Alternating current voltammetric determination of DNA damage

Chem -BtoL Interactions, 76 (1990) 111--128 Elsevier Sclentlhc Pubhshers Ireland Ltd

ALTERNATING CURRENT OF DNA DAMAGE

VOLTAMMETRIC

111

DETERMINATION

D KRZNARIC', B (~OSOVI(~", J STUBER b and R K ZAHN b~

•Center for Mamne Research Zagreb, Ruder Bo~kow~ Instztute, B~jen~ka 55, ~1000 Zagreb, {Yugoslav~ bInst~tute for Phys~olegwal Chemistry, University of Ma~nz (F R G ] and cAcademy o/ Science and L~terature, 6500 Ma~nz fFR G] and Laboratory for Mamne Molecular Bwlogy, Ma~nz and Yu 52120 Rown3 fYugoslavu~] (Received December 6th, 1989) (Revmlon received June 12th, 1990) (Accepted June 19th, 1990)

SUMMARY

The conditions for alternating current (a.c.)voltammetrlc D N A determinations have been investlgated with respect to its use wlth alkaline filterelution techniques at low D N A concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of D N A segments; peak II around -1.2 V caused by the native D N A (nDNA) form; peak III caused by denatured D N A (dDNA) at -1.4 V. Sonication of n D N A increases the peak current, however not with dDNA. Both d D N A and n D N A give linear peak current Increments with D N A increments, their regression lines cutting the concentration axis at the origin. In filterelution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of D N A . It turns out that an unknown substance, perhaps a protein or R N A , elutes from the filtersand glves rise to a current peak at about -1.3 V. This substance can interfere with the d D N A by competing for electrode surface area, since it diffuses much faster than the large molecules of the D N A Since however, d D N A has a higher affinityfor the electrode surface, after enough time, usually few minutes, the d D N A increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication,so that it will no longer Interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". W h e n E A is present at short accumulation times it prevents d D N A adsorption. By adding dDNA, the E A can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the D N A preAbbrevlatlons a c, alternating current, dDNA, denatured DNA, EA, ethanolamme, EDTA, ethylenedlamme tetraacetlcacld,nDNA, natlve DNA, VA, voltammetry, voltammetrm 0009-2797/90/$03 50 © 1990 Elsevmr Sclentlhc Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

112 sent. The conditions for voltammetric DNA determination have been investigated obeying the recognized Interactions. Avoiding organic bases and using Inorganic ones would simplify the determination procedure The reproducibility of the procedure in the range of 50--60 ng DNA/ml has been found to be __.6%.

Key words DNA damage determination -

DNA voltammetr,c determination -- DNA sonlcation -- alkaline filter elutlon of DNA -- EthanolamineDNA interaction

INTRODUCTION The actual environments of hv,ng organisms always contained, and by anthropogenIc influence additionally acquire, agents that bear considerable potential for DNA alteration [1,2]. By far the most frequent ones are single strand breaks [3]. One of the best methods for assessment of this kind of damage is by alkaline filter elutlon technique [4]. This technique requires the use of about 2 x l0 s cells/cm2 of filter surface. Deviations from these figures usually lead to bad results However, using these figures also implies that the DNA eluted from the filter comes off m concentrations changing over a wide range but always ,n very high dilution There are in the meantime quite adequate methods for DNA determination using fluorescent dyes [5]. In many cases it would be desirable to have more sensitive methods allowing for higher precision especially in the higher dilution range of the filter elutlon technique. Generally, electrochemical methods satisfy such requirements r a t h e r well. Investigations of DNA and other natural macromolecules by electrochemmal methods were extensively reviewed by PaleSek [6--9]. A.c. voltammetric measurements have been envisaged for possible use in filter elutlon techniques [10]. Among other questions the one of DNA measurement under the alkaline conditions prevailing in the elution system has been ventilated MATERIALS AND METHODS All chemicals were of the highest purity available. EthanolamIne came from Merck (Darmstadt, (F.R.G.)). All DNA used in the experiments was from Holthuma tubulosa males. It has been prepared by the method of Zahn et al [11]. It contained less than 1% of protein and RNA Measurements were carried out with a Polarecord E 506 (Metrohm Herlsau, Switzerland) in connection with a Metrohm Multi-Mode electrode (used as hanging mercury drop electrode}, an Ag/AgCl (3 mol/1) reference electrode

