Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

PHOTOCHEMISTRY OF DNA USING 193 nm EXCIMER LASER RADIATION

Photoproducts in double-stranded DNA induced by 193 nm radiation have been investigated. Double-stranded, supercoiled pBR322 DNA in buffered aqueous solution was exposed to varying fluences of 193 nm radiation from an ArF excimer laser. The quantum yields for formation of cyclobutylpyrimidine dimers, frank strand breaks and alkali labile sites were calculated from the conversion of supercoiled (Form I) DNA to relaxed (Form II) DNA after treatment with Micrococcus luteus dimer-specific endonuclease, no treatment, or treatment with alkali and heat, respectively. The quantum yields were 1.65 (+/- 0.03) X 10(-3) for pyrimidine dimers, 9.4 (+/- 3.2) X 10(-5) for frank strand breaks and 9.6 (+/- 3.6) X 10(-5) for alkali labile sites. The quantum yields for pyrimidine dimers and strand breaks and alkali labile sites were not affected by 10 nM mannitol. The relative quantum yields for these DNA photoproducts induced by 193 nm radiation differed markedly from those produced by 254 nm radiation.     

Influence of organic ions on DNA damage induced by 1 eV to 60 keV electrons

We report the results of a study on the influence of organic salts on the induction of single strand breaks (SSBs) and double strand breaks (DSBs) in DNA by electrons of 1 eV to 60 keV. Plasmid DNA films are prepared with two different concentrations of organic salts, by varying the amount of the TE buffer (Tris-HCl and EDTA) in the films with ratio of 1:1 and 6:1 Tris ions to DNA nucleotide. The films are bombarded with electrons of 1, 10, 100, and 60?000 eV under vacuum. The damage to the 3197 base-pair plasmid is analyzed ex vacuo by agarose gel electrophoresis. The highest yields are reached at 100 eV and the lowest ones at 60 keV. The ratios of SSB to DSB are surprisingly low at 10 eV (～4.3) at both salt concentrations, and comparable to the ratios measured with 100 eV electrons. At all characteristic electron energies, the yields of SSB and DSB are found to be higher for the DNA having the lowest salt concentration. However, the organic salts are more efficient at protecting DNA against the damage induced by 1 and 10 eV electrons. DNA damage and protection by organic ions are discussed in terms of mechanisms operative at each electron energy. It is suggested that these ions create additional electric fields within the groove of DNA, which modify the resonance parameter of 1 and 10 eV electrons, namely, by reducing the electron capture cross-section of basic DNA units and the lifetime of corresponding transient anions. An interstrand electron transfer mechanism is proposed to explain the low ratios for the yields of SSB to those of DSB produced by 10 eV electrons.     

MECHANISM OF DNA CLEAVAGE MEDIATED BY PHOTOEXCITED NON-STEROIDAL ANTIINFLAMMATORY DRUGS

DNA damage photoinduced by four nonsteroidal antiinflammatory drugs (NSAID) have been investigated by neutral agarose gel electrophoresis. Upon irradiation at 300 nm, in phosphate buffered solution, benoxaprofen, naproxen, ketoprofen, tiaprofenic acid photosensitized the formation of single-strand breaks (SSB) in double stranded supercoiled phi X174 DNA. The efficiency of the cleavage is higher in argon saturated solutions than in aerated solutions and it is not correlated with the quantum yield of photodegradation of the drugs. Simultaneously with the DNA strand breaks, NSAID promote a weak reduction of the electrophoretic mobility of the supercoiled form that may be attributed to the formation of pyrimidine dimers or other DNA unwinding products. These photodimerization processes suggest the involvement of a triplet-triplet energy transfer between NSAID and DNA. Addition of mannitol and superoxide dismutase decreases the efficiency of the cleavage suggesting that HO. and O2.- are involved in the DNA cleavage. Unexpectedly, addition of sodium azide quenches the cleavage both in aerated or in deaerated solutions. Substituting H2O by D2O does not change the number of SSB thus suggesting that 1O2 does not take an important place in the cleavage of DNA. From our data we tentatively assume that the cleavage occurs through a radical mechanism that may involve in a first step an energy or an electron transfer. Gel sequencing on NSAID-photoinduced DNA breakage exhibits no particular specificity except in the case of benoxaprofen where a slight selectivity for cytosine is observed.     

