[1] Nucleic acid structure analysis by polarographic techniques

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POLAROGRA PHIC TECHNIQUES

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3

Nucleic Acid Structure Analysis by Polarographic Techniques B y EMIL PALECEK

Principles of Polarography Polarography is an electrochemical method that deals with the relations between the potential of a mercury indicator electrode in an electrolytic cell and the current that flows through it. ~,2 The principle of classical directcurrent polarography is shown in Fig. 1. The mercury indicator electrode most frequently used in polarography is the dropping electrode (P); this consists of a capillary (C), one end of which is connected to the reservoir of mercury (M) and the other immersed in the solution being investigated. Because of the hydrostatic pressure of the mercury column, mercury flows through the capillary, forming droplets at the capillary tip. Thus the surface of the polarizable indicator electrode is continually renewed; the measured currents are accurately reproducible and are independent of the previous history of the experiment. As the nonpolarizable reference, electrode (R) serves either as a pool of mercury on the bottom of the vessel (V) or as a separate electrode (e.g., a calomel one) with a surface many times larger than that of the indicator electrode. A gradually increasing (or decreasing) voltage is applied to the electrodes from an external source (G), and at the same time the current is measured. As long as the potential does not reach a value at which electron transfer between the electrode and a substance in the solution starts, no substantial current appears (the so-called charging or capacity currents observed under these conditions are due to the fact that each drop of mercury falling from the capillary must be charged to a potential corresponding to the applied voltage; these currents do not, as a rule, exceed 10-7 A). When the potential reaches a value at which some substance in the solution is reduced or oxidized (such a substance is called a depolarizer), a sharp current increase occurs (Fig. lb). The increase continues until a potential is reached when all the depolarizer in the neighborhood of the electrode is exhausted and the current is limited by the transport rate of the depolarizer. A polarographic step 3 appears, the height (H) of which depends on the 1 L. Meites, "PolarographicTechniques." Wiley (Interscience), New York, 1965. J. Heyrovsk:~and J. Kfita, "l%inciplesof Polarography."Publ. House Czechoslovak Acad. Sci., Prague, 1965. 8The term "step" refers to the curve obtainedby means of direct-currentpolarography and normal pulse polarography; derivative pulse-polarographic curves are called "waves."

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TECHNIQUES FOR STRUCTURAL ANALYSIS

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B

E

(o)

(b}

Fro. 1. (a) Schematic diagram of the circuit for measuring current-voltage curves with a dropping mercury electrode. (7, linear voltage sweep generator; A, currentmeasuring device; R, reference nonpolarizable electrode; P, polarizable dropping mercury electrode; C, glass capillary; M, mercury reservoir; V, simple polarographic vessel. (b) Obtained I-E curves: ( ) background electrolyte; ( . ) depolarizer; E, potential; E1/2, half-step potential; H, height of the step; B, background discharge. concentration of the depolarizer, while the potential corresponding to the half-height of the step (half-step potential, E1/2) characterizes the qualitative nature of the investigated substance (Fig. lb). The height of the polarographic step can be calculated from the Ilkovi5 equation 1,2 for the limiting diffusion currents: Id = 0.627nFcD1/~m~lat 11e (amperes) (1) where n is the number of electrons taken up or delivered by a single depolarizer molecule during the electrode process, F the Faraday charge of 9.65 X 104 coulomb, c the concentration of the depolarizer in moles/cm 3, D the diffusion coefficient of the depolarizer in em2/sec, m the flow rate of mercury in g/sec, and t the drop time in seconds. Besides diffusion-controlled currents, there exist also other types of currents (e.g., kinetic current) that do not follow Eq. (1). The investigated solution contains, besides the depolarizer, the so-called supporting background electrolyte, which decreases the ohmic resistance of the solution. The nature, pH, and ionic strength of the background electrolyte may influence the shape of the polarographic step as well as the reducibility of the investigated shbstance. Polarographic analysis of low-molecular weight substances is carried out usually within the concentration range of 10-3 to 10-~ M; several substances in the solution can be determined simultaneously provided their half-step potentials differ from one another sufficiently.

