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Modulation Polarography of DNA and Some Types of RNA
Bioelectvocl~ernistry
Modulation
am? Bioenevgetics
Polarography
Energy
Research
(1976)
of DNA and Some Types of RNA *
by G. C. BARKER 0 and Atomic
3, 373-39’2
D.
~ICKEOWS
Establishment,
Harwell.
Didcot,
Oson,
England
The intermodulation signals produced by various poI_ynucIeotides have been studied with a 2 MHz Modulation Polarograph and, in some cases, compared with the corresponding signals observed using various modes of the HARWELL Multi-mode Polarograph. For denatured DNA the signals observed at the negative end of the adsorption range for a slightly alkaline solution seem to be non-faradaic in origin and caused by effects connected with the exchange of segments of the partly adsorbed molecules between the double layer region and a solution lamina containing those parts of the adsorbed molecules that are not in intimate contact The exchange of segments seems to be diffusionwith the interface. controlled even at a MHz and an analysis of the low and high frequency data indicates that the DNA system behaves as though the molecule were divided into independent subunits of molecular weight ca. 3000 as regards the exchange of molecular segments between the interface and . the solution_
introduction The behaviour of native and denatured DNA in acid media is fairly well understood as a result of work in several countries, i-5 but the behaviour of nucleic acids in general in slightly alkaline media is a more contentious In particular there is uncertainty about the origin of a signal topic. revealed by a.c. polarography close to what appears to be the negative end of the adsorption range for single-stranded DNA at the mercuryIn the present work we have attempted to aqueous solution interface. identify the cause of the signal mainly by comparing the analogous signal observed in 225 Hz square wave (S.\V.) polarography with the corresponding intermodulation signal (of a special type) for denatured DNA polarograph (H.F.M.P.). 6 The befound using a 2 MHz modulation
Jiilich,
* Presented at the 3rd International 27-31 Oct. 1975.
0 Present
adress : School
of Chemistry,
Symposium Bristol
on Bioelectrochemistry. University,
Bristol
374
Barker and McKeown
haviour of some tvpes of RNA also has been investigated though interpretive efforts tena to have been focussed on data for denatured DNA in slightl>- alkaline solution_ The S.W. data w-e;-e pi-ovided by the S-IV_ mode of a modern multi-mode polarograph (M.M.P.) and supporting data needed in the analysis of S.11’. and H.F.3I.P. results lvere provided b>- the radio-frequeneq(R-F.), square wave intermodulation (S.1V.I.P.) and linear scan \-oltammetric (L.S.V.) modes of the 3I.JI.P. 5 Data also were provided by the normal derivative and the integrated -derivative mode of the HARWELL t>-pe of pulse polarograph (P.P_)_ To a large extent it has been possible to avoid massive system non-linearity caused by large surface coverage. an important point when studying srgnals which are influenced in magnitude, and conceivably also in potential dependence, by strong solute adsorption and interactions between adsorbed entities_ This feature of the work is a direct consequence of the a\-ailability of novel instruments of high sensitivity.
Experimental The H.F.M.P. n-as the instrument used previously; 6 and it gave recordings showing the dependence on the mazt~ potential of a special dropping mercury electrode (D.M.E.) of the output voltage of the H.F.M.P. during a potential scan (cathodic, 0.4 1’ amplitude). synchronised with drop growth and starting 4 s after drop detachment. The output \-o!tage of the instrument (apart from small progressive changes in sensitivrty connected with drop growth durin g the potential scan) was a linear measure of the derivative (with respect to mean D.M.E. potential) of the series resisti\-e component of the interfacial impedance at 2 MHz. Thus indirectly one is able with a H.F.N.P. to study the potential dependence of one important part of the interfacial impedance at a frequency verylarge compared with the frequencies used when bridge methods are emplo>-ed. Thi s is made possible because the unusual t>-pe of intermodulation encountered with a H.F.M.P. produces a low frequency signal (~5 Hz) after demodulation which can be likened, in the contest of sensitivity, to the 10~~ frequencr signals that arise when choppers are used in the measurement of minute kc. cur-rents or voltages. The scan of the H.F.M.P. w-as of 6.6 s duration and its starting potential w-as adjusted manually (IO mV steps) with respect to a S.C.E. During the scan the D.X.E. was invariablypolarized simultaneously by a S.\V. voltage of 31 mV amplitude (peak-peak) and an unmodulated 2 MHz current of 7 mX amplitude_ A low inductance cell was used which contained a specially-constructed D.BI.E. providing, as a result of the conicall>r-tapered tip, a high degree of symmetry as regards current flow at 2 XHz in the solution surrounding the mercury- drop and also by- means of a contact to the mercur; thread at a point between the 0.1 mm capilIar>- to which the drops Lvere attached and a length of fine bore (0.04 mm) capillary to control Hg flop- (o-54 mg s-r), a low impedance connection
Modulation
Polarography
of
DX_1
and
Some
Types
of
RSX
375
to the mercury drop. The tip of the D.M.E. was hand-ground to obtain the requisite taper estending almost to the orifice of the capillary tubing. X high degree of symmetry for the D.M.E. solution is vital if nonfaradaic srgnals connected merely with the potential dependence of the capacitive part (series circuit) of the interfacial impedance are not to influence appreciably the recorded output voltage of a H.F.M.P. As in earlier \\-ork the instrument was made relatively- insensitive to such signals by making the cell circuit series resonant at 2 MHz. (see Refs. 6n and 6b). All measurements were made at ambient temperature (IS-ZOOC). An increase in recorder reading invariably indicates an increase in the value of the derivative of the interfacial resistive component Ri”, with respect to the mean potential of the D.M.E. The instrumental sensitivity was the same for all recordings given later and unit change in recorder reading denotes a change in the Lrariation in I?;,, (peak to peak) produced by the a fore-ment ioned 31 mV S.\V_ of 7-1 x IO-" !2 at the point on the recordings at which denatured DNA produced large signals connected with segmental desorption-adsorption (vi& ix&z)_ The root mean square variation in reading from trace to trace (5 scans) usually was ca. 0.5 divn. but \-ery occasionally was enhanced by radio interference. Low frequency data for the various poly-nucleotides were provided by the H_-\Ru-ELLP.P. (1953 lvintage) and by the S.\V. and L.S.V. modes of the M.M.P., 7 some use also being made of the RF (72 kHz) and S.1V.T.P. modes of the latter instrument_ Space considerations make it impossible to report the low frequency- data in detail in the present paper. They will be dealt with more fully in a later publication. Calf thymus DNA (i\lLLES Labs.), higIzZ)l ~oZyr~zerized RN-1 (E. Coli) t-RN_\ (E. COG) and Yeast RNX (ail from BDH Labs., Poole, England) were used at concentrations not esceeding 50 pg,km3 in the polarographic work. Materials to be used in wztixx form were first dissolved slowly- in 0.15 111NaCl, 0.015 41 Na Citr. Denatured materials were obtained by dissolution in a ro-fold more dilute chloride-citrate solution, heating to I00 OC for 30 min.. follo\ved by rapid coolin g to o OC and subsequent mild refrigeration.
Results and discussion
n. Pulse polarograph data Fig. I is fairly typical of the results for all the types of RNA studied when the concentration of the polynucleotide is 50 pg/cm3 and the medium is I AJ KC1 made slightly alkaline (pH z S-3) by making the soluFig. I shows the potential depention 0.1 111with respect to Na,HPO,. dence of the differential capacitance or pseudo-capacitance indicated by deri\-ati\re (with integration of the current signal prior to measurement) pulse polarography when a negative pulse (35 mV size) is employed.
Barker
3’76
and NcKeown
Here we are mainly interested in the effect of the nucleic acids at potentials more negative than cn. -o-g V (here and Iater US. S.C.E.) and it will suffice to point out that for all forms of RNA there seems to be a broad but small pseudo-capacitance peak centred at about -1.1 V which may conceivably be due to some reorientation of the adsorbed nucleotide molecule (largely single-stranded for all the types of RNA used in the present work). Of greater interest in the contest of the present paper are the much sharper pseudo-capacitance peaks observed for all t_ypes of RNA in the vicinity of -1.37 V. (It should here be noted that the P.P. data refer to an elapsed time since drop detachment of z-7 s and that there are small variations in the position of the sharp capacitance peak with nucleic acid concentration and with the nature of the nucleic acid)_ The sharp capacitance peak is not well resolved with respect to the broad but smaller peak that usually is observed at CK -1.1 V but at negati\-e potentials it seems to decay rapidly. Thus for ~zntive (implying here - as recei\-ed) ribosomal RNA from E. Coli the results in Fig. I suggest l-irtually total desorption of the polynucleotide at potentiaIs negative with respect to -1-15 V.
U (Vvs SCEI’ 1
-14
,
I
-1.0
1
-0.6
,
I
-0.2
Fig. I. Capncito,gams for native ribosomal RX-1 in I 31 KCI, 0.1 df Sa,HPO,.
The effect of denatured calf thymus DKA for the same concentration and medium closely resembles that seen in Fig. I. for E. Coli RNA withe the esception that the qhegntizfe pseudo-capacitance peak for DNA occurs at -1-47 V (after applying a correction for the influence of pulse sense and size) and consequently is better separated from the broad pseudo-capacitance peak which still is centred at crs. -1-1 V. The latter peak has been attributed by V_ALEXTA and NCRSBERG 3b to reorientation of adsorbed solute.
Modulation
Polarography
of DNA
and
Some
Types
df RNA
3f7
_ -_- :.. b. alulti-mode polarograph data The most surprising feature of the Iow frequency---data w& the large conductance peaks observed using the S.W. mode of--the, M.M.P; for all the polynucleotides (except native DNA) in the usua- slightly alkaline medium (I _U KCl, 0.1 AI Na,HPOa). These petis may -well prove to be of value in the detection and determination’of singlystranded nucleic acids at levels below I pg/cm3. Fig. 2 shows the quite sh$rp peaks for denatured DNA4 at the concentration IO, 30 and $0: pg/cnP when the instrumental sensitivity was low and the- SW. amplitude-Gas 32 mV peak to peak. The conductanc_e peak resembles that produced by
60-F .1 -
Instrument
U(Vvs -1.10
-1.20
-1.30
-1.40
SCEI
-1.50
Fig. 2. Square wax-e polaro,-ms (S.\\r_mode of XXP.) for denalrrredcalf thymus DNA in I .&I KCI, o-r &i Ns_HPO, : 3~
mV square wave. min. d.c. sensitivity, max./S a.~. sensjtivity.