113 and a glassy carbon auxiliary electrode. The surface area of the hangingmercury drop electrode was 0.0041 cm 2 Throughout the experiments a Metrohm glass cell of 10 ml has been used. The solution was stirred with a rotating teflon coated magnetic bar Unless otherwise stated, the rotation speed was 3000 rev./min. After stirring, the solution was allowed to quiet down for 30 s prior to applying potenhal scan. Experiments were carried out at room temperature. The solutions were deaerated with nitrogen prior to measurements. Unless otherwide stated, all alternating current voltammetrm measurement were carried out at a frequency of 75 Hz and an amphtude of 10 inV. The phase angle was kept at 0 ° i.e. the in-phase component of the current was measured. The working solutions were prepared from 1 or 2 ml of the eluate sample, adding to it supporting electrolyte and making it up to 10 ml with quartzdistilled water. Two DNA stock solutions were prepared: (A) in "SSC" buffer: NaC1 0.15 mol/1, Sodium Citrate 0.015 mol/1, EDTA 0 02 tool/1 (pH 7); (B) EDTA 0 02 mol/ l, NaOH to bring it to pH 13. (C) (working solution) NaCI 0.3 tool/l, NaHCO 3 0.03 mol/1 (pH 8 9) (A) has been used to keep the DNA in its native state, while m (B) it was denatured. Final DNA solutions have been prepared by using 2 ml with the chosen amount of DNA. This is then brought to 10 ml with working solution, buffer C, if necessary along with the other desired components. THE ALKALINEELUTIONTECHNIQUE In this technique suspensions from cell cultures can be used as well as homogenates from organs. Essentially the technique of Kohn et al [4] has been followed with some modlhcatmns. In brief: The suspensmn finally is made up in buffer D (EDTA 10 mmol/1, NaHSO 3 5 mmol/1, KC1 1450 mmol/l, pH 7 4). This suspension at a density of about 1-2 mllhon cells is pumped at a speed of 0.2 ml/mln onto PVF filters (Millipore, Neu-Isenburg, F R.G.) m filter holders, always taking care of keeping the whole system free of air bubbles, followed by 5 ml of ice cooled buffer E (NaCl 140 mmol/1, KH2PO 4 1.5 mmol/1, KC1 2.7 mmol/1, Na2HPO 4 8.1 mmol/1, EDTA 0.53 mmol/1, pH 7 4) Subsequently, lyslng buffer F (Sodlum-Laurylsarcoslnate solution 2% (v/v), NaC1 2 molfl, EDTA 20 mmol/l, pH 10) is pumped at a speed of 0.2 ml/mm until a total of 4.5 ml have passed. This is then followed by 10 ml of a washing buffer G (EDTA 20 mmol/1, pH 10) at a speed of 0.2 ml/mm and finally by eluhon buffer H (EDTA 20 mmol/1, Ethanolamme (EA) to bring the pH to 12.3, which has to be prepared always fresh) at a speed 0.05 ml/mln until 18 ml have passed. It is collected in 3-ml fractions. The tubes are then flushed by soluhon H. These are the fractions, the DNA concentrations of which are characterizing the extent of DNA damage. In cases where the DNA concentratmn m all fractions is low, the

114 DNA has only few single strand brakes. The amount of DNA remaining on the filters is not considered m this investigation If, however, the DNA concentratmn m the first fractions Is h~gh and strongly decreasing towards the later fractmns, then damage must be h~gh. This behavmur may be modified ff DNA is cross-hnked to proteins that stink to the filter. The m e a s ur e m ent of the DNA concentration using fluorescent dyes poses some problems In those fractmns where the concentration m the eluted alkahne solutmn is very low -- and this coincides with those that had been marginally damaged only and whmh may be the most interesting ones, e.g. below 50 ng/ml ~t may become unmeasurable Even m the higher concentratmn ranges the quahty of the outcome depends among other points, on the temporal regime Th~s makes the measurements senmtlve to de~vatmns m timing of the different reaction steps. This point has been overcome by introducing automatm handling of the alkahne eluates Therefore the next effort is directed towards possible increase in DNA concentratmn de t e r m m at m ns using vol t am m et ry -