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POLAROGRAPHIC TECHNIQUES

5

Classical Polarography of Nucleic Acids and Their Components Among usual nucleic acid constituents, only adenine and cytosine (including 5-methylcytosine and 5-hydroxymethylcytosine) are polarographically reducible.4,5 The protonized forms of both bases are reduced according to Schemes I and II. NH2 H.~NH2 Hbl~"~

N

H÷

HNI

H~ N H :

~

N

L

HN~ ~

I

R

t

R

H

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~CHEME I NH2

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I

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SCHEME II

The schemes do not include chemical reactions that follow the electroreduction (R = H, sugar or sugar phosphate). The reducibility of the bases is not substantially influenced by the attachment of the sugar or the sugar phosphate moiety.4 Single-stranded polynucleotides containing the abovementioned bases [e.g., poly(A), poly(C), and denatured DNA] are also polarographically reducible, while polynucleotides which do not contain these bases [e.g., poly(U) or poly(U,G)] are direct-current (D.C.) polarographically inactiveA 6 In double-helical polynucleotides containing adenine and cytosine, such as native DNA, poly(A), poly(U), poly(I), poly(C), etc., the polarographie reducibility is more or less suppressed. In neutral medium, native DNA is completely D.C. polarographically inactive7 ,s while poly(A), poly(U) and poly(I)- poly(C) yield relatively low currentsA g Because of low values of diffusion coefficients of polynucleotides1°,11 (for native DNA with a sedimentation coefficient of 22 S, a diffusion coeffi4 B. 6 E. e E. 7 E. s E. ' E. xo D. xl D.

J a n t k a n d P. J. Elving, Chem. Rev. 68, 295 (1968). Pale~ek, ProFr. Nucl. Acid. Res. Mol. Biol. 9, 31 (1969). Pale~ek, J. Electroanal. Chem. 22, 347 (1969). Paledek, J. Mol. Biol. 20, 263 (1966). PaleSek a n d V. Vetterl, Biopolymers 6, 917 (1968). Pale5ek, Experientia 25, 13 (1969). Bach and I. R. Miller, Biopolymers 5, 161 (1967). Lang and P. Coates, J. Mol. Biol. 36, 137 (1968).

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TECHNIQUES FOR STRUCTURAL ANALYSIS

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eient of 1.3 X 10-8 cm2/sec was reported, 1° and that for denatured DNA was given as 7 X 10- 8 cm2/sec), it is necessary to work with relatively concentrated solutions if reliable values are to be obtained by means of D.C. polarography. For this reason the D.C. polarographie method is not suitable for most of polynucleotide studies. Modern Methods Derived from Classical Polarography

The sensitivity of the classic polarographic method is limited by the magnitude of the capacity current. If the faradaic current is of the same order of magnitude as the capacity current, or is less, then its exact measurement is impossible. Therefore the increase in sensitivity of the polarographic estimations could not be gained only by application of modern amplifiers; only the experiments aimed at modifying the method of the indicator electrode polarization have achieved success in this respect. A number of modern methods have been introduced differing in principle more or less from the classical polarography. TM From among modern polarographic methods namely pulse polarography~3 and oscillographic polarography at controlled alternating current (A.C.) in connection with the socalled "first curve technique ''~4 have been successfully applied for the study of polynucleotides. Oscillographic Polarography at Controlled Alternating Current When A.C. passes through an electrolytic cell, each of the electrodes becomes alternately cathode and anode for every half-period of the current. In the combination of a polarizable indicator electrode with an unpolarizable one, the latter keeps its potential constant irrespective of current direction, while the indicator electrode subject to alternate cathodic and anodic polarization changes its potential accordingly. In oscillographic polarography at controlled A.C. the indicator mercury electrode is polarized with alternating sine-wave or square-wave current (Fig. 2a) and the potential E is followed as a function of time t (Fig. 2c) by means of an oscilloscopeJ4,~5 If a solution containing the background electrolyte, e.g., 1 M NaC1, is electrolyzed, the indicator electrode, during the half-period of A.C., is charged to negative potentials until it has attained the reduction potential of sodium ions. Here the electrode potential cannot be changed any 1~H. Schmidt and M. yon Stackelberg, "Modern Polarographic Methods." Academic Press, New York, 1963. 13G. C. Barker and A. W. Gardner, Fresenius Z. Anal. Chem. 173, 79 (1960). 14R. Kalvoda, "Techniques of Oscillographic Polarography." Elsevier, Amsterdam, 1965. 16M. tteyrovsk:~and K. Micka, in "ElectroanalyticalChemistry" (A. J. Bard, ed.), Vol. 2, p. 193. Dekker, New York, 1967.