a z-electron
faradaic reaction as regards width and lack of dependence of position on concentration. Also for concentrations up to 20 pg/cw, the size of the conductance peak is linearly related to polynucletitide concentration. Then, of course, for the drop age in question the-surface concentration just before the conductance peak (remembering that-the S.W. polarogram refers to signals observed during a 6 s duration- 0.5 V I potential scan starting 4 s after the start of drop growth) is-expected to be linearly related to concentration from the work of VALESTX and NijRx~ BERG. 3db The corresponding apparent conductance observed using -the normal derivative mode of the P.P. is about 20 times smaller after making allowance for the differences in flow rate and drop age. This -decrease in conductance (in the P.P. case the average value for the elapsed time range 0.02-0.04 s), which makes pulse polarography somewhat unsuitable- for the detection of DNA (and other pol_ynucleotides also) in alkaline media,
37s
Barker
and
SlcIieown
is much larger than the corresponding decrease for a normal faradaic reaction (reversible or irreversible) uncomplicated by reactant adsorption. It will become evident later that the decrease is a consequence of a close approach to equilibrium as regards segmental desorption at the interface when the elapsed time after perturbation of the potential exceeds 0.02 s. The results reported for denatured DNA by VALESTX and NCRSBERG 3b indicate that at low frequencies the equivalent circuit for the interface is large must be in the potential range in Lvhich the S.\V. conductance dominated as regards the impedance which shunts the double layer capacitance by an adsorption-desorption capacitance. The latter component is not dependent in value on frequency and can be equated approsimate!y \vith the pseudo-capacitance obserl-ed on puke capacitograms I. Ho\ve\-er, it also can be seen from the large of the type seen in Fig. x-alues of S.\Y_ conductance found for denatured DN_\ (and also for the various t>-pes of RX_&) that the circuit limb w-hich shunts the double lax-er capacitance (infinite frequenq-) must also include resisti\-e elements which become relatively unimportant at large \-alues of elapsed time though of paramount importance in producin, m the peak on the S.\V. polarogram \\-hen the recording refers to repetitil-e measurements made at an e!apsed to suppose that these resisti\-e eletime close to 2 ms. Tt is reasonabIe ments are in some wa\- connected with mass transfer of nucleic acid in the solution close to tge electrode surface. Thus in what folio\\-s it will be implicitl>- assumed, partly because of the big difference betlveen the S.11’. and P-P_ conductances (normalized to constant drop area and conbe accounted for in terms stant elapsed time) \vhkh cannot satisfactoril\of an effect caused by the use of a fast cathodic potential scan in the M.3I.P. esperimcnts and a ver>- slowlchanging scan in the P.P. work, that the sharp S.Ii’_ conductance peak for denatured DNA is basically in double ia3-er non-faradaic in origin being caused by slow changes structure which inevitably accompan)adsorption or desorption of nucleic acid following a sma!l potential changeThe S-IV. peaks thus are assumed similar to the adsorption-desorption (tensammetric, dcsad) peaks observed with solutions containin, m strongladsorbed organic solHo\\-e\-er, usualI>- decad peaks utes such as long chain aliphatic alcohols. for relatil-ely simple organic molecules, if large and narroxv. are appreat which the ciabl>- dependent on concentration as regards the potential That the latter feature is not evident apparent conductance is largest. in the case of a nucleic acid peak at first encourages one to think that the peak is of faradaic origin but such a view can be totally incorrect as it is a thermod>-namic requirement in the case of very strong solute adsorption from a solution containing little of the solute that, for lox surfze covernge. the transition from adsorption to no adsorption at the D.M.E., when the will occur in a potential range potential .is rapidly made more negatil-e, If the latter state of affairs independent of the solute concentration. esists inevitably the signals observed in KC. polarography or S.\I’- polarography will resemble faradaic signals as regards their invariant position on the potential ask. It is. however, very seldom that potentialindependent non-faradaic waves are observed for simple organic solutes
Mociulation
Polarography
of DNA
and
Some
Types
of RNA
379
of quasi-a or 34imensional shape as normally the signals for slight surface coverage are smaI1. partly because the adsorption coefficient for the solute is not markedly potential dependent for low surface coverage_ For the nucleic acids the results of many investigations suggest that at negative potentials the adsorbed molecules are quite rapidly desorbed in a rather small potential interval as the potential becomes more negative. Thus large SW. peaks are not so remarkable and one expects from studies of the P-P., S.AV. and H.F.M.P. signals produced by the nucleic acids to learn something about the kinetics of desorption, and the effective size of the segments of the molecule which become attached to, or detached from, the interface when the potential is slightly changed. In what follows we make use of the concept of segmental desorption-adsorption which has been employed by MILLER * and by other workers S-lo when discussing the electrochemistry of polyelectrolytes or polynucleotides. Septenfnl
desor$tiosmzdsorpfion
(S D-A}
It can be shown that provided at the electrode-solution interface the adsorption-desorption reaction namic viewpoint, as a special type is, for a simple solute, possible to
adsorption and desot-ption of a solute can be regarded as indivisible steps can be thought of, from a thermodyof charge transfer reaction. Thus it write for the non-faradaic reaction rr
S -+ n,e- f
S,d
(a)
whet-e S is the solute, Sd is the adsorbed form of S and n, is the number of electrons supplied to the electrode at comtant potential when one molWe assume that the surface covecule of S is adsorbed at the interface. erage is small and that, in the supposedly small potential interval in which the transition from strong adsorption to little adsorption is mainly concentrated, parameter n, is not markedly potential dependent and can the influence of be regarded as a constant indicatin, * fairly quantitatively S& on the double layer structure. In reality n, for simple organic solutes is quite likely to be markedly potenrial-dependent if the low surface coverage requirement is satisfied, but occasionally it is fairly constant at the extreme end of the potential range in which an inorgamc OP organic solute is adsorbed. Parameter n, has a crucial effect on the desad impedance at high frequencies and its evaluation is essential in the analysis of impedance or Two methods can be used_’ The conductance data for desad systems. first is to study the potential dependence of the zero-frequency adsorption capacitance ‘for low7 surface coverage an6 constant solute concentration This capacitance Cd is defined appro_xiin the solution at the interface. mately by
(1)
Barker and NcKeown
3so
if n, can be treated as a potential-independent parameter and K is a constant. This equation cannot easily be appbed to nucIeic acid data but an analogous equation involving the S.W. conductance in place of Cod can be employed if the frequency is sufficiently large for the measured conductance to be largely diffusion-controlled_ This version of equation (I) can be applied to conductance data for the shoulders of conductances peaks such as are reproduced in Fig. 3. Ntematively n, can be calculated from measurements of the width of the S.W. conductance peak at half the maximum value making use of the approsimate expression for the measured conductance Gs D-A kP &D-A
=
(I +P)'
where k is a constant and P=esp
(u--uJn,P RT
Again it is assumed that S D-A is diffusion-controlled in the S.W. case (this is not a strictly justifiable assumption for a S.\V. frequency of 335 Hz, but it is one which must be made for reasons of simplicity in the first analysis of esperimental data)_ U, is the potential at which Gs D-A is largest and can be roughly identified in the S-W. case with the potential at which approsimately half the denatured DNA adsorbed prior to the potential scan, and during the earlier part of this scan, has been segmentally desorbed from the Interface. In the case of simple adsorption-desorption (e.g. the specific adsorption of I- on mercury) the phy-sical significance of n, is clear if equation (I) is employed_ It represents, if appropriate esperimental conditions have been employed, the number of electrons supplied to the electrode at constant potential per iodide ion adsorbed. The application of equation (I) to the corresponding pseudo-capacitance data for denatured DNA at the negative end of the adsorption range (if accurate data were available) would without any doubt lead to a value for n, close to -2. However L.S.V. data for the same system, although not of great accuracy, suggest that the double layer charge-density for the potential region in question changes by about -0 .a electrons per base adsorbed at constant potential (here ruse must be made of L.S.V. data for slightly acidified media as well as those for the pH S.a medium). A comparison of these electron transfer numbers, taking account of the monomeric molecular weight. leads to an apparent molecular weight for the process of segmental desorption-adsorption of the order of 3000, a value very much smaller than the true molecular weight of the single-stranded nucleotide (in escess of 10~). This difference highlights the difficulty of applying conventional thermodvnamic equations to systems containing molecules of great length (possibly also to molecuIes that are large in more than I di-
Moduiation
Polaroeaphy
of DKX
and
Some
Types
of RX-1
381
mension such as cytochrome c)_ Clearly in the case of a long molecule whether or not a small segment is adsorbed at the interface is not influenced by the state or energy level of segments of the molecule some distance away from the segment in question_ In other words, the molecule will behave as though it were composed of independent subunits of much smaller molecular weight than the true molecular weight of the polynucleotide. The apparent molecular weight mentioned above points to segmental desorption of subunits whose Zen&b on the average is similar in magnitude to the diameter of the nucleotide with the bases stacked_ Following earlier workers such as MILLER 8 it may be assumed that, as the potential becomes more negative, complete desorption of the denatured DNA molecule is preceded by segmental desorption-adsorption, leading to partial release into the solution of adsorbed material which at more positive potentials had been in intimate contact with the electrode. The material released, one imagines, will be largely confined in a solution lamina bounded by the interface and it is reasonable to suppose that the release of segments will be accompanied by a gradual increase in the thickness of this lamina, the thickness tending to infinity when adsorp- tion ceases completely_ For the denatured DNA system the S-IV. conductance falls away rapidly at potentials positive with respect to U, and it is probably not far from the truth to assume virtually no segmental desorption at potentials more positive than -1.3s V. For this potential range we envisage thus only signals produced by slow reorientation. 3b Converting the above model into an electrical equivalent circuit, making use of arguments used earlier rr but introducing the new concept of an e/jective molecular weight for the segments eschanged between the interface and also the above-mentioned solution lamina of varying thickness, one arrives at the equivalent circuit for the S D-A part of the interfacial impedance shown in Fig. 3. In this circuit it is implicit that segmental desorption-adsorption proceeds reversibly at the boundary be-
I
TTTTT
Fig. 3_ Equi\-alent electrical circuits for the S D-_A part of the interfacial impedance ; a aperiodic circuit, b reduced form of the circuit at high frequency when the S D-X impedance becomes R,(r + I/?>, see ec,uation I.
