Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Charge transfer in peptides. Pulse radiolysis investigation of one-electron reactions in dipeptides of tryptophan and tyrosine.

One-electron oxidation of TyrOH-TrpH or TrpH-TyrOH in aqueous solutions by N3 radicals occurs predominantly at the tryptophyl residue. The corresponding indolyl radicals (absorbing at 510 nm) are subsequently transformed into phenoxyl radicals (absorbing at 390/405 nm): TyrOH-Trp leads to TyrO-TrpH, k5 = 5.4 x 10(4)s-1, (5), Trp-TyrOH leads to TrpH-TyrO, k7 = 7.3 x 10(4)s-1. (7) The first-order radical transformation rates are independent of the (initial) concentration of N3 or peptide and unaffected by urea (as a modifier of hydrogen bond structures). Intermolecular conversion of indolyl into phenoxyl radicals, e.g. by reaction of GlyH-Trp with TyrOH-GlyH, is very slow and inefficient. It is concluded that reactions (5) and (7) occur by intramolecular charge transfer across the peptide bond.     

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 study of the interaction of retinoids with peroxyl radicals

Vitamin A (retinol) and its derivatives-retinal and retinoic acid-are known for their ability to inhibit lipid peroxidation. Antioxidant actions of retinoids have been attributed to chain-breaking by scavenging of peroxyl radicals. Based on chemical analysis of retinoic acid degradation products formed during microsomal lipid peroxidation, it was previously suggested that retinoids interact with peroxyl radicals forming free carbon-centered radical adducts. However, it can be argued that such a mode of antioxidant action of retinoids is not sufficient to fully explain their effectiveness at inhibiting lipid peroxidation, which in many systems is comparable to, or even exceeds, that of alpha-tocopherol. In order to elucidate the mechanism of interaction of retinoids with peroxyl radicals, (trichloromethyl)peroxyl radical was generated by pulse radiolysis, and its interactions with retinoids solubilized in Triton X-100 micelles were followed by kinetic absorption spectroscopy. All retinoids--retinol, retinal, and retinoic acid--interacted with the peroxyl radical, and at least two transient products were detected. One of these products, absorbing at 590 nm, was identified as retinoid cation radical. Therefore, we postulate that, apart from formation of radical adducts, retinoids may also scavenge peroxyl radicals by electron transfer.     

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.     

One-electron reduction of D-amino acid oxidase. Kinetics of conversion from the red semiquinone to the blue semiquinone

The reduction of D-amino acid oxidase (DAAO) by hydrated electrons (eaq-) has been studied in the absence and presence of benzoate by pulse radiolysis. The eaq-did not reduce the flavin moiety in DAAO and reacted with the amino acid residues in the protein. In the presence of benzoate, eaq- first reacted with benzoate to yield benzoate anion radical. Subsequently, the benzoate anion radical transferred an electron to the complex of DAAO-benzoate to form the red semiquinone of the enzyme with a second-order rate constant of 1.2 X 10(9) M-1 s-1 at pH 8.3. After the first phase of the reduction, conversion of the red semiquinone to the blue semiquinone was observed in the presence of high concentration of benzoate. This process obeyed first-order kinetics, and the rate increased with an increase of the concentration of benzoate. In addition, the rate was found to be identical with that of the formation of the complex between benzoate and the red semiquinone of DAAO as measured by a stopped-flow method. This suggests that bound benzoate dissociates after the reduction of the benzoate-DAAO complex by benzoate anion radical and that free benzoate subsequently recombines with the red semiquinone of the enzyme to form the blue semiquinone.     

Quaternary structure of methemoglobin. Pulse radiolysis study of the binding of oxygen to the valence hybrid.

The pulse radiolysis of solutions of adult human methemogolbin was used in order to reduce a single heme iron within the protein tetramers. The valence hybrids thus formed were reacted with oxygen. Kinetics of the reactions were studied. The effects of pH and inositol hexaphosphate were examined. The kinetics of the ligation of oxygen to stripped valence hybrids showed a single phase behavior at the pH range 6.5 to 9. As the pH was lowered below 6.5, a second, slower phase became apparent. In the presence of inositol hexaphosphate, above pH 8, the kinetics of oxygen binding was of a single phase. As the pH was lowered, a transition to a second, slower phase was noticed. Below pH 7, the slower phase was the only detectable one. The analysis of the relative contribution of the faster phase to the total reaction as a function of the pH showed a typical transition curve characterized by a pK = 7.5 and a Hill parameter n = 2.9. On this basis, it is concluded that human adult stripped methemoglobin resides in an R quarternary structure, while the presence of IHP stabilizes the T structure at pH below 7.5. This transition between the quaternary structures of methemoglobin cannot be accounted for by the switch between the high spin and the low spin states of the ferric iron. This switch of spin state takes place at pH greater than 8.2.     