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POLAROGRAPHIC TECHNIQUES Response o b t a i n e d

Signal a p p l i e d (a)

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Time

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FIG. 2. Summary of the signals applied and the responses obtained in oscillographic polarography at controlled square-wave alternating current. Signal applied: (a) usual polarization with multiple current cycles; (b) the "first curve technique," I, polarization with a single current cycle per each drop of mercury; II, polarization with two current cycles per each drop of mercury; f, time at which the drop falls. Response obtained: (c) oscillogram E versus t. , background electrolyte; , depolarizer. (d) Oscillogram dE~dr versus E. The nature of the substance under investigation is characterized by the potential of the indentation (El), and its concentration by the depth (D) of the indentation; S~ the curve of background electrolyte; C, cathodic part of the oscillogram; A~ anodic part of the oscillogram; MP, left marginal point (at the potential of this, anodic dissolution of mercury takes place); MP, right marginal point (at the potential of this point, background electrolyte discharge occurs). In the figure, conventionally used times and current values are given. more b y continued supply of negative charge, since the latter is consumed in electrolytic reduction of sodium ions with subsequent formation of a sodium amalgam. As soon as the current changes its direction, t h a t is, the anodic half-cycle begins, the a m a l g a m starts to dissolve. After complete dissolution of the a m a l g a m the electrode is charged to more positive potentials, until it reaches the dissolution potential of mercury. I f the current passes continuously, the whole process is repeated at a rate depending on the current intensity and frequency. When the background electrolyte contains a depolarizer, e.g., c a d m i u m ions, then, after a t t a i n m e n t of the reduction potential of cadmium during the cathodic half-cycle the current will be consumed in reduction of cadmium ions while the electrode potential

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TECHNIQUES FOR STRUCTURAL ANALYSIS

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will remain nearly constant. Only after all cadmium ions on the electrode surface are consumed by electrolysis, the electrode will be charged to more negative potentials and a short delay or inflection on the potential-time curve will appear (Fig. 2c). The position of the inflection is determined by the nature of the depolarizer, while its length is proportional to the depolarizer concentration in the solution. In practicM analytical work, a derivative curve dE/dr versus E is usually followed (Fig. 2d). In this case the presence of a depolarizer is manifested by the appearance of a well-measurable indentation instead of a poorly developed inflection on the E versus t curve (Fig. 2c). The cathodic part (C) of the oscillogram can be compared to the D.C. polarogram; on the other hand, no analogy can be found in classical polarography for the anodie part (A) of the oscillogram. Therefore the anodie part may yield some additional information on the electrode process; e.g., the appearance of the cathodic and the anodic indentations at the same potential can be used as a simple test of reversibility of the electrode reaction. Substances producing neither a D.C. polarographie step nor an oscillopolarographic cathodic indentation may undergo reduction at the potential of background electrolyte discharge, and yield an anodic indentation due to oxidation of the reduction product (such anodic indentations are produced, e.g., by guanine ~,16-x8 and also by polynucleotides containing guanineS.19-~°). D.C. polarographically inactive substances may be also estimated by means of oseillopolarography if they adsorb on the electrode, and change the capacity of the electrode double layer; in principle, similar results can be obtained as with the aid of tensammetric method. 2x Compared with D.C. polarography, oscillopolarographic analysis is roughly of the same sensitivity, but it is somewhat less accurate. Contrary to D.C. polarography and pulse polarography the presence of oxygen does not interfere with oscillopolarographic estimations. Oscillopolarographic analysis is substantially faster than other polarographic methods. If the "first curve technique" is used, a single A.C. cycle is applied to each mercury drop at a definite time after the fall of the preceding drop (Fig. 2b). With the aid of this technique primary processes can be distinguished from those conditioned by the product of electrolysis arising during the polarization by the first cycle. Moreover, if the depolarizer adsorbs on the electrode at potentials more positive than the potential at xe E. Paledek, Collect. Czech. Chem. Commun. 25, 2283 (1960). x~B. Janik and E. PaleSek, Z. Naturforsch. B 21, 1117, (1966). 18B. Janik, Z. Naturforsch. B 24, 539 (1969). x9E. PaleSek, Collect. Czech. Chem. Commun. 31, 2360 (1966). 20E. Pale~ek, Abh. Deut. Akad. Wiss. Berlin, Kl. Med. p. 501 (1966). 2xB. Breyer and It. Bauer, "Alternating Current Polarography and Tensammetry." Wiley (Interscience), New York, 1963.