tween the double layer and the solution lamina. No allowance for eschange of segments of molecules which in the S D-A reLgion are totally unadsorbed between the solution lamina and the bulk of the solution is made in the equivalent circuit, largely because when the S.1V. mode of the M.M.P. is employed such segments contribute little to the effective concentration of segments within the lamina. It is assumed that the se,o;ments not in
3%
Barker
and 3Ic Iieown
intimate contact with the interface move within the solution lamina which contains them by linear diffusion_ This questionable assumption is permissible for the purposes of semi-quantitative data analysis. The equil-alent circuit in Fig. 3 differs onlr in structure from the circuit foi- normal adsorption-desorption with mass transfer of the solute by linear diffusion in that diffusion is limited to the solution lamina and is described in the S D-A circuit b>- the motion of charge in a transmission line of fizzite instead of semi-infinite length. C, in the circuit is the capacitance connected with the dependence of the double layer charge component connected with the presence of adsorbed segments on potential for constant segmental concentration in the solution at the interface. It is thus a special Q-pe of adsorption capacitance. The pseudo-capacitance, which in principle can be measured, is to be identified loosely with the series combination of C, and the shunt capacitance of the section of resistive transmission line. At high frequencies if the lamina thickness (line length), n, is not too small the resisti1.e part of the input impedance of the S D-A circuit becomes
(4) where CL)is the angular frequent>-. likewise and then the resistance are gi\-en by l 1
R, refers to unit area if the circuit does and capacitance per unit length of line
J&L = _!?TIL’ F” C, D,
(5)
and C
7-L.-
n,“P’C,
RT
(6)
\vhere C, is the effecti\-e concentration for the diffusing segments in the solution lamina and D, is the relevant diffusion coefficient. From equaeschange of tions (4). (5) and (6) it follows that, for diffusion-controlled segments betiveen the solution lamina and the interface, at high frequencies the resisti\-e component of the S D-_-X impedance should be approsimateI!- given by
RT
(7)
The concentration C, presumably will at first increase progressivelv .. --_ in the S D-A region as the mean potential becomes more negative but ultimatel>it will fall steadily in \-alue due to the progressive increase in parameter rl. It should be mentioned that the peak observed on the S.\V_ polarogram, with denatured DN:\ present, did not change in size when the
Modulation
Polarography
of DK_\
and
Some
T_vpes qf RX-1
353
amplitude of the potential scan was reduced to 0.2 V, provided the starting potential was adjusted so that the masimum S.!V. conductance occurred at the same value of elapsed time. This simple but. somewhat unespected, fact becomes comprehensible if allowance is made for (n) the fact that DNA adsorption from the bulk of the solution in the S-IV. experiments remained virtually diffusion-controlled until the potential entered the potential range in which S D-X is largely concentrated and (b) the fact that equilibrium as regards segmental desorption in the S D-A region is established with a time constant of the order of 0.01 s. The latter state of affairs results in a value for C, at the point of masimum S.\V. signal which is virtuall~~ independent of the rate of change of mean potential proxrided that this is not such as to pass through the S D-A region in a time less than 0.1 s. Although at first sight this lack of a dependence of S-IV. signal on scan rate seemed to point away- from a non-faradaic origin for the signal (here it should be noted that theory in the case of simple adsorptiondesorption peaks produced b>- a solute of normal size predicts that both the size and position of the desad peaks for low surface co\rerage should vary- with the rate of change of mean potential), it is in reality strong evidence for the occurrence of seamental desorption in which on157 parts of the adsorbed molecules are released from the interface in a region lying between diffusion-controlled adsorption from the solution and no adsorption of the DX.1 strands at the electrode surface. Xot unespectedly the S.\\‘. conductance \vas much smaller if an anodic potential scan was employed in place of the more usual cathodic scan. This reduction is connected with the suppression of accumulation of DNA at the interface prior to and durin g the early part of the scan when starting from a potential just outside the adsorption range.
If the circuit in Fig. 3 applies, it is found that the intermodulation signal produced by reversible S D-A should vary approsimatelr u-ith mean potential according to the derilwative of P/(I t I?)% with respect to the potential. Thus the wax-e produced b>- S D--A on the H.F.M.P. polarogram should resemble in shape to the second derivative of a normal n.c. polarographic \vave and should change sign when the potential is very c&e to c/‘, for the S.IV_ conductance peak. In practice the behaviour of denatured DNA and of the various types of RNA conforms to these espectations fairl; \ve!l but quite large H.F.XP. signals also are observed on the positive side of the S D-,1 region that presumably are connected with *the =Joested b\- \'_ALEST.A and ~\'~;RsBERG.~~ These reorientation process subb signals we shall discuss elsewhere.n. Denatured calf thymus DN..X for denatured DX-X at the concenFig. 4 shows results obtained Fair linearity trations I0 , 30 and 50 pg/cm” in the usual pH S-2 medium. as regards the dependence of the intermodulaticn signal on concentration
Barker and AIcKeown
3%
is observed throughout the studied potential range. To within the experimental error the null points for the signal vary little with concentration, the variation of the null point for the S D-L% region being connected with the uncertainty in starting potential of as much as IO mV. The S D-A
40 I
-1.30
-1.10
Native DNA (Calf Thymus) 20
Blank
50yg/cm3
0
U (KS SCE) I
-1.10
I
I
-1
30
I
I
-1.50
Fig. _I_
H.F.M.P. results for denat~w~d calf thymus DNA ; 31 mV square wave. max./4 instrumental sensiti\-ity ; medium r M 1X1. 0.1 31 KL_HPO,.
wax-es estend
from about -1.30 V to a potential approaching at least and ultimately the various curves merge into one another at the most negative potentials indicatin g the ce.ssqGon of DNA adsorption. The waves are noticeably asymmetric with respect to the null point at about -1.47 I’, an effect not connected with large surface coverage but
-1-55
V
presumably connected indirectly with the variation with potential of the lamina thickness n and the influence of this variation on the effective se,mental concentration within the lamina. b. Ribosomal RNA (E. CoZi) The results for highly polymerized RNA from E. Coli in vaative (as received) and denatured form are given in Figs. 5 and 6. Apart from the movement of the S D-A wave to a more positive potential the curves resemble the correspondin g 50 pg/cm3 curve in Fig. 1. For both forms of the 25. Coli material, the S D-A signal changes sign at ca. -I.# V which is virtually the potential at which the pseudo-capacitance (Fig. I) for the ~~rnir_Je material is largest. (a correction for pulse height and sense must be applied to the Fig. I results). For both types of E. Coli RNA the masimum value of S.\k_ conductance is observed at a potential of -x_#g V and the agreement of-this value with the potential of the H.F.M.P. null point suggests that signals due probably to reorientation have largely disappeared in the region of segmental desorption-adsorption. The results for this material su ggest that RNA adsorption persists to more neg-
Modulation
Polarography
of DNA
and
Some
Types
bf RNA
3%
ative potentials than might be thought from a study of the variation of the S-W- conductance with potential. Thermal denatnration of the E. Coli RNA had little effect on the well-defined S.W. peak as regards position or size, but seemed to produce a small but detectable change in the size and shape of the H.F.M.P. wave for S D-A. The results i: Fig. 5 and 6 suggest that this wave exhibits asymmetry akin to that found for denatured DPU’A. 60-
5oJJg/cn?
Bknk ,
0
5.
H.F.XP-
ditions
Fig.
, Denatured
-1.30
DNA ( CalF thymus
results for native ribosomal RNA as for Fig. _c. 50 pgjcrns RNA.
6.
H.F.M.P.
conditions
results for denafured ribosomal as for Fig. 4. 50 pg/cm* RNA.
1
-1.50
-1.30
-1.10
Fig.
-1.10
(E. CA)
RX-4
; con-
(E. Coli).
Barker
96
and 3kKeown
c. RNA (Yeast) This inespensi\-e material, of uncertain purity, ga\:e somewhat unsatisfactory H.F.31-P. results, the linearity of the system as regards the dependence of the intermodulation signal on concentration being poor, as the results in Fig. 7 clearI>- sho\v, though the two null points surprisingly were not concentration-dependent_ At the IO &cm3 level this material in the pH S-3 medium produced a quasi-symmetrical peak of half-peak width 70 mV (corrected for the effect of S-IV. amplitude) the masimum conductance bei?g obserl-ed at -I.+o V, a potential \-alue \vhich does not agree well with the \-alue of -1.36 V for the null point of the cor-
Blank
,
I
I
1
-1.30
-1.10
Oenztured RNA (RtboscmaLfColi)
Fina- iH-F-11-P.
results
for
tznfi~.~ \-east
RS.1
; conditions
as
for
responding H.F.3I.P. n-ave. This discrepancy is much larger than the possible error in potential measurement and probably reflects the occurrence of additional signals in the H.F.N.P. case produced by substances other than RX:,% Cleat-l>- high frequency techniques that produce large non-faradaic signals, such as H.F.M.P., may in the future prove useful for stud>lng the purity of materials like DNA and RNA. Again organic adsorptlon seems to persist to unespectedly negative potentials judging from the fact that the \-arious H.F.‘I\I.P. cur\-es in Fig. 7 onl_smerge together at potentials more negatil-e than -1-55 V, a fact which was not revealed by low frequent>- measurements (S-IV., P-P.. L-S-V.. R.F.). d. Transfer RNA (E. Coli) Surprisinglythis material (go T$ RSA, +I yO volatile matter) in the pH S-2 medium gal-e H.F.N. P. signals that x-aried almost linearly \vith concentration up to 30 Fg/cm3 for values of the mean potential more negative than --1.1 V (Fig. S). The potential of the null point for the V in good agreement with the value of U, S D-A region is cn. -1.3s
Modulation
Polarography
of DN_A
and
Some
Types
of RX-4
3%
of -1.355 V for the S.W_ peak. This material is unique, among .the nucleic acids- studied thus far with the M.M.P., in producing an almost symmetrical peak on the S.\V. polarogram when the concentration is low (IO &cm”), though this qymmetry rapidly deteriorates’as the concentration is raised and for 50 pg/cm3 is more pronounced than that_ohserved for denatured DNA. Yet again the H.F.M.P. results prove that organic adsorption - not necessarily of t-RNA - persists to a potential more negative than -1-55 V. Denaturation, not unespectedly, had~ no noticeable effect on the signals obtained by any of the available experimental methods.
60
40
,-P,
20
E I8 0’ .z cc
I
1
I
I
-1.30
-1 10
z-0.90
t-
RNA(E.Co/i)
Blank 0 Fig_ S. H.F.M.P. for Fig.
results for transfer RX-a
(E.
Coli)
; conditions
as
a_
about the effective diffusion coefficients of Without information would be unprofitable to discuss the relative the various nucleic acids it sizes of the various signals produced by the different nucleotides at fised However it should be mentioned that the S.W. concentration (wtjcm3). peaks although not invariant in height do not show great dependence- in size, shape or width on the nature of the nucleotide and to some extent the same is obviously true for the H.F.M.P. signals observed in the poThe tential range in which segmental desorption-adsorption is observed. largest differences perhaps are between the behaviour of denatured DNA on the one hand and of t-RNA on the other hand. At the IO pg/cm3 level the materials give S-W. peaks of almost the same size though they do not occur at the same potential. The H.F.M.P. signals for t-RNA, however, although imperfectly resolved from orie~tcatio~~signal seem to be
3ss
Barker and McKeown
about twice as large as the corresponding denatured DNA signals. The difference really is rather small relative to the espected large difference between the diffusion coefficients of the two materials in the bulk of the solutiotk. One suspects the existence of an unidentified compensating factor tending to make relatively uniform the electrochemical behaviour of nucleic acids of widely varyin,= molecular weight. Clearly there is a need for some work on the effect of sonification on the S.W. and other signals.