SINGLE- and DOUBLE-STRAND BREAK FORMATION IN DOUBLE-STRANDED DNA UPON NANOSECOND LASER-INDUCED PHOTOIONIZATION

Double-stranded (ds) calf thymus DNA (0.4 mM), excited by 20 ns laser pulses at 248 nm, was studied in deoxygenated aqueous solution at room temperature and pH 6.7 in the presence of a sodium salt (10 mM). The quantum yields for the formation of hydrated electrons (phi c-), single-strand breaks (phi ssb) and double-strand breaks (phi dsb) were determined for various laser pulse intensities (IL). phi c- and phi ssb increase linearly with increasing IL; however, phi ssb has a tendency to reach saturation at high IL (greater than 5 X 10(6) Wcm-2). The ratio phi ssb/phi c-, representing the number of ssb per radical cation, is about 0.08 at IL less than or equal to 5 X 10(6) Wcm-2. For comparison, the number of ssb per OH radical reacting with dsDNA is 0.22. On going from argon to N2O saturation, phi ssb and phi dsb become larger by factors of approximately 5 and 10-15, respectively. This enhancement is produced by attack on DNA bases by OH radicals generated by N2O-scavenging of the photoelectrons. While phi ssb is essentially independent of the dose (Etot), phi dsb depends linearly on Etot in both argon- and N2O-saturated solutions. The linear dependence of phi dsb implies a square dependence of the number of dsb on Etot. This portion of dsb formation is explained by the occurrence of two random ssb, generated within a critical distance (h) in opposite strands. For both argon- and N2O-saturated solutions h was found to be of the order of 40-70 phosphoric acid diester bonds. On addition of electron scavengers such as 2-chloroethanol (or N2O plus t-butanol), phi dsb is similar to that in neat, argon-saturated solutions. Thus, hydrated electrons are not involved in the chemical pathway leading to laser-pulse-induced dsb of DNA.     

Comparison between X-ray photon and secondary electron damage to DNA in vacuum

Both monolayer and thick (20 microm) films of dry pGEM-3Zf(-) plasmid DNA deposited on tantalum foil were exposed to Al Kalpha X-rays (1.5 keV) for various times in an ultrahigh vacuum chamber. For monolayer DNA, the damage was induced mainly by low energy secondary electrons (SEs) emitted from the tantalum. For the thick films, DNA damage was induced chiefly by X-ray photons. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis. The exposure curves for the formation of nicked circular (single strand break, SSB), linear (double strand break, DSB), and interduplex cross-link forms 1 and 2 were obtained for both monolayer and thick films of DNA, respectively. The lower limits of G values for SSB and DSB induced by SEs were derived to be 86 +/- 2 and 8 +/- 2 nmol J(-1), respectively. These values are 1.5 and 1.6 times larger than those obtained with 1.5 keV photons. The projected X-ray energy dependence of the low energy electron (LEE) enhancement factor for the SSB and DSB in monolayer DNA is also discussed. This new method of investigation of the SE-induced damage to large biomolecules allows direct comparison of the yield of products induced by high energy photons and LEEs under identical experimental conditions.     

Multiphoton near-infrared femtosecond laser pulse-induced DNA damage with and without the photosensitizer proflavine

The excitation of pBr322 supercoiled plasmid DNA with intense near-IR 810 nm fs laser pulses by a simultaneous multiphoton absorption mechanism results in single-strand breaks after treatment of the irradiated samples with Micrococcus luteus UV endonuclease. This enzyme cleaves DNA strands at sites of cyclobutane dimers that are formed by the simultaneous absorption of three (or more) 810 nm IR photons (pulse width approximately 140 fs, 76 MHz pulse repetition, average power output focused through 10x microscope objective is approximately 1.2 MW/cm2). Direct single-strand breaks (without treatment with M. luteus) were not observed under these conditions. However, in the presence of 6 microM of the intercalator proflavine (PF), both direct single- and double-strand breaks are observed under conditions where substantial fractions of undamaged supercoiled DNA molecules are still present. The fraction of direct double-strand breaks is 30 +/- 5% of all measurable strand cleavage events, is independent of dosage (up to 6.4 GJ/cm2) and is proportional to In, where I is the average power/area of the 810 nm fs laser pulses, and n = 3 +/- 1. The nicking of two DNA strands in the immediate vicinity of the excited PF molecules gives rise to this double-strand cleavage. In contrast, excitation of the same samples under low-power, single-photon absorption conditions (approximately 400-500 nm) gives rise predominantly to single-strand breaks, but some double-strand breaks are observed at the higher dosages. Thus, single-photon excitation with 400-500 nm light and multiphoton activation of PF by near-IR fs laser pulses produces different distributions of single- and double-strand breaks. These results suggest that DNA strand cleavage originates from unrelaxed, higher excited states when PF is excited by simultaneous IR multiphoton absorption processes.     