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POLAROGRAPHIC TECHNIQUES

9

which the depolarizer is reduced, the sensitivity of the oscillopolarographic method can be increased. 2~,2aThis increase is achieved by leaving the mercury drop in solution for a period necessary for the accumulation of the depolarizer in the electrode surface. Only then, a single cycle of A.C. is applied to the electrode, causing the reduction of almost all adsorbed depolarizer molecules (the delay period of about 5-15 seconds has been used in experiments with polynucleotides). Thus the difficulty that follows from the small diffusion rate of polynucleotides is eliminated. Owing to the strong adsorption of polynucleotides around the potential of electrocapillary zero, the estimation of polynucleotides is more sensitive than the estimation of more weakly adsorbed polynucleotide monomeric units. Poly(C) and poly(A) can be estimated by this technique even in the concentration of 10-6 M (relative to the phosphorus content). If the potential at which the depolarizer is adsorbed does not agree with the potential to which mercury immersed into the analyzed solution is charged, the potential corresponding to the adsorption potential of the depolarizer can be applied to the electrode during the quiescent period from an external source, u The study of nucleic acids has been carried out mostly with oscillopolarography at controlled current. 5 It may be expected, however, that similar results can be obtained also by means of the voltage-controlled oscillographic polarography. Instrumentation

The commercially available instrument Model LP 600 polaroscope is made by Laboratornt pristroje n.p., Prague (earlier types of polaroscope, Models P 524 and P 576, were manufactured by Krizik n.p., Prague). The instrument works with a current of sinusoidal shape at the frequency of 50 cycles/second and records the dependence dE~dr versus E. The LP 600 Polaroscope contains circuits that make it possible to compensate ohmic potential drop in solution and to perform various methods of quantitative analysis. An electromagnetic drop time controller belongs to the standard accessory of this instrument. The applicability of the new Model LP 601 polaroscope is enlarged by incorporation of the "first curve technique" accessories. Instruments suitable for the "first curve technique" and working at different A.C. frequencies and with currents of sinusoidal or rectangular shape have been designed and built in several laboratories. 14,~-~ 2~R. Kalvoda and G. Budnikov, Collect. Czech. Chem. Commun. 28, 838 (1963). 28E. Pale~ek, Biochim. Biophys. Acta 94, 293 (1965). 2~R. Kalvoda, Collect. Czech. Chem. Commun. 34, 1076 (1969). 25R. Kalvoda and J. Mackfi, Collect. Czech. Chem. Commun. 21,493 (1956). 2eL. Moln~r and E. BirS, Chem. Zvesti 14, 849 (1960). 27F. ~ev~ik and K. Metzl, Chem. Zvesti 18, 458 (1964).

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TECHNIQUES FOR STRUCTURAL ANALYSIS

Pulse Polarography In pulse polarography, 12,13 a single square-wave voltage impulse is applied to each drop of mercury at a definite time after the fall of the preceding drop. The method has two variants, the normal and the derivative. In normal pulse polarography, the amplitudes of the successive pulses are increased linearly with time (Fig. 3a). The resulting curve is similar in shape to the usual D.C. polarographic step (Fig. 3d). In the derivative method, pulses Signal applied and response obtained E'

(a)

_I'l I

I;~secIf

I-I

', nl

II

Response obtained

rB

I

I kl_.l t_.l

i

I

I

I

I i I I

~OmSecl I

(d)

(b)

8

i ~5mVI ~ Prvr~;~

r !