S D-A equivalent
electrical circuit for denatured DNA at Up
Of the various results those for denatured DNA in I M KCI, 0.1 M Na,HPO, are most suitable for examination in depth to show that the S-IV_ conductance is consistent with the H.F.M.P. data, assuming reversible segmental desorption-adsorption described by the equivalent circuit in Fig. 3_ n. Efiective value of .IG~ in the S D-A region. Parameter n, is needed to assign values to components in the equivalent circuit though it needs not be known to compare the size of the S.\V. signal at U, with the correspondin,= H.F.M.P. signals-in the S IX-4 region, provided the signal in the S-IV. case is largely diffusion-controlled (i.e. not limited by C, or the finite length of the transmission line). Equation (z) has been applied to data obtained from the shoulder of S-IV. peaks such as are shown in Fig. 2 giving values for n, of -1.7 and -2.1 respectively for the negative and positive shoulders. The mean of these two tentative values agrees quite well with the value of -1.8 computed from the half-peak width (corrected for the infiuence of S-IV. amplitude) using equation (3) again. Thus the value n, = -123 has been employed when evaluating R-rr. and CT= for the resistive transmission line in Fig. 3_ b. Variation of the S D-A impedance with frequency Taking the circuit in Fig. 3 and assuming a frequency sufficiently large for C, to be treated as a short-circuit, and a variation of Csr the efiective concentration of diffusing segments in the solution lamina, with potential governed mainly by the potential variation of (I + P)-l where P is given by equation (3). it is found that the H.F.M.P. signal (at the cell) for unit area of interface should be given with fair accuracy by
where AU is the amplitude of the H.F.M.P. square wave, &I is the double layer impedance (low coverage with DNA) at 2 MHz and I?, refers to 2 MHz and is given by equation (7). This equation predicts a variation
Modulation
Polarography
of DX’A
and
Some
Types
of RX-1
389
of H.F.M.P. signal with potential in the S D-A region which has a specia1 type of symmetry (reffected normally in the x direction and also simultaneously reflected in the ‘y direction) about the potential at which P = I (U= Up). In practice the S D-A waxre is appreciably asymmetric about this potential but such asymmetry can be allowed for by taking the geometric mean of the numerical values of the maximum and minimum values cf A&,, when using equation (S) to calculate the vaIue of R, at 2 MHz for U = U,. Taking data from the 50 yg/cm3 curve in Fig. 4 such a caIcuIation leads to a value for R, (applying small corrections connected with the finite S.\V. amplitude of 31 mV peak to peak and the finite 2 MHz current amplitude) at 2 MHz of o.S, Q for unit area of interface, U = U,, and 50 pglcrn3 DNA. It readily foIIows that at 235 Hz the corresponding value for R, should be cn. 7s Q for unit area, if, at this frequency, segmental exhange between the interface and the lamina is still diffusion-controlled_ This result points to a square wave conductance for the measurement conditions which prevailed in the present work of 4x ro-” mhos cm+ for U = Up whereas the observed value of the conductance was 1.3 mhos cm-“. The discrepancy is not large bearing in mind the large change in frequency and the simplifying assumptions made in the model on which the circuit in Fig. 3 is based. Nevertheless it is of interest to note that a substantial part of the discrepancy vanishes if, using data appearin,m later, a more accurate calculation is made of the square wa\-e conductance taken, u account of the limiting effects of Ca, and of the finite length of the line, on the measured conductance. Then the calculated conductance based on the H.F.M.P. data and adsorption data (mainly of L.S.V. origin) is about 1.5 times larger than the esperimental value. Such agreement. is indeed rather better than might reaIt does support very strongly the view that the sonably be expected. well-developed SW. peaks for DNA (probably also for RNA) are of non-faradaic origin and caused by the special type of adsorptiondesorption which we have here termed segmental desorption-adsorption, but which has been recognised for many years by workers in the polyelectrol_yte field.8 \Ve think it impossible to esplain the effect of frequency in terms of a faradaic mechanism without invoking a reaction involving reversible electron transfer at a MHz and lower frequencies to adsorbed soIute which, in its reduced form, is virtually unadsorbed by the electrede. For such a seemingly improbable reaction the equivalent circuit could have exactly the same structure and component composition as the one advanced here for the process of S D-A, even to the inclusion of a line of finite length. It is, however, difficult to believe that electron transfek, presumably to a few of the reducible bases, would be a reversible process at 2 MHz. However there is no experimental evidence to show conclusively that the latter cannot be the case, though experience suggests that a faradaic origin for the H.F.N.P. signals is highly improbable. thickness of the Iamina containing the unadsorbed c. Approximate segments of DNA at potential U,. The parameter d can be appro_ximately evaluated if R, is known
390
Barker
and
Mck’eown
and some assumption about the effective diffusion coefficient for segmental diffusion is made as well as a more certain assumption concerning the fraction of the partly adsorbed DNA moiecule which is unadsorbed at potential Up, making use of equations (7) and (S) and experimental data reproduced in Fig. + For a solution the bulk of which contains 50 pg,/cm3 DN,4 it is found, taking an effective molecular weight for the se,aments in\-olved in S D-X of 3000, that for potential U, the value of C,, the adsorption capacitance in the S D-A equivalent circuit is approsimately 22 ?F. making use here of L.S.V. data for a siightly acidified solution and assuming that adsorption of denatured DNA for both slightly alkaline and slightly acidified solutions is virtually diffusioncontrolled [in 3I.JI.P. experiments up to the S D--4 region in the alkaline solution case and to the nearby region in which base reduction occurs when the solution is slightly acid (pH s-s--6.0)]. Making the assumption that at potential Up about half the molecule has been released into the solution one finds that the effective value of C, must then be of the order of a x 10-8 mol cm-” if D, is of the order of 3 x IO- 6 cm” s-l, a I-alue roughly equal to the geometric mean of the espected value for diffusion of a monomeric unit and the value reported by VALESTA and GRAHJLASS 3=for the apparent diffusion coefficient of monomeric units forming part of the calf thvmus DNA strand. It is then a trivial step to show that the series resistance for the transmission line must at potential Up-have a value of the order of SOO !A if the circuit refers to unit surface area, the corresponding value for the shunt capacitance of the line being about jo pF. The fine iength, i.e. the solution lamina thickness for potential U, is found to be of the order of 2 x IO-~ cm. The component values mentioned above, taken together with the value of cn. ~2 FF for C,, point to an approach to equilibrium when the potential is slightly disturbed which is comples as regards time dependance but roughly can be treated as esponential with.a time constant of about IO ms. Such a conclusion is consistent with the large reduction in apparent conductance for denatured DNX in the S D-X region w-hen pulse polarography replaces square However it also has to wave polarograph>- that was mentioned earlier. be noted that in the P.P. case the mean potential is virtually constant during the drop life and this leads to less accumuiation of DX..A at the interface at the potential of masimum conductance than in the S.IV. case (with a pote n tia 1 scan sy-nchronised with drop growth) after due allowance for differences in drop age and size at the measurement time. Currently, however, it is believed that the absence of a rapid delayed cathodic potential scan can only be the cause of a reduction in apparent conductance in the P.P. case of the order of a. If this belief is correct the decline in sensitil-ity with increasing elapsed time above I ms must, as indicated above, be attributed to the tendency for the system to come to equilibrium as regards the release of se,ments from the double layer region into the solution lamina when the potential is made more negative. Surprisingly the theory of the system is slightIy more complex in the P.P. case than when the S.IV. mode of the Jl.~~.P. is employed largely because in the latter case, by a fortunate accident, DNA4 adsorption tends
Modulation Polarography of DSA
and Some Types of RS-1
391
to be virtualIy- diffusion-controlled
until almost the value of drop age at which the S D-A conductance appears. Though some of the steps taken in the evaluation of parameters such as the lamina thickness, d, can be criticized on the ground of oversimplification of a very comples adsorption-diffusion system involving a solute of great complesity, the only assumption that may prove difficult to justify, even qualitatively. is the assumption that segments are eschanged between the interface (i.e. the double layer region) and the solution in much the same manner as if they did not form part of a long If this assumption is made all the esperimental results for molecule. denatured DXA can be semi-quantitatively esplained but this does not justify the assumption though some such assumption is made fairly plausible by qualitative reasoning given earlier. Despite such doubts we have no doubt that the large S.\V. conductance that is observed close to what is the negative end of the adsorption range for denatured DNA in slightly alkaline solution is non-faradaic in origin. and caused by the exchange of segments of the DKA strands betlveen the double layer region and the solution close to the electrode surface, this partial eschange being the precursor of total desorption as the potential is made more negative.
AcknowIedgements IIre are grateful to Prof. NtiRSBERG for the gift of a sample of calf thymus DNX. Also we acknowledge the considerable benefits gained from discussions with Prof. N~;.RSBERG and Dr. VALEST_-\, with Prof. BERG and with Dr. P_ALECEK on matters which until \-ery recently were entirely foreign to us.
References 1a E. b
C 23 b c 32 b 0
PXLECEK and V. VETTERL, I3iopo~)~mev.s6, gr7 (196s) Bioplr>vs. _-Ida 262, 125 (IgTz) E. PALE~EK and V. BRABEC. Biochim. For additional Refs. see E. PALE~EK. Coil. Czeclr.C/rem. Cor~~ruzr,t. 39, 3.+.+g
(r9i4) H. BERG, H. BXR and A. GOLMICK, Biopolymers 5, 6r (1967) G. HORS, J_ Eleciroanal. H. BERG, B. TRASSELT, J. FLEJIJIISG, H. BX~and Cfreirr. Iderfrtcial Electvoclrem. 21. IS I (1969) For additional Refs. see J. FLENMISG and H. BERG, Bioefecfroc?rem.Bioeireq. 1, $60 (1974) P. V'XLESTA and
P. GRAHMASS.
J.
Etectronnal.
cheirr. 49, 41 (1971) J. P. VALESTA and H.\V. &71'i;.~~~~~~, trochein. 49, 55 (1974)
Electronnat.
Chem. Chew
Interfacial Interfacial
ElectroEtec-
Barker
392 C
43
b 53
b 63 b
3 9
10
11
For additional
and
NC Keown
Refs. see P. VALEXTA and H.\V. X~~RXBERG. Biophys. Slrrrct. (1971) J_ Elecfvoannl. CJxm. Iderfucinl Electvochenr. 46, Sg (1973) Bioelectroclrenz. Bioeneq. 1, REYSAUD and P-J. SICARD. B. >r_XLFO\-, J.X. 136’ (1974) B. JAEIK and P. J_ EL\-ISG, Chem. Rev. 68, zg=j (1965) For additional Refs. see P. J_ ELX-ISG, S. J. PACE and J.E. O’REILLY, J. Am. Chem. SOG. 95, 647 (1973) CJJem. G.C. BARKER, J.-A_ BOLZAS and _1.\1-. GARDSER, J_ Eleclyonnnl. Inferfacinl Elecivoclrenz. 52, 193 (1974) G.C. BARKER and D. MCKEOWS, J_ Electvonnal. CJzenz. Idevfmial E!eclvoclrem. 59, 293 (1975) G-C. BARKER, _A.\\‘. G_XRDSER and 31. J_ LX-ILLIAMS. J_ EZeclroanal. CJrenz. I~rterfczcinl. ECectrocJzern. 42, app 3-r (1973) 1-R. MILLER. Tmm. Fnrtrday Sot. 57, 301 (rg6r) E. PALE~EK. Symposium OIC Eleclycchem. -4 nal. of Xzrcieic ,-1cids. Czechoslovakia. Ma!- r 975 H.\V. X~RSBERG. ibid. G.C. BARKER. Pzrye _APPZ. Chew. 15, 239 (1967) &!f?cJL 1, 17 D. THEVESOT.