-

RESULTS AND DISCUSSION

The form of the potentzal-current relatwnsh~p of denatured D N A m 0 3 mol/l NaC~ 0 03 mol/l NaHCO s pH 8 9 Figure 1 gives typmal alternating current (a c.) voltammogram Two sharp peaks, one around - 1 . 4 V charactemstic of denatured DNA, peak III is m accordance with our previous paper [10] A smaller peak around - 1 . 1 V can be observed for both nDNA and dDNA and is ascribed either to a slow desporptlon of segments of DNA adsorbed mainly via sugar-phosphate backbone [7] or to the reormntatlon of the constituents of DNA [12]. This peak proved to be r a t h e r variable It has not been investigated any further A c voltammetmc compamson of native and denatured D N A zn ethanolamme containing solutwns Figure 2 demonstrates the dlfference in the potential vs current curves for native (A) and denatured DNA (B) dDNA was prepared by placing nDNA m a solution of the final composition: NaCl 0 3 mol/l, NaHCO 3 0.03 mol/1, EDTA 0.002 mol/1, ethanolamlne 0.002 mol/l (pH 12 3) for 10 mm This pH denatured nDNA. The pH has been lowered to pH 8 9. The same procedure was applied m the preparation of nDNA solution, except that nDNA was added at the end when pH was already 8 9 Native DNA gives t hr e e moderate peaks, denatured DNA two prominent ones. Theoretical speculation as well as expemmental observation (see later) implied the peak II of curve A being characteristic of nDNA, while ~ts peak III belongs to some denatured fractmn coming along with it Peak III ~s characteristm for dDNA Both peaks II and III from both curves can be used to derive a measure for DNA denaturation and DNA concentrations. Lmeamty

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Fig. 2 A c voltammetrzc curves for 350 ~g/l (A) native and (B) denatured DNA m 0 3 mol/l NaCI, 0.03 mol/l NaHC08, 0 002 mol/] EDTA and apprommately 0 002 mol/ (C2Hs)~NOH Accumulation time 2 min, accumulation potential - 0 8 V, pH 8 9

116 of p e a k I I I height and c o n c e n t r a t i o n of D N A is d e m o n s t r a t e d in p r e v , o u s p a p e r [10] By c o m p a r i n g the p e a k s I I I f r o m Figs. 1 and 2, it can be d e m v e d t h a t the p r e s e n c e of e t h a n o l a m l n e in Fig. 2 c o n m d e r a b l y lowers p e a k III. If increasing a m o u n t s of elutlon buffer ( E D T A 0.02 mol/l and E A to b r i n g p H to 12.3) w e r e added to the solutmn of c o n s t a n t c o n c e n t r a t m n of d e n a t u r e d DNA, p e a k III, c h a r a c t e r i s t i c for d D N A , d e c r e a s e s c o n s i d e r a b l y and b e c o m e s b r o a d e r . Without E A the s a m e e x p e r i m e n t does not show such an effect We thus conclude t h a t the p r e s e n c e of E A in a c o n c e n t r a t , o n d e p e n d e n t m a n n e r lowers the D N A d e p e n d e n t height of p e a k I I I A p e a k m a l k a h n e E A e l u a t e s a r o u n d - 1 3 V zs n o t d u e to D N A

Alkaline eluates with E A show often only a b r o a d p e a k a r o u n d - 1 . 3 (Fig 3, c u r v e 1) if to 1 ml of eluate, solution C is a d d e d up to 10 ml for m e a s u r e m e n t . In such a case the addition of small c o n c e n t r a t i o n s of internal s t a n d a r d d D N A i n c r e a s e s t h e p e a k at - 1 . 3 V, although less t h a n e x p e c t e d e v e n w h e n E A effects are t a k e n into consideration. This i n c r e a s e of - 1 . 3 V p e a k could easily be t h e r e a s o n for m l s j u d g e m e n t of this p e a k as b e i n g due to dDNA, especially since often no o t h e r D N A p e a k is o b s e r v a b l e u n d e r such condl-

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117 tlons. The dependence of such - 1 . 3 V peak height vs. amount of added dDNA is usually linear, however with a very small slope. Using this slope for extrapolation of the peak height without addition of internal standard would bring dDNA concentrations of 2--15 mg/1 instead of around 100 pg/1. Therefore this - 1 3 V peak is not attr:buted to dDNA The additmn of ,nternal standard rather superposes onto an unrelated preexisting peak. At the moment ,t is not clear why should the two peaks superpose at low additmns of DNA, since the standard peak III of dDNA is about - 1 4 V. Posruble explanation is that, at first, added DNA undertakes some sort of interaction, other than simple displacement, with the unknown substance adsorbed at the electrode resulting in an increase of peak at - 1.3 V. When all unknown substance is used up or simply starts to displace from the electrode, the standard peak III or dDNA starts to appear, With increasing accumulatmn time this - 1 . 3 V peak hrst increases and after 2--3 mm starts to decrease. This decrease may be attributed to competmon for adsorpt,on sites on the electrode surface. The peak probably comes from a substance passing through the filter m the course of the alkaline elutlon process along with the dDNA. The form and potentml of the peak point towards RNA [10]. Since dDNA adsorbs more strongly, probably existing however at a lower concentration and having a lower diffusion coefficient than the - 1 . 3 V peak substance, this will reach the electrode first. Yet with increasing accumulation times the DNA will displace it The same is observed ff dDNA concentration ~s increased. It is fortunate that the two peaks (at - 1.3 V and - 1.4 V) are suffic,ently separated once the peak III of dDNA appears. As can be seen from Fig 3, peak III height determination presents no difficulty. The interaction of the - 1 3 V substance w~th D N A