Hydrolytic cleavage of DNA by quercetin manganese(II) complexes

Quercetin manganese(II) complexes were investigated focusing on its DNA hydrolytic activity. The complexes successfully promote the cleavage of plasmid DNA, producing single and double DNA strand breaks. The amount of conversion of supercoiled form (SC) of plasmid DNA to the nicked circular form (NC) depends on the concentration of the complex as well as the duration of incubation of the complexes with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.2 in the presence of 100 μM of the complexes is found to be 1.32 × 10⁻⁴ s⁻¹. The hydrolytic cleavage of DNA by the complexes was supported by the evidence from free radical quenching, thiobarbituric acid-reactive substances (TBARS) assay and T4 ligase ligation.     

SINGLET OXYGEN INDUCES FRANK STRAND BREAKS AS WELL AS ALKALI- AND PIPERIDINE-LABILE SITES IN SUPERCOILED PLASMID DNA

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.     

PSORALENS CLEAVE pBR322 DNA UNDER ULTRAVIOLET RADIATION

Supercoiled (SC) pBR322 was used to probe the recent claim that 5-geranoxylpsoralen (5-GOP) did not photoreact with DNA. Contrary to expectations, 5-GOP was found to damage DNA in the presence of UV-A through two competing pathways: (a) single strand breaks, identified by the conversion of supercoiled into open circular and linear DNA, and (b) cross-linking, revealed by the fluence-dependent decrease in the extent of denaturation of the double stranded supercoiled DNA to single stranded circular DNA. In addition, a fluence-dependent modification reduced the ability of the restriction enzyme EcoR I to linearize the photosensitized DNA, and alkali-labile lesions were generated. Psoralen, 5-methoxypsoralen, and 8-methoxypsoralen, which are well-known to undergo cycloaddition to DNA, had a more pronounced effect on supercoiled DNA. Single strand breaks occurred more readily than with 5-GOP, and the surviving SC form remaining had reduced electrophoretic mobility in agarose gels. In all cases, the DNA damage was more prominent when oxygen was absent.     

Low-energy electron-induced single strand breaks in 2'-deoxycytidine-3'-monophosphate using the local complex potential based time-dependent wave packet approach

Recent experimental and theoretical investigations on resonant electron scattering off DNA and DNA fragments using low-energy electrons (LEEs), to propose the mechanism for single strand breaks (SSBs) and double strand breaks (DSBs), have received considerable attention. It is our purpose here to understand theoretically the comprehensive route to SSB in a selected DNA fragment, namely, 2'-deoxycytidine-3'-monophosphate (3'-dCMPH), induced by LEE (0-3 eV) scattering using the local complex potential based time-dependent wave packet (LCP-TDWP) approach. To the best of our knowledge, there is no time-dependent quantum mechanical study that has been reported in the literature for this DNA fragment to date. Initial results obtained from our calculation in the gas phase provide a good agreement with experimental observation and show the plausibility of SSB at 0.75 eV, which is very close to the highest SSB yield reported from the experimental measurement (0.8 eV) on plasmid DNA in the condensed phase.     

Photogenotoxicity of Skin Phototumorigenic Fluoroquinolone Antibiotics Detected Using the Comet Assay

Abstract— The fluoroquinolone(FQ) antibiotics photosensitize human skin to solar UV radiation and are reported to photosensitize tumor formation in mouse skin. As tumor initiation will not occur without genotoxic insult, we examined the potential of ciprofloxacin, lomefloxacin, fle-roxacin, BAYy3118 (a recently developed monofluori-nated quinolone) and nalidixic acid to photosensitize DNA damage in V79 hamster fibroblasts in vitro. Cells were exposed to 37.5 kj/m₂ UVA (320-400 nm; glass filtered Sylvania psoralen + UVA (PUVA) tubes; calibrated Waldmann radiometer) at 4A_oC in the presence of FQ and immediately afterwards embedded in agarose, lysed and placed in an electrophoretic field at pH 12. Under these denaturing conditions, the presence of DNA single-strand breaks (SSB), alkali-labile sites (ALS) and double-strand breaks (DSB) can be visualized as DNA migrating away from the nucleus (characteristic "comet" appearance) after staining with a specific fluorochrome. At FQ concentrations that induced minimal loss of cell viability (neutral red uptake assay) the compounds tested induced comets with a rank order of BAYy3118 norfloxacin ciprofloxacin lomefloxacin fleroxacin nalidixic acid. If cells were incubated after treatment for 1 h at 37_oC, the comet score decreased, suggesting efficient removal of SSB/ALS/DSB. Addition of the DNA polymerase, inhibitor, aphidicolin, to cells treated with either ciprofloxacin alone or ciprofloxacin + UVA resulted in an accumulation of SSB due to the endo/exonuclease steps of excision repair. We have demonstrated that the FQ are photogenotoxic in mammalian cells but that FQ-pho-tosensitized SSB are efficiently repaired. Preliminary evidence that ciprofloxacin photosensitizes the formation of DNA lesions warranting excision repair may indicate production of more mutagenic lesions.     