Modulation
am? Bioenevgetics
Polarography
Energy
Research
(1976)
of DNA and Some Types of RNA *
by G. C. BARKER 0 and Atomic
3, 373-39’2
D.
~ICKEOWS
Establishment,
Harwell.
Didcot,
Oson,
England
The intermodulation signals produced by various poI_ynucIeotides have been studied with a 2 MHz Modulation Polarograph and, in some cases, compared with the corresponding signals observed using various modes of the HARWELL Multi-mode Polarograph. For denatured DNA the signals observed at the negative end of the adsorption range for a slightly alkaline solution seem to be non-faradaic in origin and caused by effects connected with the exchange of segments of the partly adsorbed molecules between the double layer region and a solution lamina containing those parts of the adsorbed molecules that are not in intimate contact The exchange of segments seems to be diffusionwith the interface. controlled even at a MHz and an analysis of the low and high frequency data indicates that the DNA system behaves as though the molecule were divided into independent subunits of molecular weight ca. 3000 as regards the exchange of molecular segments between the interface and . the solution_
introduction The behaviour of native and denatured DNA in acid media is fairly well understood as a result of work in several countries, i-5 but the behaviour of nucleic acids in general in slightly alkaline media is a more contentious In particular there is uncertainty about the origin of a signal topic. revealed by a.c. polarography close to what appears to be the negative end of the adsorption range for single-stranded DNA at the mercuryIn the present work we have attempted to aqueous solution interface. identify the cause of the signal mainly by comparing the analogous signal observed in 225 Hz square wave (S.\V.) polarography with the corresponding intermodulation signal (of a special type) for denatured DNA polarograph (H.F.M.P.). 6 The befound using a 2 MHz modulation
Jiilich,
* Presented at the 3rd International 27-31 Oct. 1975.
0 Present
adress : School
of Chemistry,
Symposium Bristol
on Bioelectrochemistry. University,
Bristol
374
Barker and McKeown
haviour of some tvpes of RNA also has been investigated though interpretive efforts tena to have been focussed on data for denatured DNA in slightl>- alkaline solution_ The S.W. data w-e;-e pi-ovided by the S-IV_ mode of a modern multi-mode polarograph (M.M.P.) and supporting data needed in the analysis of S.11’. and H.F.3I.P. results lvere provided b>- the radio-frequeneq(R-F.), square wave intermodulation (S.1V.I.P.) and linear scan \-oltammetric (L.S.V.) modes of the 3I.JI.P. 5 Data also were provided by the normal derivative and the integrated -derivative mode of the HARWELL t>-pe of pulse polarograph (P.P_)_ To a large extent it has been possible to avoid massive system non-linearity caused by large surface coverage. an important point when studying srgnals which are influenced in magnitude, and conceivably also in potential dependence, by strong solute adsorption and interactions between adsorbed entities_ This feature of the work is a direct consequence of the a\-ailability of novel instruments of high sensitivity.
Experimental The H.F.M.P. n-as the instrument used previously; 6 and it gave recordings showing the dependence on the mazt~ potential of a special dropping mercury electrode (D.M.E.) of the output voltage of the H.F.M.P. during a potential scan (cathodic, 0.4 1’ amplitude). synchronised with drop growth and starting 4 s after drop detachment. The output \-o!tage of the instrument (apart from small progressive changes in sensitivrty connected with drop growth durin g the potential scan) was a linear measure of the derivative (with respect to mean D.M.E. potential) of the series resisti\-e component of the interfacial impedance at 2 MHz. Thus indirectly one is able with a H.F.N.P. to study the potential dependence of one important part of the interfacial impedance at a frequency verylarge compared with the frequencies used when bridge methods are emplo>-ed. Thi s is made possible because the unusual t>-pe of intermodulation encountered with a H.F.M.P. produces a low frequency signal (~5 Hz) after demodulation which can be likened, in the contest of sensitivity, to the 10~~ frequencr signals that arise when choppers are used in the measurement of minute kc. cur-rents or voltages. The scan of the H.F.M.P. w-as of 6.6 s duration and its starting potential w-as adjusted manually (IO mV steps) with respect to a S.C.E. During the scan the D.X.E. was invariablypolarized simultaneously by a S.\V. voltage of 31 mV amplitude (peak-peak) and an unmodulated 2 MHz current of 7 mX amplitude_ A low inductance cell was used which contained a specially-constructed D.BI.E. providing, as a result of the conicall>r-tapered tip, a high degree of symmetry as regards current flow at 2 XHz in the solution surrounding the mercury- drop and also by- means of a contact to the mercur; thread at a point between the 0.1 mm capilIar>- to which the drops Lvere attached and a length of fine bore (0.04 mm) capillary to control Hg flop- (o-54 mg s-r), a low impedance connection
Modulation
Polarography
of
DX_1
and
Some
Types
of
RSX
375
to the mercury drop. The tip of the D.M.E. was hand-ground to obtain the requisite taper estending almost to the orifice of the capillary tubing. X high degree of symmetry for the D.M.E. solution is vital if nonfaradaic srgnals connected merely with the potential dependence of the capacitive part (series circuit) of the interfacial impedance are not to influence appreciably the recorded output voltage of a H.F.M.P. As in earlier \\-ork the instrument was made relatively- insensitive to such signals by making the cell circuit series resonant at 2 MHz. (see Refs. 6n and 6b). All measurements were made at ambient temperature (IS-ZOOC). An increase in recorder reading invariably indicates an increase in the value of the derivative of the interfacial resistive component Ri”, with respect to the mean potential of the D.M.E. The instrumental sensitivity was the same for all recordings given later and unit change in recorder reading denotes a change in the Lrariation in I?;,, (peak to peak) produced by the a fore-ment ioned 31 mV S.\V_ of 7-1 x IO-" !2 at the point on the recordings at which denatured DNA produced large signals connected with segmental desorption-adsorption (vi& ix&z)_ The root mean square variation in reading from trace to trace (5 scans) usually was ca. 0.5 divn. but \-ery occasionally was enhanced by radio interference. Low frequency data for the various poly-nucleotides were provided by the H_-\Ru-ELLP.P. (1953 lvintage) and by the S.\V. and L.S.V. modes of the M.M.P., 7 some use also being made of the RF (72 kHz) and S.1V.T.P. modes of the latter instrument_ Space considerations make it impossible to report the low frequency- data in detail in the present paper. They will be dealt with more fully in a later publication. Calf thymus DNA (i\lLLES Labs.), higIzZ)l ~oZyr~zerized RN-1 (E. Coli) t-RN_\ (E. COG) and Yeast RNX (ail from BDH Labs., Poole, England) were used at concentrations not esceeding 50 pg,km3 in the polarographic work. Materials to be used in wztixx form were first dissolved slowly- in 0.15 111NaCl, 0.015 41 Na Citr. Denatured materials were obtained by dissolution in a ro-fold more dilute chloride-citrate solution, heating to I00 OC for 30 min.. follo\ved by rapid coolin g to o OC and subsequent mild refrigeration.
Results and discussion
n. Pulse polarograph data Fig. I is fairly typical of the results for all the types of RNA studied when the concentration of the polynucleotide is 50 pg/cm3 and the medium is I AJ KC1 made slightly alkaline (pH z S-3) by making the soluFig. I shows the potential depention 0.1 111with respect to Na,HPO,. dence of the differential capacitance or pseudo-capacitance indicated by deri\-ati\re (with integration of the current signal prior to measurement) pulse polarography when a negative pulse (35 mV size) is employed.
Barker
3’76
and NcKeown
Here we are mainly interested in the effect of the nucleic acids at potentials more negative than cn. -o-g V (here and Iater US. S.C.E.) and it will suffice to point out that for all forms of RNA there seems to be a broad but small pseudo-capacitance peak centred at about -1.1 V which may conceivably be due to some reorientation of the adsorbed nucleotide molecule (largely single-stranded for all the types of RNA used in the present work). Of greater interest in the contest of the present paper are the much sharper pseudo-capacitance peaks observed for all t_ypes of RNA in the vicinity of -1.37 V. (It should here be noted that the P.P. data refer to an elapsed time since drop detachment of z-7 s and that there are small variations in the position of the sharp capacitance peak with nucleic acid concentration and with the nature of the nucleic acid)_ The sharp capacitance peak is not well resolved with respect to the broad but smaller peak that usually is observed at CK -1.1 V but at negati\-e potentials it seems to decay rapidly. Thus for ~zntive (implying here - as recei\-ed) ribosomal RNA from E. Coli the results in Fig. I suggest l-irtually total desorption of the polynucleotide at potentiaIs negative with respect to -1-15 V.
U (Vvs SCEI’ 1
-14
,
I
-1.0
1
-0.6
,
I
-0.2
Fig. I. Capncito,gams for native ribosomal RX-1 in I 31 KCI, 0.1 df Sa,HPO,.
The effect of denatured calf thymus DKA for the same concentration and medium closely resembles that seen in Fig. I. for E. Coli RNA withe the esception that the qhegntizfe pseudo-capacitance peak for DNA occurs at -1-47 V (after applying a correction for the influence of pulse sense and size) and consequently is better separated from the broad pseudo-capacitance peak which still is centred at crs. -1-1 V. The latter peak has been attributed by V_ALEXTA and NCRSBERG 3b to reorientation of adsorbed solute.
Modulation
Polarography
of DNA
and
Some
Types
df RNA
3f7
_ -_- :.. b. alulti-mode polarograph data The most surprising feature of the Iow frequency---data w& the large conductance peaks observed using the S.W. mode of--the, M.M.P; for all the polynucleotides (except native DNA) in the usua- slightly alkaline medium (I _U KCl, 0.1 AI Na,HPOa). These petis may -well prove to be of value in the detection and determination’of singlystranded nucleic acids at levels below I pg/cm3. Fig. 2 shows the quite sh$rp peaks for denatured DNA4 at the concentration IO, 30 and $0: pg/cnP when the instrumental sensitivity was low and the- SW. amplitude-Gas 32 mV peak to peak. The conductanc_e peak resembles that produced by
60-F .1 -
Instrument
U(Vvs -1.10
-1.20
-1.30
-1.40
SCEI
-1.50
Fig. 2. Square wax-e polaro,-ms (S.\\r_mode of XXP.) for denalrrredcalf thymus DNA in I .&I KCI, o-r &i Ns_HPO, : 3~
mV square wave. min. d.c. sensitivity, max./S a.~. sensjtivity.