Proof that dDNA with increasing concentrations indeed displaces the - 1 3 V compound ~s m F~g. 3. A solutmn conta,nmg 1 ml of EA eluate made up to 10 ml with solution C gives one single prom,nent peak at - 1.3 V. With the addltmn of increasing amounts of dDNA th,s peak more and more decreases, while a new, a dDNA peak builds up around - 1 . 4 V, slgmfymg displacement of the umdentifmd - 1 . 3 V substance from the electrode surface. Substituting EA by NaOH m the alkahne elutlon buffer considerably increases the dDNA peak amplitude by factors of 5 - 1 0 w~thout ehmmatmg the interferences w:th adsorptmn competitors such as orgamc molecules in the filter eluates. Basically the alteratmn pattern of the dDNA peaks w,th increasing accumulation t,me ~s the same for the eluates with NaOH as w~th EA. In both cases the - 1 . 3 V and - 1 . 4 V peaks increase and at longer accumulation times decrease. More details are g~ven later. The onset of the - 1.4 V peak decrease occurs the sooner the more orgamc material other than DNA ,s involved including EA.

118

D~fferences among fractwns from the same filter elutwn Often, however, when filter eluates are measured, two peaks appear: the mentioned one around - 1 3 V and the standard peak III around - 1 . 4 V, charactemstm of dDNA. At high concentratmns of orgamc materml the peak III decrease can start already after short accumulatmn times, e.g. after 1 mm. This was often observed with fractmn number 1 of a filter eluate serms, whmh usually contains higher amounts of orgamc materml other than DNA. However, later fractmns of the same channel, containing less orgamc matereal, would give results with less interferences for the same accumulatmn t~me. It has been vemfied experimentally that by using short enough accumulatmn times (1 mm or less), expected values, as demved by other methods (fluorescent dyes}, may be approached more closely. The first fractions of different channels m filter elutmns qmte often ymld DNA values w~th high standard dewatmns. This Is paralleled by h~gh concentratmns of orgamc materml. Peak current dependence of d~fferent concentrations of added d D N A on accumulatwn tzme The interaction of the - 1.3 V substances and the peak III (around - 1.4 V) caused by dDNA has been discussed before. F~gure 4 gives another example for what can be detected by comparing different accumulation t~mes when h~gher dDNA concentrations are added to EA eluates. Since the - 1 . 3 V peak and peak III frequently supemmpose, addition of denatured DNA causing displacement of the - 1 3 V substance from the electrode, diminishes the - 1 . 3 V peak. This decrease however occurs faster, than the increase of the dDNA peak with the msmg DNA concentration, resulting m an overall decrease of the compound peak. Yet this hardly can explain the decrease of the compound peak to zero (Fig. 4, 4 ram). Here the effect of EA as the alkahmzatmn agent used m filter elutlon techmques comes into play. As demonstrated m Fig. 5 (curve 3) with short accumulatmn times, the DNA peak current m EA solutions, as occurring m filter elutlons, does not start to rose upon dDNA additmn unless more than 0.2 mg dDNA/1 are reached. Even then the slope is qmte shallow. Th~s is quite different with NaOH as alkah (Fig. 5, curve 2). Here the peak amphtude increase IS a steep function of the dDNA concentration m the solutmn, with the regressmn hnes intersecting at the origin. Practmally an ldentmal regressmn hne is obtained when no elutmn buffer was present m the solutmn (Fig. 5, curve 1) The effect of EA on the voltammetrm actlwty of dDNA may be explained by an anteractmn m solutmn resulting m a form that does not ymld peak III. Only after all free EA has been scavanged by dDNA, further dDNA addltmn gives peak III activity. The dependence of peak currents on accumulatmn times (F~g. 6) further illustrates that for short times the - 1 3 V substance within the first mm is increasing and then dropping to zero within the next three mm (curve 1),

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CONCENTRATION OF ADDED ONA ( p g / t ) F~g 4 Dependence of a c voltammetrm peak current (at - 1 3 V) on the concentratmn of added denatured DNA at varmus accumulation txmes DNA was added to a sample of 2 ml of eluate with (C2H6)4NOH (filled up to 10 ml of solution), 0 3 tool/1 NaC], 0 03 tool/1 NaHC08 (pH 8 9) Accumulatmn potentxal - 1 0 V