Effects of high-energy-pulse-electron beam radiation on biomacromolecules

To study the molecular mechanism of high mutation frequency induced by high-energy-pulse-electron (HEPE) beam radiation, the effects of HEPE radiation on yeast cells, plasma membrane, plasmid DNA, and protein activity were investigated by means of cell counting, gel electrophoresis, AO/EB double fluorescent staining, etc. The results showed that the viability of yeast cells declined statistically with increase of absorbed doses. The half lethal dose (LD50) was 134 Gy. HEPE beam radiation had little influence on the function of plasma membrane and protein, while it could induce much DNA damage of single strand breaks (SSB) and double strand breaks (DSB) that were required for gene mutation. The G-value for DSB formation of HEPE beam radiation in aqueous solution was 5.7 times higher than that caused by 60Co gamma rays. HEPE can be a new effective method for induced mutation breeding and deserves further research in the future.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

Double-strand hydrolysis of plasmid DNA by dicerium complexes at 37 degrees C.

Significant effort has been made to develop synthetic metal complexes that hydrolyze DNA. Here we report a new dicerium complex, Ce(2)(HXTA) (HXTA = 5-methyl-2-hydroxy-1,3-xylene-alpha,alpha-diamine-N,N,N',N'-tetraacetic acid), which can hydrolyze DNA at pH 8 and 37 degrees C. This complex hydrolyzes DNA restriction fragments to give products with high regioselectivity, affording >90% 5'-OPO(3) and 3'-OH ends, like the products of DNA hydrolyzing enzymes. Ce(2)(HXTA) also hydrolyzes Litmus 29 plasmid DNA to afford both nicked and linear DNA. Analysis of the relative amounts of supercoiled, nicked, and linear DNA present show that there is one double-strand cleavage per ten single-strand cleavages, indicating that the linear DNA formed cannot be the result of two random single-strand cleavage events. The kinetics of nicked and linear DNA formation are comparable, both being associated with apparent first-order rate constants of approximately 1 x 10(-)(4) s(-)(1) for complex concentrations of 10(-)(5)-10(-)(4) M. These observations suggest that similar factors affect the hydrolysis of the first and second DNA strands and that cleaving the phosphodiester bond is likely the rate determining step in both cases. This is the first detailed study of a metal complex shown to mimic DNA hydrolases in their capability to effect double-strand DNA hydrolysis regioselectively at the 3'-O-P bond.     

Vacuum UV laser induced scission of simian virus 40 DNA

Abstract— The effect of vacuum UV-laser irradiation on Simian virus 40 (SV40) DNA I was studied in vitro. Following exposure of SV40 DNA I to an argon fluoride (ArF) laser (LD = 193 nm), single-strand breaks occur in either strand of the double-stranded superhelical molecule (DNA I) and convert it to the DNA II conformation. Upon increased irradiation times, additional breaks occur in already scissioned strands resulting in the production of smaller fragments as analyzed by alkaline sucrose centrifugation. These results suggest that short periods of irradiation lead to conformational alterations of SV40 DNA I.     

Strand breakage in poly(C), poly(A), single- and double-stranded DNA induced by nanosecond laser excitation at 193 nm.