a z-electron
faradaic reaction as regards width and lack of dependence of position on concentration. Also for concentrations up to 20 pg/cw, the size of the conductance peak is linearly related to polynucletitide concentration. Then, of course, for the drop age in question the-surface concentration just before the conductance peak (remembering that-the S.W. polarogram refers to signals observed during a 6 s duration- 0.5 V I potential scan starting 4 s after the start of drop growth) is-expected to be linearly related to concentration from the work of VALESTX and NijRx~ BERG. 3db The corresponding apparent conductance observed using -the normal derivative mode of the P.P. is about 20 times smaller after making allowance for the differences in flow rate and drop age. This -decrease in conductance (in the P.P. case the average value for the elapsed time range 0.02-0.04 s), which makes pulse polarography somewhat unsuitable- for the detection of DNA (and other pol_ynucleotides also) in alkaline media,
37s
Barker
and
SlcIieown
is much larger than the corresponding decrease for a normal faradaic reaction (reversible or irreversible) uncomplicated by reactant adsorption. It will become evident later that the decrease is a consequence of a close approach to equilibrium as regards segmental desorption at the interface when the elapsed time after perturbation of the potential exceeds 0.02 s. The results reported for denatured DNA by VALESTX and NCRSBERG 3b indicate that at low frequencies the equivalent circuit for the interface is large must be in the potential range in Lvhich the S.\V. conductance dominated as regards the impedance which shunts the double layer capacitance by an adsorption-desorption capacitance. The latter component is not dependent in value on frequency and can be equated approsimate!y \vith the pseudo-capacitance obserl-ed on puke capacitograms I. Ho\ve\-er, it also can be seen from the large of the type seen in Fig. x-alues of S.\Y_ conductance found for denatured DN_\ (and also for the various t>-pes of RX_&) that the circuit limb w-hich shunts the double lax-er capacitance (infinite frequenq-) must also include resisti\-e elements which become relatively unimportant at large \-alues of elapsed time though of paramount importance in producin, m the peak on the S.\V. polarogram \\-hen the recording refers to repetitil-e measurements made at an e!apsed to suppose that these resisti\-e eletime close to 2 ms. Tt is reasonabIe ments are in some wa\- connected with mass transfer of nucleic acid in the solution close to tge electrode surface. Thus in what folio\\-s it will be implicitl>- assumed, partly because of the big difference betlveen the S.11’. and P-P_ conductances (normalized to constant drop area and conbe accounted for in terms stant elapsed time) \vhkh cannot satisfactoril\of an effect caused by the use of a fast cathodic potential scan in the M.3I.P. esperimcnts and a ver>- slowlchanging scan in the P.P. work, that the sharp S.Ii’_ conductance peak for denatured DNA is basically in double ia3-er non-faradaic in origin being caused by slow changes structure which inevitably accompan)adsorption or desorption of nucleic acid following a sma!l potential changeThe S-IV. peaks thus are assumed similar to the adsorption-desorption (tensammetric, dcsad) peaks observed with solutions containin, m strongladsorbed organic solHo\\-e\-er, usualI>- decad peaks utes such as long chain aliphatic alcohols. for relatil-ely simple organic molecules, if large and narroxv. are appreat which the ciabl>- dependent on concentration as regards the potential That the latter feature is not evident apparent conductance is largest. in the case of a nucleic acid peak at first encourages one to think that the peak is of faradaic origin but such a view can be totally incorrect as it is a thermod>-namic requirement in the case of very strong solute adsorption from a solution containing little of the solute that, for lox surfze covernge. the transition from adsorption to no adsorption at the D.M.E., when the will occur in a potential range potential .is rapidly made more negatil-e, If the latter state of affairs independent of the solute concentration. esists inevitably the signals observed in KC. polarography or S.\I’- polarography will resemble faradaic signals as regards their invariant position on the potential ask. It is. however, very seldom that potentialindependent non-faradaic waves are observed for simple organic solutes
Mociulation
Polarography
of DNA
and
Some
Types
of RNA
379
of quasi-a or 34imensional shape as normally the signals for slight surface coverage are smaI1. partly because the adsorption coefficient for the solute is not markedly potential dependent for low surface coverage_ For the nucleic acids the results of many investigations suggest that at negative potentials the adsorbed molecules are quite rapidly desorbed in a rather small potential interval as the potential becomes more negative. Thus large SW. peaks are not so remarkable and one expects from studies of the P-P., S.AV. and H.F.M.P. signals produced by the nucleic acids to learn something about the kinetics of desorption, and the effective size of the segments of the molecule which become attached to, or detached from, the interface when the potential is slightly changed. In what follows we make use of the concept of segmental desorption-adsorption which has been employed by MILLER * and by other workers S-lo when discussing the electrochemistry of polyelectrolytes or polynucleotides. Septenfnl
desor$tiosmzdsorpfion
(S D-A}
It can be shown that provided at the electrode-solution interface the adsorption-desorption reaction namic viewpoint, as a special type is, for a simple solute, possible to
adsorption and desot-ption of a solute can be regarded as indivisible steps can be thought of, from a thermodyof charge transfer reaction. Thus it write for the non-faradaic reaction rr
S -+ n,e- f
S,d
(a)
whet-e S is the solute, Sd is the adsorbed form of S and n, is the number of electrons supplied to the electrode at comtant potential when one molWe assume that the surface covecule of S is adsorbed at the interface. erage is small and that, in the supposedly small potential interval in which the transition from strong adsorption to little adsorption is mainly concentrated, parameter n, is not markedly potential dependent and can the influence of be regarded as a constant indicatin, * fairly quantitatively S& on the double layer structure. In reality n, for simple organic solutes is quite likely to be markedly potenrial-dependent if the low surface coverage requirement is satisfied, but occasionally it is fairly constant at the extreme end of the potential range in which an inorgamc OP organic solute is adsorbed. Parameter n, has a crucial effect on the desad impedance at high frequencies and its evaluation is essential in the analysis of impedance or Two methods can be used_’ The conductance data for desad systems. first is to study the potential dependence of the zero-frequency adsorption capacitance ‘for low7 surface coverage an6 constant solute concentration This capacitance Cd is defined appro_xiin the solution at the interface. mately by
(1)
Barker and NcKeown
3so
if n, can be treated as a potential-independent parameter and K is a constant. This equation cannot easily be appbed to nucIeic acid data but an analogous equation involving the S.W. conductance in place of Cod can be employed if the frequency is sufficiently large for the measured conductance to be largely diffusion-controlled_ This version of equation (I) can be applied to conductance data for the shoulders of conductances peaks such as are reproduced in Fig. 3. Ntematively n, can be calculated from measurements of the width of the S.W. conductance peak at half the maximum value making use of the approsimate expression for the measured conductance Gs D-A kP &D-A
=
(I +P)'
where k is a constant and P=esp
(u--uJn,P RT
Again it is assumed that S D-A is diffusion-controlled in the S.W. case (this is not a strictly justifiable assumption for a S.\V. frequency of 335 Hz, but it is one which must be made for reasons of simplicity in the first analysis of esperimental data)_ U, is the potential at which Gs D-A is largest and can be roughly identified in the S-W. case with the potential at which approsimately half the denatured DNA adsorbed prior to the potential scan, and during the earlier part of this scan, has been segmentally desorbed from the Interface. In the case of simple adsorption-desorption (e.g. the specific adsorption of I- on mercury) the phy-sical significance of n, is clear if equation (I) is employed_ It represents, if appropriate esperimental conditions have been employed, the number of electrons supplied to the electrode at constant potential per iodide ion adsorbed. The application of equation (I) to the corresponding pseudo-capacitance data for denatured DNA at the negative end of the adsorption range (if accurate data were available) would without any doubt lead to a value for n, close to -2. However L.S.V. data for the same system, although not of great accuracy, suggest that the double layer charge-density for the potential region in question changes by about -0 .a electrons per base adsorbed at constant potential (here ruse must be made of L.S.V. data for slightly acidified media as well as those for the pH S.a medium). A comparison of these electron transfer numbers, taking account of the monomeric molecular weight. leads to an apparent molecular weight for the process of segmental desorption-adsorption of the order of 3000, a value very much smaller than the true molecular weight of the single-stranded nucleotide (in escess of 10~). This difference highlights the difficulty of applying conventional thermodvnamic equations to systems containing molecules of great length (possibly also to molecuIes that are large in more than I di-
Moduiation
Polaroeaphy
of DKX
and
Some
Types
of RX-1
381
mension such as cytochrome c)_ Clearly in the case of a long molecule whether or not a small segment is adsorbed at the interface is not influenced by the state or energy level of segments of the molecule some distance away from the segment in question_ In other words, the molecule will behave as though it were composed of independent subunits of much smaller molecular weight than the true molecular weight of the polynucleotide. The apparent molecular weight mentioned above points to segmental desorption of subunits whose Zen&b on the average is similar in magnitude to the diameter of the nucleotide with the bases stacked_ Following earlier workers such as MILLER 8 it may be assumed that, as the potential becomes more negative, complete desorption of the denatured DNA molecule is preceded by segmental desorption-adsorption, leading to partial release into the solution of adsorbed material which at more positive potentials had been in intimate contact with the electrode. The material released, one imagines, will be largely confined in a solution lamina bounded by the interface and it is reasonable to suppose that the release of segments will be accompanied by a gradual increase in the thickness of this lamina, the thickness tending to infinity when adsorp- tion ceases completely_ For the denatured DNA system the S-IV. conductance falls away rapidly at potentials positive with respect to U, and it is probably not far from the truth to assume virtually no segmental desorption at potentials more positive than -1.3s V. For this potential range we envisage thus only signals produced by slow reorientation. 3b Converting the above model into an electrical equivalent circuit, making use of arguments used earlier rr but introducing the new concept of an e/jective molecular weight for the segments eschanged between the interface and also the above-mentioned solution lamina of varying thickness, one arrives at the equivalent circuit for the S D-A part of the interfacial impedance shown in Fig. 3. In this circuit it is implicit that segmental desorption-adsorption proceeds reversibly at the boundary be-
I
TTTTT
Fig. 3_ Equi\-alent electrical circuits for the S D-_A part of the interfacial impedance ; a aperiodic circuit, b reduced form of the circuit at high frequency when the S D-X impedance becomes R,(r + I/?>, see ec,uation I.