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CONCENTRATION OF ADDED DNA ( p g / I ) Fig 5 Dependence of a c voltammetrlc DNA peak III current on the concentration of added denatured DNA (1) 0 3 tool/1 NaCI, 0 03 mol/l NaHCO 8, (2) 0 3 molfl NaCI, 0 03 mol/l NaHCO 8, 0.004 mol/l EDTA, about 0.001 tool/1 NaOH, (3) 0 3 molfi NaCI, 0 03 mol/l NaHCO 3, 0 004 tool/1 EDTA, about 0 001 mol/1 (C2Hs)~NOH Accumulation time 0 5 rain, accumulatmn potential - 1 0 V

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6 Dependence o f a c voltammetrm peak at - 1 3 V (curves 1 and 3) and peak Ill of denatured DNA (curves 2 and 4) on the accumulatmn time 1 ml of alkahne eluate with (C2Hs)4NOH (filled up to 10 ml of working solutmn), 0 3 tool/1 NaC1, 0 03 mol/1 NaHCO 3 (pH 8 9) Accumulatmn potentml - 1 0 V Somcatlon time (1) and (2) 0, (3) and (4) 40 s Fig

while m the meantime after 2 mm starting from zero the dDNA peak is steadily rising (curve 2). When the whole sample has been somcated, the 1.3 V peak currents were lowered considerably (curve 3), the onset of the rose from zero of the dDNA peak current already started at 1 mm and was steeper (curve 4) This shows that the - 1 . 3 V compound is sensitive to somcatlon as is the dDNA, which is cut into smaller and faster diffusing ent~tms, so that it displaces the unknown compound faster from the electrode surface. Another posslbihty would be that the faster diffusing dDNA does not allow the unknown compound to accumulate to the same extent as the unsomcated dDNA does, since it Is diffusing so much faster Since peak III (curves 2 and 4) considerably shifts towards positive potentials with increased accumulation times, it is not clear whether it really corresponds to peak III charactemstm for dDNA or not. When however to the same solutions dDNA is added, then the peak m question rises, whmh slgmties that this peak corresponds to the one charactemstm for dDNA. Since these somcatlon effects have been observed m mixtures where different specms of orgamc molecules were present, they had to be repeated without such orgamc components, Le., with native and denatured DNA m solutmn C. -

121

Dependence of peak III current on sonwatwn t~me Two solutions of native and denatured DNA, m supporting electrolyte C, were sonlcated and peak III heights were compared to the ones before sorecation (Fig 7). dDNA shows only a neghgable increase, whereas nDNA gives large increases, which level off with increasing somcatlon t]mes. Obviously some of the native DNA gets denatured under the somcatlon t r e a t m e n t This increasing denaturation can be explained by the increasing number of frayed ends with increasing sclsslons of the DNA molecules. The effectivehess of sclssions on the other hand can be expected to decrease when the DNA molecules are getting shorter and shorter. On the other hand, peak II, charactemstm for native DNA, is increasing with sonicatmn time also (not shown here), conflicting with the Idea that more nDNA gets denatured and therefore the peak II should decrease A possible explanatmn can be seen m the idea that nDNA, which is rat her mgid, cannot adsorb so well to the electrode as dDNA. However, after sorecation, when double stranded nDNA molecules are getting shorter they can stack b ette r on the electrode surface. In addition, by being shorter their diffusion coefficients also increase. It is reassuring that both peak potentials do not change with somcation, mdmatmg that basically the same type of substance is responsible for the peaks. When DNA is denatured m the presence of EA by brmgmg it to pH 12.3 for 15 min and then back to pH 8.9, peak III currents, without as well as with 20 and 40 s of sonication, show ]dentmal Increases with increasing accumulation times, which are linear up to 2 min (Fig 8) This can be expected, ff essentmlly all DNA in the samples had been denatured.

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Fig 7 D e p e n d e n c e of relative i n c r e a s e of a c v o l t a m m e t r ] c D N A peak III h e i g h t (as c o m p a r e d to n o n s o m c a t e d solution) on t h e t i m e of s o m c a t l o n of solution 350 pg/l (1) d e n a t u r e d , (2) native D N A m 0 3 tool/1 NaCI and 0 03 tool/1 N a H C O 3 (pH 8 9) Accumulat]on potential - 0 8 V, accumulation time 1 m m