Single- and double-stranded calf thymus DNA and two polynucleotides (0.4 mM) were studied in aqueous solution at pH approximately 7 using pulsed, 20 ns laser excitation at 193 nm. Monophotonic ionization of the nucleic acids is suggested from the linear dependences of the concentration of ejected electrons and the number of single- and double-strand breaks (ssb, dsb, respectively) on laser intensity (IL) in the range (0.2-3) x 10(6) W cm-2. The quantum yields of formation of hydrated electrons (phi e-) and ssb and dsb (phi ssb and phi dsb) are therefore independent of IL. In contrast, under 248 nm excitation these quantum yields increase linearly with IL under otherwise comparable conditions. Nevertheless, several effects and mechanistic implications are analogous using lambda exc = 193 and 248 nm. For polycytidylic acid, poly(C), in Ar-saturated solution for example, the efficiency of ssb per radical cation (eta RC = phi ssb/phi e-) is similar to the efficiency of ssb per OH radical (eta OH). For polyadenylic acid, poly(A), and single- and double-stranded DNA eta RC (lambda exc = 193 nm) is significantly smaller than eta OH. The ratio phi ssb (N2O)/phi ssb (Ar) is approximately 2 for poly(C), approximately 4 for poly(A) approximately 10 for DNA; the conversion of hydrated electrons into OH radicals in N2O-saturated solution and smaller eta RC than eta OH values in the case of DNA account for these results. For double-stranded DNA phi dsb does not depend on IL but increases linearly with the dose, indicating an accumulative effect of two ssb to generate one dsb. The critical distance for this event is 60-85 phosphoric acid diester bonds.     

CORRELATION BETWEEN CELL SURVIVAL AND DNA SINGLE-STRAND BREAK REPAIR PROFICIENCY IN THE CHINESE HAMSTER OVARY CELL LINES AA8 AND EM9 IRRADIATED WITH 365-nm ULTRAVIOLET-A RADIATION

Cell survival parameters and the induction and repair of DNA single-strand breaks were measured in two Chinese hamster ovary cell lines after irradiation with monochromatic UVA radiation of wavelength 365 nm. The radiosensitive mutant cell line EM9 is known to repair ionizing-radiation-induced single-strand breaks (SSB) more slowly than the parent line AA8. EM9 was determined to be 1.7-fold more sensitive to killing by 365-nm radiation than AA8 at the 10% survival level, and EM9 had a smaller shoulder region on the survival curve (alpha = 1.76) than AA8 (alpha = 0.62). No significant differences were found between the cell lines in the initial yields of SSB induced either by gamma-radiation (as determined by alkaline sucrose gradient sedimentation) or by 365-nm UVA (as determined by alkaline elution). For measurement of initial SSB, cells were irradiated at 0.5 degrees C to minimize DNA repair processes. Rejoining of 365-nm induced SSB was measured by irradiating cells at 0.5 degrees C, allowing them to repair at 37 degrees C in full culture medium, and then quantitating the remaining SSB by alkaline elution. The repair of these breaks followed biphasic kinetics in both cell lines. EM9 repaired the breaks more slowly (t1/2 values of 1.3 and 61.3 min) than did AA8 (t1/2 values of 0.9 and 53.3 min), and EM9 also left more breaks unrepaired 90 min after irradiation (24% vs 8% for AA8). Thus, the sensitivity of EM9 to 365-nm radiation correlated with its deficiency in repairing DNA lesions revealed as SSB in alkaline elution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.     

LASER FLASH PHOTOLYSIS AND PHOTOINACTIVATION OF SUBTILISIN BPN'

Abstract— Laser flash photolysis of subtilisin BPN'at 265 nm has shown that photoionization of tryptophanyl (Trp) and tyrosinyl (Tyr) residues are the principal initial photochemical reactions. The initial products are the corresponding oxidized radicals. Trp and Tyr, and hydrated electrons (e_aq) which react with the enzyme at: k (e_aq+ subt. BPN') = 2.1 × 10¹⁰ M⁻¹ s⁻¹. The photoionization quantum yield was 0.032 ± 0.005 at 265 nm, which was enhanced 3.5-fold by simultaneous excitation at 265 and 530 nm. The photoionization yields were unchanged by 3 M bromide ion and 8 M urea. which did affect the enzyme fluorescence excited at 265 and 295 nm. A similar lack of correlation between the effects of perturbants on the photionization yields and fluorescence yields was found for subtilisin Carlsherg. The results indicate that the monophotonic and biphotonic ionization of the Trp residues does not involve the thermally-equilibrated. lowest excited singlet state and that singlet energy transfer from Tyr to Trp does not contribute to Trp photoionization. The photoinactivation quantum yield was 0.014 for 265 nm laser excitation. which was not changed by simultaneous 530 nm excitation. The corresponding quantum yield was 0.009 for low intensity 254 nm radiation, indicative of a biphotonic contribution to photoinactivation. The results are explained by postulating that photolysis of Trp-113 leads to disruption of hydrogen bonding to Asn-117 and a shift in the primary chain sequence associated with the aromatic substrate binding sites. The photoionization quantum yields in subtilisin BPN'and subtilisin Carlsberg agree with a model based on the assumption that exposed Trp and Tyr residues contribute independently at intrinsic photoionization efficiencies characteristic of the chromophores.