tween the double layer and the solution lamina. No allowance for eschange of segments of molecules which in the S D-A reLgion are totally unadsorbed between the solution lamina and the bulk of the solution is made in the equivalent circuit, largely because when the S.1V. mode of the M.M.P. is employed such segments contribute little to the effective concentration of segments within the lamina. It is assumed that the se,o;ments not in
3%
Barker
and 3Ic Iieown
intimate contact with the interface move within the solution lamina which contains them by linear diffusion_ This questionable assumption is permissible for the purposes of semi-quantitative data analysis. The equil-alent circuit in Fig. 3 differs onlr in structure from the circuit foi- normal adsorption-desorption with mass transfer of the solute by linear diffusion in that diffusion is limited to the solution lamina and is described in the S D-A circuit b>- the motion of charge in a transmission line of fizzite instead of semi-infinite length. C, in the circuit is the capacitance connected with the dependence of the double layer charge component connected with the presence of adsorbed segments on potential for constant segmental concentration in the solution at the interface. It is thus a special Q-pe of adsorption capacitance. The pseudo-capacitance, which in principle can be measured, is to be identified loosely with the series combination of C, and the shunt capacitance of the section of resistive transmission line. At high frequencies if the lamina thickness (line length), n, is not too small the resisti1.e part of the input impedance of the S D-A circuit becomes
(4) where CL)is the angular frequent>-. likewise and then the resistance are gi\-en by l 1
R, refers to unit area if the circuit does and capacitance per unit length of line
J&L = _!?TIL’ F” C, D,
(5)
and C
7-L.-
n,“P’C,
RT
(6)
\vhere C, is the effecti\-e concentration for the diffusing segments in the solution lamina and D, is the relevant diffusion coefficient. From equaeschange of tions (4). (5) and (6) it follows that, for diffusion-controlled segments betiveen the solution lamina and the interface, at high frequencies the resisti\-e component of the S D-_-X impedance should be approsimateI!- given by
RT
(7)
The concentration C, presumably will at first increase progressivelv .. --_ in the S D-A region as the mean potential becomes more negative but ultimatel>it will fall steadily in \-alue due to the progressive increase in parameter rl. It should be mentioned that the peak observed on the S.\V_ polarogram, with denatured DN:\ present, did not change in size when the
Modulation
Polarography
of DK_\
and
Some
T_vpes qf RX-1
353
amplitude of the potential scan was reduced to 0.2 V, provided the starting potential was adjusted so that the masimum S.!V. conductance occurred at the same value of elapsed time. This simple but. somewhat unespected, fact becomes comprehensible if allowance is made for (n) the fact that DNA adsorption from the bulk of the solution in the S-IV. experiments remained virtually diffusion-controlled until the potential entered the potential range in which S D-X is largely concentrated and (b) the fact that equilibrium as regards segmental desorption in the S D-A region is established with a time constant of the order of 0.01 s. The latter state of affairs results in a value for C, at the point of masimum S.\V. signal which is virtuall~~ independent of the rate of change of mean potential proxrided that this is not such as to pass through the S D-A region in a time less than 0.1 s. Although at first sight this lack of a dependence of S-IV. signal on scan rate seemed to point away- from a non-faradaic origin for the signal (here it should be noted that theory in the case of simple adsorptiondesorption peaks produced b>- a solute of normal size predicts that both the size and position of the desad peaks for low surface co\rerage should vary- with the rate of change of mean potential), it is in reality strong evidence for the occurrence of seamental desorption in which on157 parts of the adsorbed molecules are released from the interface in a region lying between diffusion-controlled adsorption from the solution and no adsorption of the DX.1 strands at the electrode surface. Xot unespectedly the S.\\‘. conductance \vas much smaller if an anodic potential scan was employed in place of the more usual cathodic scan. This reduction is connected with the suppression of accumulation of DNA at the interface prior to and durin g the early part of the scan when starting from a potential just outside the adsorption range.
If the circuit in Fig. 3 applies, it is found that the intermodulation signal produced by reversible S D-A should vary approsimatelr u-ith mean potential according to the derilwative of P/(I t I?)% with respect to the potential. Thus the wax-e produced b>- S D--A on the H.F.M.P. polarogram should resemble in shape to the second derivative of a normal n.c. polarographic \vave and should change sign when the potential is very c&e to c/‘, for the S.IV_ conductance peak. In practice the behaviour of denatured DNA and of the various types of RNA conforms to these espectations fairl; \ve!l but quite large H.F.XP. signals also are observed on the positive side of the S D-,1 region that presumably are connected with *the =Joested b\- \'_ALEST.A and ~\'~;RsBERG.~~ These reorientation process subb signals we shall discuss elsewhere.n. Denatured calf thymus DN..X for denatured DX-X at the concenFig. 4 shows results obtained Fair linearity trations I0 , 30 and 50 pg/cm” in the usual pH S-2 medium. as regards the dependence of the intermodulaticn signal on concentration
Barker and AIcKeown
3%
is observed throughout the studied potential range. To within the experimental error the null points for the signal vary little with concentration, the variation of the null point for the S D-L% region being connected with the uncertainty in starting potential of as much as IO mV. The S D-A
40 I
-1.30
-1.10
Native DNA (Calf Thymus) 20
Blank
50yg/cm3
0
U (KS SCE) I
-1.10
I
I
-1
30
I
I
-1.50
Fig. _I_
H.F.M.P. results for denat~w~d calf thymus DNA ; 31 mV square wave. max./4 instrumental sensiti\-ity ; medium r M 1X1. 0.1 31 KL_HPO,.
wax-es estend
from about -1.30 V to a potential approaching at least and ultimately the various curves merge into one another at the most negative potentials indicatin g the ce.ssqGon of DNA adsorption. The waves are noticeably asymmetric with respect to the null point at about -1.47 I’, an effect not connected with large surface coverage but
-1-55
V
presumably connected indirectly with the variation with potential of the lamina thickness n and the influence of this variation on the effective se,mental concentration within the lamina. b. Ribosomal RNA (E. CoZi) The results for highly polymerized RNA from E. Coli in vaative (as received) and denatured form are given in Figs. 5 and 6. Apart from the movement of the S D-A wave to a more positive potential the curves resemble the correspondin g 50 pg/cm3 curve in Fig. 1. For both forms of the 25. Coli material, the S D-A signal changes sign at ca. -I.# V which is virtually the potential at which the pseudo-capacitance (Fig. I) for the ~~rnir_Je material is largest. (a correction for pulse height and sense must be applied to the Fig. I results). For both types of E. Coli RNA the masimum value of S.\k_ conductance is observed at a potential of -x_#g V and the agreement of-this value with the potential of the H.F.M.P. null point suggests that signals due probably to reorientation have largely disappeared in the region of segmental desorption-adsorption. The results for this material su ggest that RNA adsorption persists to more neg-
Modulation
Polarography
of DNA
and
Some
Types
bf RNA
3%
ative potentials than might be thought from a study of the variation of the S-W- conductance with potential. Thermal denatnration of the E. Coli RNA had little effect on the well-defined S.W. peak as regards position or size, but seemed to produce a small but detectable change in the size and shape of the H.F.M.P. wave for S D-A. The results i: Fig. 5 and 6 suggest that this wave exhibits asymmetry akin to that found for denatured DPU’A. 60-
5oJJg/cn?
Bknk ,
0
5.
H.F.XP-
ditions
Fig.
, Denatured
-1.30
DNA ( CalF thymus
results for native ribosomal RNA as for Fig. _c. 50 pgjcrns RNA.
6.
H.F.M.P.
conditions
results for denafured ribosomal as for Fig. 4. 50 pg/cm* RNA.
1
-1.50
-1.30
-1.10
Fig.
-1.10
(E. CA)
RX-4
; con-
(E. Coli).
Barker
96
and 3kKeown
c. RNA (Yeast) This inespensi\-e material, of uncertain purity, ga\:e somewhat unsatisfactory H.F.31-P. results, the linearity of the system as regards the dependence of the intermodulation signal on concentration being poor, as the results in Fig. 7 clearI>- sho\v, though the two null points surprisingly were not concentration-dependent_ At the IO &cm3 level this material in the pH S-3 medium produced a quasi-symmetrical peak of half-peak width 70 mV (corrected for the effect of S-IV. amplitude) the masimum conductance bei?g obserl-ed at -I.+o V, a potential \-alue \vhich does not agree well with the \-alue of -1.36 V for the null point of the cor-
Blank
,
I
I
1
-1.30
-1.10
Oenztured RNA (RtboscmaLfColi)
Fina- iH-F-11-P.
results
for
tznfi~.~ \-east
RS.1
; conditions
as
for
responding H.F.3I.P. n-ave. This discrepancy is much larger than the possible error in potential measurement and probably reflects the occurrence of additional signals in the H.F.N.P. case produced by substances other than RX:,% Cleat-l>- high frequency techniques that produce large non-faradaic signals, such as H.F.M.P., may in the future prove useful for stud>lng the purity of materials like DNA and RNA. Again organic adsorptlon seems to persist to unespectedly negative potentials judging from the fact that the \-arious H.F.‘I\I.P. cur\-es in Fig. 7 onl_smerge together at potentials more negatil-e than -1-55 V, a fact which was not revealed by low frequent>- measurements (S-IV., P-P.. L-S-V.. R.F.). d. Transfer RNA (E. Coli) Surprisinglythis material (go T$ RSA, +I yO volatile matter) in the pH S-2 medium gal-e H.F.N. P. signals that x-aried almost linearly \vith concentration up to 30 Fg/cm3 for values of the mean potential more negative than --1.1 V (Fig. S). The potential of the null point for the V in good agreement with the value of U, S D-A region is cn. -1.3s
Modulation
Polarography
of DN_A
and
Some
Types
of RX-4
3%
of -1.355 V for the S.W_ peak. This material is unique, among .the nucleic acids- studied thus far with the M.M.P., in producing an almost symmetrical peak on the S.\V. polarogram when the concentration is low (IO &cm”), though this qymmetry rapidly deteriorates’as the concentration is raised and for 50 pg/cm3 is more pronounced than that_ohserved for denatured DNA. Yet again the H.F.M.P. results prove that organic adsorption - not necessarily of t-RNA - persists to a potential more negative than -1-55 V. Denaturation, not unespectedly, had~ no noticeable effect on the signals obtained by any of the available experimental methods.
60
40
,-P,
20
E I8 0’ .z cc
I
1
I
I
-1.30
-1 10
z-0.90
t-
RNA(E.Co/i)
Blank 0 Fig_ S. H.F.M.P. for Fig.
results for transfer RX-a
(E.
Coli)
; conditions
as
a_
about the effective diffusion coefficients of Without information would be unprofitable to discuss the relative the various nucleic acids it sizes of the various signals produced by the different nucleotides at fised However it should be mentioned that the S.W. concentration (wtjcm3). peaks although not invariant in height do not show great dependence- in size, shape or width on the nature of the nucleotide and to some extent the same is obviously true for the H.F.M.P. signals observed in the poThe tential range in which segmental desorption-adsorption is observed. largest differences perhaps are between the behaviour of denatured DNA on the one hand and of t-RNA on the other hand. At the IO pg/cm3 level the materials give S-W. peaks of almost the same size though they do not occur at the same potential. The H.F.M.P. signals for t-RNA, however, although imperfectly resolved from orie~tcatio~~signal seem to be
3ss
Barker and McKeown
about twice as large as the corresponding denatured DNA signals. The difference really is rather small relative to the espected large difference between the diffusion coefficients of the two materials in the bulk of the solutiotk. One suspects the existence of an unidentified compensating factor tending to make relatively uniform the electrochemical behaviour of nucleic acids of widely varyin,= molecular weight. Clearly there is a need for some work on the effect of sonification on the S.W. and other signals.