J PCwl i ~ I I 1 I

I I

I I

I I

I I

E

Time It

(cl

P o

I If

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!~ 20'

I I imsec

--~1 I q ~ I Itl

(e)

I,--

ii

i

I Itl

Time

--i.I I~-

Itl

~

B

S liE s I

,,

E

FIG. 3. Summary of the signals applied and the responses obtained in pulse polarography. Signal applied: periodicity of the polarizing voltage (a) in normal pulse polarography, (b) in derivagive pulse poiarography. Response obtained: (c) T, the total current comprising the background current together with that part of current contributed by the impulse (both capacity and faradaic current). The current after eliminating the background current: C, faradaic current only; F, capacity current only; g, time at which current is measured; f, time at which the drop falls. (d) The pulse polarographic step obtained by means of normal method. (e) The pulse polarographic wave obtained with the aid of derivative method. The half-step potential (E]n) or the summit potential of the wave (E,) is characteristic for the nature of the substance investigated and the step or the wave height (H) for its concentration. 8, supporting background electrolyte; B, background discharge. In the figure, conventionally used times, potentials~ and current values are given.

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POLAROGRAPHIC TECHNIQUES

11

of constant amplitude are superimposed on a steadily increasing (or decreasing) voltage (Fig. 3b) and either a wave or a peak curve is obtained (Fig. 3e). In both methods the current is measured over the second half of the pulse duration, when the value of the capacity current decreasing rapidly with time is almost zero (Fig. 3c). The sensitivity of normal pulse polarographic methods is the same for both reversible and irreversible processes; the limit of detection of low-molecular weight depolarizers is 10- ~ M. The derivative method is slightly more sensitive, allowing the determination of low-molecular weight substances even in a concentration of 10-8 M for reversible and 5 × 10-s M for irreversible processes; reducible synthetic polynucleotides can be estimated by this method even in concentration of about 1 ~g/ml. Even after correction of the Ilkovi6 equation (1) for an expanding spherical electrode,2s the experimental currents in classical polarography did not agree perfectly with theory. It became clear that remaining discrepancies were caused mainly by the (a) inconstancy of mercury flow rate, (b) depletion of the solution in the vicinity of the capillary orifice (due to the electrolysis at preceding mercury drops), and (c) partial collapse of the diffusion layer. Although not designed for this purpose, normal pulse polarography is so far the nearest approach to an "ideal" polarography. Application of a polarizing pulse during a short time interval toward the end of the drop life prevents effects a-c. 29 Recently, exact equations have been derived for the instantaneous and average currents in normal pulse polarography, including both reversible and irreversible diffusion-controlled processes, and catalytic and kinetic reactions. More information on theory of pulse polarography can be found in the literature? 9-33 Instrumentation

The pulse polarograph was designed by G. C. Barker ~8,3~at the Atomic Energy Research Establishment, Harwell. Since 1962, the Model A 1700 Southern-Harwell pulse polarograph has been manufactured by Southern Analytical Ltd, Camberley, Surrey, England. The instrument is suitable for both normal and derivative pulse polarography. Besides the 35 mV pulse amplitude given in Fig. 3b, also pulses of 7 mV amplitude can be super2sj. Kouteck:~,Czech. J. Phys. 2, 50 (1963). ~9A. A. A. M. Brinkman, "Theory of Pulse Polarography with an Application to the Hydration of Formaldehyde."Bronder-Offset,Rotterdam, 1968. 30A. A. A. M. Brinkman and J. M. Los, J. Electroanal. Chem. 14, 269 (1967). 31A. A. A. M. Brinkman and J. M. Los, J. Electroanal. Chem. 14, 285 (1967). 33E. P. Parry and R. A. Osteryoung,Anal. Chem. 37, 1634 (1965). 33G. C. Barker and J. A. Bolzan, Fresenius Z. Anal. Chem. 216, 215 (1966). 34G. C. Barker, U. K. At. Energy Res. Estab., C/R 1553 (1956).

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TECHNIQUES FOR STRUCTURAL ANALYSIS