Pulse radiolysis study of a yeast cytochrome c from Hansenula anomala

The reduction of Hansenula anomala yeast cytochrome c by e-aq and CO-.2 was investigated by pulse radiolysis, at a high reductant to protein concentration ratio. The reactivity of the radicals was studied by observing absorbance changes in the cytochrome c spectrum over the wavelength range 280-600 nm. At pH 7, over the time scale of the radical decays (i.e. 0-4 microseconds for e-aq; 0-40 microseconds for CO-.2s) and beyond, the hemoprotein was reduced without any spectrally detected intermediate between ferri-and ferro-forms. This conclusion was reached by simulation studies based on the direct reduction of the yeast cytochrome c from the ferri- to the ferro-form, yielding a correct fit between experimental and calculated absorbance curves. The reduction rate constants were determined to be 1.0 +/- 01 X 10(10) M-1 S-1 for e-aq and 0.7 +/- 0.05 X 10(9) M-1 S-1 for CO-.2 at 0.16 M ionic strength, pH 7.0 and 20 degrees C, thus not significantly different from other values reported for horse heart cytochrome c. However, in the 360-390 nm region the generation of an additional radical species was noticed. The present experimental data were compared with previously published reports.     

Intramolecular electron transfer in proteins. Radiolysis study of the reductive activation of daunorubicin complexed in egg white apo-riboflavin binding protein

Daunorubicin, an anthracycline antitumor antibiotic, can be complexed in egg white apo-riboflavin binding protein. The reduction of this complex was studied by gamma-radiolysis and pulse radiolysis using COO.- free radicals as reductants. The final products are 7-deoxydaunomycinone intercalated in the protein and thiol groups coming from the reduction of disulfide bonds of the protein, in the respective proportions of 90% and 10%. One-electron reduction of the complex gives daunorubicin semiquinone radical and a disulfide anion. The rate constants of the reactions of COO.- ions with the complex and with the disulfide bond in the protein alone are respectively equal to 2.4 x 10(8) mol-1.L.s-1 and 6.4 x 10(7) mol-1.L.s-1. Daunorubicin semiquinone decays by a first-order process, the rate constant of which is independent of the initial protein and radical concentrations. Without protein, daunorubicin semiquinone undergoes a disproportionation-comproportionation equilibrium [Houée-Levin, C., Gardès-Albert, M., Ferradini, C., Faraggi, M., & Klapper, M. (1985) FEBS Lett. 179, 46-50]. We propose that a protein residue reduces semiquinone by an intramolecular path. This creates an electron hole in the protein which may alter its function. This reduction process is very different from the reduction mechanism of riboflavin binding protein by the same reductant [Faraggi, M., Steiner, J.P., & Klapper, M.H. (1985) Biochemistry 24, 3273-3279]. These results suggest a new deleterious pathway to explain the antitumor and/or cytotoxic effect of this drug.     

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.     

One-electron reduction of adriamycin: Properties of the semiquinone

Pulse radiolysis of aqueous solutions containing adriamycin and redox indicators of known one-electron reduction potential (E¹) shows that its E¹ at pH 7 is ?328 mV (vs NHE). The variation E¹ with pH in the range 6–12 shows that the net charge on the semiquinone at pH 7 is zero. As well as the pK_a values of 2.9 and ≥ 14 established independently, the semiquinone has a pK_a close to 9.2. The new data enable the structure and likely reactivity of the semiquinone to be specified.     

Role of membrane charge and semiquinone structure on naphthosemiquinone derivatives and 1,4-benzosemiquinone disproportionation and membrane-buffer distribution coefficients.

Semiquinone membrane/buffer partition coefficients have been determined for 1,2-naphthosemiquinone (ONQ.-), 1,4-naphthosemiquinone (NQ.-) and two of its hydroxylated derivatives, 5,8-dihydroxy-1,4-naphthosemiquinone (NZQ.-) and 5-hydroxy-1,4-naphthosemiquinone (JQ.-) as a function of membrane charge in multilamellar vesicles of phosphatidylcholine (PC) and equimolar mixtures of this lipid and phosphatidic acid (PC:PA) and cetyltrimethylammonium bromide (PC:CTAB) at physiological pH with the exception of values corresponding to PC:PA mixtures which were obtained at pH 9. These coefficients follow the order PC:PA < PC < PC:CTAB in agreement with the negative charge of the semiquinones. The disproportionation equilibria of the naphthosemiquinone derivatives are shifted to the semiquinone in the presence of neutral and positive membranes, being more pronounced in the latter. However, very low partition coefficients as well as small shifts in the semiquinone disproportionation equilibrium were observed for ONQ.- as compared to the other semiquinones. No partition of 1,4-benzosemiquinone (BQ.-) into the lipid phase was detected for either charged or neutral lipid membranes. The presence of lipid membranes decreases the BQ.- equilibrium concentration in the presence of all the types of membranes considered here.