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A very small change m peak III hezght, as a consequence of different times of somcation, i.e., dzfferent molecule size, indicates that the molecule size has only a slight influence upon the a.c. voltammetrzc response A small increase m peak III height m Fig. 7. curve 1 is attr]buted to the increase of dzffusion coefhczent due to decrease of DNA molecule szze wzth somcatzon. Furthermore, this mean that the addzhon of internal standard to a sample of DNA whose size zs unknown should present no major problem According to our experzmental results, the error due to the different szze of DNA molecules in the sample and m the internal standard never exceeded 10O/o and m most cases was considerably less. The current dependence of peak II on accumulation times under somcatlon can be studzed (Fig. 9) by adding nDNA at pH 8.9 before somcahon as before. As can be derived from Fig. 9, both peaks II and III can be measured and both increase with increasing accumulation hines, peak II m a linear fashion up to about 1.5 mm and with dzmimshing slope at higher times, peak III growing hnearly beyond 4 mm. This mdzcates that the electrode has not yet been fully covered by the dDNA molecules which continue to add on, while nDNA molecules are displaced by the stronger adsorbing dDNA. For equal DNA concentrahons peak II for nDNA is about ten times lower than peak III for dDNA. Thus sonlcahon obviously produces only small amounts of denaturation, since peak III only grows less than 50O/o by somcatzon

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Fig 9 Dependence of a c voltammetrm peak III height (O, e, × ) and peak II height (E],LZX)on accumulation time 350 ~g/1 nDNA, 0 3 mol/I NaC1, 0 03 mol/1 NaHCOs, 0 002 mol/l EDTA and approximately 0 001 mol/t (C2Hs)~NOH(pH 8 9) Somcatmn time (O,[3) 0 s, (o,B) 20 s, (× ,A) 40 s Aecumulatmn potential - 0 8 V

The behawour of the charactemstw D N A peaks m filter eluate solutwns Before (Fig. 8) it has b e e n s h o w n t h a t t h e r e was no increase m t h e p e a k I I I c u r r e n t a m p l i t u d e b y s o m c a t l o n of dDNA. The situation ,s different m solutions with filter eluates (Fig 10). H e r e the p e a k I I I c u r r e n t s of d D N A show l a r g e i n c r e a s e s w , t h somcation times, whmh cannot s t e m f r o m an increase in a m o u n t of dDNA. As discussed earlier, a v o l t a m m e t r m p e a k at - 1.3 V found m filter e l u a t e s ~s r e p l a c e d b y p e a k I I I with increasing sonication times, m u c h in the s a m e w a y as if s o m e compounds, e.g. p r o t e , n or s o m e a g e n t s f o r m i n g c o m p l e x e s with DNA, would be d e s t r o y e d by the t r e a t m e n t . In the first case son,cation would a l t e r the compound so as to m a k e it less a d s o r a b a b l e and less c o m p e t i t i v e w~th d D N A for the a d s o r p t i o n sites. In the second case sonicatlon would b r e a k t h e complex, s e t t i n g d D N A free to a d s o r b to t h e electrode and to increase p e a k III. This r e a s o n i n g covers t h e e v e n t s for the first m i n u t e of s o m c a t i o n d u r i n g which t h e r e is a s t e e p rose in p e a k I I I height. Shallower i n c r e m e n t s m p e a k rme o b s e r v e d a f t e r p r o l o n g e d s o m c a t l o n m a y be the consequence of appreciable s h o r t e m n g of the D N A chains. I n t e r e s t i n g l y m sonicated alkahne eluate samples, a f t e r s t a n d i n g for 12 h or m o r e at p H 8.9 the p e a k I I I increase does v a m s h a l m o s t completely. This has to be i n v e s t i g a t e d f u r t h e r .

124

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SONICATION TIME (rain}

Fig 10 Dependence of a c voltammetric peak III height on the somcatmn time 1 ml of eluate with (1) (C~Hs)4NOH,(2) NaOH (made up to 10 ml of working solution), 0 3 mol/l NaC1, 0 03 mol/l NaHCO3 (pH 8 9) Accumulation potentml - 1 V, accumulation time (1) 3 min, (2) 1 mm

Two EA fdter eluate components may block D N A peak currents To an u n s o m c a t e d E A filter eluate internal s t a n d a r d d D N A ~s added and its peak I I I c u r r e n t plotted against D N A c o n c e n t r a t i o n (Fig. 11) A h n e a r r e g r e s s i o n hne e m e r g e s whmh i n t e r s e c t s with the c o n c e n t r a t i o n axis on the positive side (Fig 11, c u r v e 1) M a t h e m a t i c a l l y formal this means t h a t t h e r e exists n e g a t i v e c o n c e n t r a t m n s of D N A m the eluate sample. Chemmally ~t shows t h a t D N A is blocked m some w a y from s t a c k i n g to the electrode surface as long as its c o n c e n t r a t i o n does not s u r p a s s a crltmal value. A f t e r somcatlon for 3 m m {curve 2) a n o t h e r linear r e g r e s s i o n hne, r u n n i n g parallel to c u r v e 1 but shifted to considerably more negatlve values is obtained. P r e s u m a b l y by p r o l o n g i n g somcatlon f u r t h e r shifts could be reached, so t h a t t h e r e ~s no g u a r a n t e e t h a t the l n t e r s e c t m n value allows for the d e t e r m i n a t i o n of the t r u e D N A value. This m i g h t be achmved best by m t e r c a h b r a t l o n with o t h e r i n d e p e n d e n t m e t h o d s for D N A d e t e r m i n a t i o n (see later). If instead of an eluate, E A elutlon buffer is used m the same t y p e of e x p e m m e n t as before, the results as m Fig. 12 are obtained. Different somcatlon times do not change slopes or m t e r s e c t m n s of the d D N A c o n c e n t r a t m n vs peak I I I c u r r e n t r e g r e s s m n hnes (curves 1--3) slgmficantly H o w e v e r , due to the presence of E A the l n t e r s e c t m n with the c o n c e n t r a t m n axis still

