reacts with the ferrimyoglobin substrate to produce ferrimyoglobin peroxide with a G value of 0.7 (±0.1) in the zero-dose limit. At doses of >1 krad μm^?1 of myoglobin present, the composition of the three myoglobin derivatives reaches a radiolysis steady state. In this moderate-dose plateau region, this composition is 44% ferri, 18% deoxy, and 38% ferri peroxide. The · OH-induced hemoprotein radicals that do not initiate 1-eq redox conversions undergo reactions that generate dimer and other globin-modified material.