S D-A equivalent
electrical circuit for denatured DNA at Up
Of the various results those for denatured DNA in I M KCI, 0.1 M Na,HPO, are most suitable for examination in depth to show that the S-IV_ conductance is consistent with the H.F.M.P. data, assuming reversible segmental desorption-adsorption described by the equivalent circuit in Fig. 3_ n. Efiective value of .IG~ in the S D-A region. Parameter n, is needed to assign values to components in the equivalent circuit though it needs not be known to compare the size of the S.\V. signal at U, with the correspondin,= H.F.M.P. signals-in the S IX-4 region, provided the signal in the S-IV. case is largely diffusion-controlled (i.e. not limited by C, or the finite length of the transmission line). Equation (z) has been applied to data obtained from the shoulder of S-IV. peaks such as are shown in Fig. 2 giving values for n, of -1.7 and -2.1 respectively for the negative and positive shoulders. The mean of these two tentative values agrees quite well with the value of -1.8 computed from the half-peak width (corrected for the infiuence of S-IV. amplitude) using equation (3) again. Thus the value n, = -123 has been employed when evaluating R-rr. and CT= for the resistive transmission line in Fig. 3_ b. Variation of the S D-A impedance with frequency Taking the circuit in Fig. 3 and assuming a frequency sufficiently large for C, to be treated as a short-circuit, and a variation of Csr the efiective concentration of diffusing segments in the solution lamina, with potential governed mainly by the potential variation of (I + P)-l where P is given by equation (3). it is found that the H.F.M.P. signal (at the cell) for unit area of interface should be given with fair accuracy by
where AU is the amplitude of the H.F.M.P. square wave, &I is the double layer impedance (low coverage with DNA) at 2 MHz and I?, refers to 2 MHz and is given by equation (7). This equation predicts a variation
Modulation
Polarography
of DX’A
and
Some
Types
of RX-1
389
of H.F.M.P. signal with potential in the S D-A region which has a specia1 type of symmetry (reffected normally in the x direction and also simultaneously reflected in the ‘y direction) about the potential at which P = I (U= Up). In practice the S D-A waxre is appreciably asymmetric about this potential but such asymmetry can be allowed for by taking the geometric mean of the numerical values of the maximum and minimum values cf A&,, when using equation (S) to calculate the vaIue of R, at 2 MHz for U = U,. Taking data from the 50 yg/cm3 curve in Fig. 4 such a caIcuIation leads to a value for R, (applying small corrections connected with the finite S.\V. amplitude of 31 mV peak to peak and the finite 2 MHz current amplitude) at 2 MHz of o.S, Q for unit area of interface, U = U,, and 50 pglcrn3 DNA. It readily foIIows that at 235 Hz the corresponding value for R, should be cn. 7s Q for unit area, if, at this frequency, segmental exhange between the interface and the lamina is still diffusion-controlled_ This result points to a square wave conductance for the measurement conditions which prevailed in the present work of 4x ro-” mhos cm+ for U = Up whereas the observed value of the conductance was 1.3 mhos cm-“. The discrepancy is not large bearing in mind the large change in frequency and the simplifying assumptions made in the model on which the circuit in Fig. 3 is based. Nevertheless it is of interest to note that a substantial part of the discrepancy vanishes if, using data appearin,m later, a more accurate calculation is made of the square wa\-e conductance taken, u account of the limiting effects of Ca, and of the finite length of the line, on the measured conductance. Then the calculated conductance based on the H.F.M.P. data and adsorption data (mainly of L.S.V. origin) is about 1.5 times larger than the esperimental value. Such agreement. is indeed rather better than might reaIt does support very strongly the view that the sonably be expected. well-developed SW. peaks for DNA (probably also for RNA) are of non-faradaic origin and caused by the special type of adsorptiondesorption which we have here termed segmental desorption-adsorption, but which has been recognised for many years by workers in the polyelectrol_yte field.8 \Ve think it impossible to esplain the effect of frequency in terms of a faradaic mechanism without invoking a reaction involving reversible electron transfer at a MHz and lower frequencies to adsorbed soIute which, in its reduced form, is virtually unadsorbed by the electrede. For such a seemingly improbable reaction the equivalent circuit could have exactly the same structure and component composition as the one advanced here for the process of S D-A, even to the inclusion of a line of finite length. It is, however, difficult to believe that electron transfek, presumably to a few of the reducible bases, would be a reversible process at 2 MHz. However there is no experimental evidence to show conclusively that the latter cannot be the case, though experience suggests that a faradaic origin for the H.F.N.P. signals is highly improbable. thickness of the Iamina containing the unadsorbed c. Approximate segments of DNA at potential U,. The parameter d can be appro_ximately evaluated if R, is known
390
Barker
and
Mck’eown
and some assumption about the effective diffusion coefficient for segmental diffusion is made as well as a more certain assumption concerning the fraction of the partly adsorbed DNA moiecule which is unadsorbed at potential Up, making use of equations (7) and (S) and experimental data reproduced in Fig. + For a solution the bulk of which contains 50 pg,/cm3 DN,4 it is found, taking an effective molecular weight for the se,aments in\-olved in S D-X of 3000, that for potential U, the value of C,, the adsorption capacitance in the S D-A equivalent circuit is approsimately 22 ?F. making use here of L.S.V. data for a siightly acidified solution and assuming that adsorption of denatured DNA for both slightly alkaline and slightly acidified solutions is virtually diffusioncontrolled [in 3I.JI.P. experiments up to the S D--4 region in the alkaline solution case and to the nearby region in which base reduction occurs when the solution is slightly acid (pH s-s--6.0)]. Making the assumption that at potential Up about half the molecule has been released into the solution one finds that the effective value of C, must then be of the order of a x 10-8 mol cm-” if D, is of the order of 3 x IO- 6 cm” s-l, a I-alue roughly equal to the geometric mean of the espected value for diffusion of a monomeric unit and the value reported by VALESTA and GRAHJLASS 3=for the apparent diffusion coefficient of monomeric units forming part of the calf thvmus DNA strand. It is then a trivial step to show that the series resistance for the transmission line must at potential Up-have a value of the order of SOO !A if the circuit refers to unit surface area, the corresponding value for the shunt capacitance of the line being about jo pF. The fine iength, i.e. the solution lamina thickness for potential U, is found to be of the order of 2 x IO-~ cm. The component values mentioned above, taken together with the value of cn. ~2 FF for C,, point to an approach to equilibrium when the potential is slightly disturbed which is comples as regards time dependance but roughly can be treated as esponential with.a time constant of about IO ms. Such a conclusion is consistent with the large reduction in apparent conductance for denatured DNX in the S D-X region w-hen pulse polarography replaces square However it also has to wave polarograph>- that was mentioned earlier. be noted that in the P.P. case the mean potential is virtually constant during the drop life and this leads to less accumuiation of DX..A at the interface at the potential of masimum conductance than in the S.IV. case (with a pote n tia 1 scan sy-nchronised with drop growth) after due allowance for differences in drop age and size at the measurement time. Currently, however, it is believed that the absence of a rapid delayed cathodic potential scan can only be the cause of a reduction in apparent conductance in the P.P. case of the order of a. If this belief is correct the decline in sensitil-ity with increasing elapsed time above I ms must, as indicated above, be attributed to the tendency for the system to come to equilibrium as regards the release of se,ments from the double layer region into the solution lamina when the potential is made more negative. Surprisingly the theory of the system is slightIy more complex in the P.P. case than when the S.IV. mode of the Jl.~~.P. is employed largely because in the latter case, by a fortunate accident, DNA4 adsorption tends
Modulation Polarography of DSA
and Some Types of RS-1
391
to be virtualIy- diffusion-controlled
until almost the value of drop age at which the S D-A conductance appears. Though some of the steps taken in the evaluation of parameters such as the lamina thickness, d, can be criticized on the ground of oversimplification of a very comples adsorption-diffusion system involving a solute of great complesity, the only assumption that may prove difficult to justify, even qualitatively. is the assumption that segments are eschanged between the interface (i.e. the double layer region) and the solution in much the same manner as if they did not form part of a long If this assumption is made all the esperimental results for molecule. denatured DXA can be semi-quantitatively esplained but this does not justify the assumption though some such assumption is made fairly plausible by qualitative reasoning given earlier. Despite such doubts we have no doubt that the large S.\V. conductance that is observed close to what is the negative end of the adsorption range for denatured DNA in slightly alkaline solution is non-faradaic in origin. and caused by the exchange of segments of the DKA strands betlveen the double layer region and the solution close to the electrode surface, this partial eschange being the precursor of total desorption as the potential is made more negative.
AcknowIedgements IIre are grateful to Prof. NtiRSBERG for the gift of a sample of calf thymus DNX. Also we acknowledge the considerable benefits gained from discussions with Prof. N~;.RSBERG and Dr. VALEST_-\, with Prof. BERG and with Dr. P_ALECEK on matters which until \-ery recently were entirely foreign to us.
References 1a E. b
C 23 b c 32 b 0
PXLECEK and V. VETTERL, I3iopo~)~mev.s6, gr7 (196s) Bioplr>vs. _-Ida 262, 125 (IgTz) E. PALE~EK and V. BRABEC. Biochim. For additional Refs. see E. PALE~EK. Coil. Czeclr.C/rem. Cor~~ruzr,t. 39, 3.+.+g
(r9i4) H. BERG, H. BXR and A. GOLMICK, Biopolymers 5, 6r (1967) G. HORS, J_ Eleciroanal. H. BERG, B. TRASSELT, J. FLEJIJIISG, H. BX~and Cfreirr. Iderfrtcial Electvoclrem. 21. IS I (1969) For additional Refs. see J. FLENMISG and H. BERG, Bioefecfroc?rem.Bioeireq. 1, $60 (1974) P. V'XLESTA and
P. GRAHMASS.
J.
Etectronnal.
cheirr. 49, 41 (1971) J. P. VALESTA and H.\V. &71'i;.~~~~~~, trochein. 49, 55 (1974)
Electronnat.
Chem. Chew
Interfacial Interfacial
ElectroEtec-
Barker
392 C
43
b 53
b 63 b
3 9
10
11
For additional
and
NC Keown
Refs. see P. VALEXTA and H.\V. X~~RXBERG. Biophys. Slrrrct. (1971) J_ Elecfvoannl. CJxm. Iderfucinl Electvochenr. 46, Sg (1973) Bioelectroclrenz. Bioeneq. 1, REYSAUD and P-J. SICARD. B. >r_XLFO\-, J.X. 136’ (1974) B. JAEIK and P. J_ EL\-ISG, Chem. Rev. 68, zg=j (1965) For additional Refs. see P. J_ ELX-ISG, S. J. PACE and J.E. O’REILLY, J. Am. Chem. SOG. 95, 647 (1973) CJJem. G.C. BARKER, J.-A_ BOLZAS and _1.\1-. GARDSER, J_ Eleclyonnnl. Inferfacinl Elecivoclrenz. 52, 193 (1974) G.C. BARKER and D. MCKEOWS, J_ Electvonnal. CJzenz. Idevfmial E!eclvoclrem. 59, 293 (1975) G-C. BARKER, _A.\\‘. G_XRDSER and 31. J_ LX-ILLIAMS. J_ EZeclroanal. CJrenz. I~rterfczcinl. ECectrocJzern. 42, app 3-r (1973) 1-R. MILLER. Tmm. Fnrtrday Sot. 57, 301 (rg6r) E. PALE~EK. Symposium OIC Eleclycchem. -4 nal. of Xzrcieic ,-1cids. Czechoslovakia. Ma!- r 975 H.\V. X~RSBERG. ibid. G.C. BARKER. Pzrye _APPZ. Chew. 15, 239 (1967) &!f?cJL 1, 17 D. THEVESOT.