125

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CONCENTRATION

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Fig II Dependence of D N A peak Ill current on the concentrahon of d D N A added 1 ml of eluate wxth (C~Hs)~NOH (made up to 10 ml of worknng solutlon). 0 3 mol/l NaCI, 0 03 mol/] N a H C O a (pH 8 9) Sonncahon time (I) 0, (2) 3 m m Accumulahon potential - 1 V, accumulahon hme 1 mm

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100 CONCENTRATION

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Fig 12 Dependence of D N A peak III current on the concentration of d D N A added (1,2,3) I ml of eluate buffer i e 0 02 mol/l E D T A and (C2Hs)4NOH up to p H 12 3 (made up to I0 ml of working solution), 0 8 mol/l NaCI. 0 03 mol/l N a H C O ~ (pH 8 9). (4) 0 3 mol/l NaC|, 0 03 mol/l N a H C O 3 (pH 8 9) somcatlon tlme (I).(4) 0 ram, (2) I m m , (3) 2 m m Accumulation potential - I V, accumulation time 1 m m

126 shows blocked dDNA below about 50 ~g/1 When, on the other hand, EA :s replaced by inorganic salt, this does not happen any more (curve 4) From this it may be concluded that in EA containing alkaline filter elutmns th er e are at least two components that can block dDNA, so that below critical concentrations they become undetectable by a c. voltammetry, i.e, the intersectmns of the curves of the type in Figs. 11 and 12 move to higher x-values. One of these components has been recogmzed (Fig. 5) as EA. The EA cannot be destroyed by somcatmn and its effects remain unchanged (Fig. 12). In an EA filter eluate sample the intersections are changed when somcated due to a second component Therefore, if DNA concentration in a filter eluate has to be determined, the following procedure should be followed: (a) the sample should be somcated (time of somcation depends on the type of apparatus used); (b) the intersection of the DNA additmn versus peak III current regression w~th the concentratmn axis should be determined for this sample, (c) the same regresstun should be estabhshed using the eluate buffer (without the eluate), (d) this (c) value should be subtracted from the (b) value to arrive at the DNA concentratmn Correlatwn of a~c voltammetmc and fluorescence measurements The 8 fractions from one filter eluate channel with EA were sonlcated for 3 mln and t l t r at ed with dDNA internal standards. The apparent DNA concentratlons were determined from the intersections of the regression lines with the concentration axis These concentrations were plotted against the fluorescence values obtained from ahquots of the same filter elutlons by the method of Erlckson et al. [5] (Fig. 13). Leaving out fraction 1 of the eluates, the correlation gave a coefficient of r 2 = 0.986. Theoretically the regression line: DNA fluorescence vs. voltammetrlc DNA concentration should not intersect the fluorescence axis at positive values, since negative concentration values are not possible. This comes from the DNA blocking effect of EA and the unknown orgamc components washed out from the sample by the elutlon buffer, as dlscused before (Figs. 11 and 12). Somcatlon of 3 min may be assumed to be sufflcmnt for dest roym g the blocking effect of the unknown substance This still leaves the EA which is insensitive to sonication and this causes concentrations to assume negative values. In order to arrive at a correctmn, the amount of blocking of EA has to be determined by measuring the EA elutmn buffer as before (Figs. 5 and 12). The change in measured DNA concentration due to EA has to be added to the values m Fig 13 to arrive at the corrected "real" DNA concentratmns (dotted hne in Fig 13). For this filter elutmn channel the fluorescence background value was 42, whereas the dotted line cuts the fluorescence axis at 47, which is in reasonable a g r e e m e n t This slightly higher voltammetric value may signify that a 3 mm sonIcatmn may not be enough to destroy

127 ~

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1

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I

I

I

J

70

80

90

100

; 110

I t20

FLUORESCENCE Fig 13 Dependence of DNA concentrahon, determined by a c voltammetry, on the fluorescence for the eluate fractions of one channel with (C=Hs)4NOH 1 ml of eluate sample (made up to 10 ml of working solution), 0 3 tool/1 NaCI, 0 03 tool/1 NaHC0 s (pH 8 9) Somcatlon 3 ram, accumulabon potential - 1 V, accumulation brae 1 mm Dotted hne is eorrecbon for the blank blocking effect of EA

completely the DNA blocking activity by the unknown orgamc compound washed from the filter. Figure 14 gives the regression for eluates with EA being replaced by NaOH buffer, thus rendering the EA correction unnecessary. The correlation factor m this case is r 2 = 0.944.

03

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0

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1 o

o

I

I

I

l

I

I

I

i

50

60

70

80

90

100

110

120

FLUORESCENCE Fig 14 Dependence of DNA concentration, determined by a c voltammetry, on the fluorescence for the eluate fraction of one channel with Na0H 1 ml of eluate (made up to 10 ml of working sohtlon), 0 3 mol/l NaC1, 0 03 tool/NaHCO 3 (pH 8 9) Somcatlon 2 mm, accumulation potential - 1 V, accumulation time 0 5 mm

128

Further conszderatwns In E A e l u a t e s a m p l e s w i t h 3 m m s o m c a t m n t h e h v e b d l t y , m t h e r a n g e of D N A c o n c e n t r a t m n s a r o u n d 59 to be + 6%. The strongest dewatmns from hnear regressmn, a l w a y s m f r a c t m n 1 of an e l u a t e c h a n n e l T h i s can b e l y s m g and w a s h i n g with l y s m g solutmn

fold o v e r a l l r e p r o d u c l n g / m l , h as b e e n f o u n d ff o c c u r m g a t all, Is avoided by increasing

ACKNOWLEDGEMENTS W e g r a t e f u l l y a c k n o w l e d g e t h e s u p p o r t of t h e I n t e r n a t m o n a l e s B u r o G e e s t h a c h t of t h e G e r m a n B u n d e s m m l s t e r m m f u r F o r s c h u n g u n d T e c h n o l o g m a n d of t h e C r o a t m n F u n d of S c m n t l h c R e s e a r c h I n t e r e s t REFERENCES 1 B N Ames, Identifying environmental chemicals causing mutations and cancer, Scmnce, 204 (1979) 587 -- 593 2 H F Stmh (Ed), Carcinogens and Mutagens in the Environment, CRC Press, Inc Boca Raton, FL, Vol 1--III, 1983 3 P Henson, The presence of single-stranded regions m mammahan DNA, J Mol Biol, 119 (1978) 487 -- 492 4 KW Kohn, L C Erlckson, R A G Ewig and C A Frmdman, Fractionatlon of DNA from mammahan cells by alkahne elution, Biochemistry, 15 (1976) 4629--4637 5 L C Ermkson, R Osmka, N A Sharkey and K W Kohn, Measurement of DNA damage in unlabeled mammahan cells analyzed by alkaline elutlon and a fluorimetric DNA assay, Anal Blochem, 106 (1980) 169-174 6 E Palecek, in Progress m nuclem acid research and molecular biology, m J N Davldson and W E Cohn (Eds), Vol 9, Academm Press, New York, 1969, p 31 7 E Palecek, in Electroanalysis in hygiene, environmental, chnical and pharmaceutmal chem lstry, m W F Smyth (Ed), Elsevmr, Amsterdam/Oxford/New York, 1969, p 79 8 E Palecek, Modern polarographlc (voltammetric) techmques m biochemistry and molecular biology Part II Analysis of macromolecules, m G Mllazzo (Ed), Topms in Bloelectrochemistry and Bloenergetics, Vol 5, John Wiley and Sons Ltd, 1983, p 65 9 E Palecek, Electrochemical behaviour of bmlogical macromolecules, Bioelectrochem Brae nerg, 15 (1986) 275--295 10 D Krznarl6 and B Cosovi6, Alternating current voltammetric determination of DNA con centratmns at a microgram per hter level, Anal Bmchem, 156 (1986) 454-462 11 R K Zahn, E Tmsler, A Klemschmldt and D Ling, Em Konserwerungs- und Darstellungsverfahren fur Desoxyribonucleinsauren und lhre Ausgangsmatermhen, Biochem Zeitschr, 336 (1962) 281- 298 12 P Valenta, H W Nurnberg and P Klahre, Electrochemmal behawour of natural and biosynthetic polynucleotldes at the mercury electrode III A comparative study on the electrochemmal properties of native and denatured DNA, Bloetectrochem Bioenerg, 1 (1974) 4875O5