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imposed on a gradually increasing voltage. Measurements can be performed both with or without a mechanical drop time controller, and the voltage sweep can be applied at three different rates: 3 minutes, 7.5 minutes, and 15 minutes per volt. If required, the current is integrated over 3 or 9 drop lives. The height of the step or wave can be measured by the instrument with high accuracy necessary for analytical purposes. On the other hand, the measurement of potentials is burdened by a considerable error (about ± 2 0 - 5 0 mV) if no reference substance (having a known potential value) can be added to the analyzed solution. The electrode stand makes it possible to measure samples that have volumes not less than 3-4 ml. If smaller volumes have to be measured (around 1 ml), it is necessary to use special vessels and usually to adapt the vessel holder. The electrode stand is construeted for work with vessels having a mercury pool at the bottom of the vessel as a reference electrode. In the case of measurements against the separated reference electrode, it is necessary to work with an inadequate volume of the analyzed solution or to use another type of vessel and adapt or remove the stand (when the stand is eliminated, it is not possible to control the drop fall mechanically). The polarographic vessel is placed in the stand in a water bath controlled by a thermostat. For measurements at higher temperatures (e.g., when the thermal denaturation of polynucleotides is followed), it is necessary, however, to use a special thermostatted vessel (not delivered by the manufacturer) ; this is schematically presented in Fig. 4. The vessel has to be well closed to prevent oxygen from entering it; however, the cover (B) may not interfere with the capillary (G) movements that tear off the mercury drops. If temperature is increased in about 5° steps, thermal equilibrium in this vessel is reached within 1-2 minutes. Quite recently Southern Analytical Ltd. introduced a new Model A 3100 pulse polarograph; this differs from the preceding model by greater variability of some parameters. The delay time, i.e., the period between the birth of the drop and the application of the pulse, is variable from 0.5 to 5 seconds (in Model A 1700, I second only). The voltage sweep is extended from 1 V to 2.5 V, and its rate can be changed from 1 V per minute to 1 V per hour. The pulse amplitude in the derivative method is variable from 2 to 100 mV, and the pulses may be applied either in the same direction as the voltage ramp or in the opposite direction to it. Also, the duration of the pulse as well as the period of current measurement t (Fig. 3e) may be varied independently. The instrument is built in a single floor-standing rack (57 X 91 × 171 cm) designed to be near a laboratory bench upon which the stand could be placed. The electrode stand does not differ from that delivered with Model A 1700. A wide range of variable parameters in the Model A 3100 pulse polarograph may be well utilized in electrochemical research, e.g., in electrode reaction kinetic studies.

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POLAROGRAPHIC TECHNIQUES

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H~O FIG. 4. Vessel for pulse-polarographic measurement at elevated temperature. A, mechanical drop controller; B, stopper; C, rubber tubing; D, silicone oil; E, mercury reference electrode; F, coat; G, capillary; H, thermometer.

CPA-3 Polarographic Analyzer produced by Melabs, Palo Alto, California contains four modes of operation: classical D.C. polarography, single sweep method, derivative and normal pulse polarography. In derivative pulse polarography the pulses of 10, 30, and 100 mV amplitude may be applied in both directions to the voltage ramp. The pulse duration is not variable. The mercury electrode stand is equipped with a water bath controlled by a thermostat within 20°-40°C. The mechanically controlled drop time is variable from 0.5 to 2 seconds. Quite recently production of polyfunctional electrochemical instruments has been started by Princeton Applied Research Corporation, Princeton, New Jersey. Their apparatuses PAR 170 and PAR 171 have the following capabilities: D.C. polarography, phase-sensitive A.C. polarography, normal and derivative pulse polarography, direct potentiometry, controlled-potential electrolysis, anodic stripping analysis, specific ion and pH measurements. The more complex PAR 170 offers further possibilities: cyclic voltammetry, controlled-current electrolysis, chronopotentiometry,

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TECHNIQUES FOR ST~UCTURhL ANALYSXS

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chronoamperometry, coulometry at controlled potentials, coulometry at controlled current, chronocoulometry and pulse response studies. The description of these instruments will be limited here to their pulse-polarographic units. In the derivative method, pulses of 50 mV fixed amplitude are used. In PAR 170 the pulse duration is 45-65 msec (56 msec in PAR 17l) and the current is measured over the last 5-20 msec (16 msec in PAR 171). The method of measurements of potentials in PAR instruments secures a high level of accuracy. There is a little doubt that the large number of electrochemical methods offered by PAR electrochemistry systems will be of great use in chemical analysis and especially in the study of the electrode processes. Use of Polarographic Methods in the Study of the Nucleic Acid Secondary Structure The fact that the polarographic behavior of single-stranded polynucleotides differs from the behavior of double-stranded ones makes polarographic methods useful in nucleic acid structure research. The significance of polarographic methods lies mainly in their sensitivity to small structural changes in a polynucleotide and also in the fact that they differ in principle from techniques hitherto used for nucleic acid structure studies. It is necessary to note that polarography cannot yield absolute data on polynucleotide structure such as can be obtained by X-ray analysis; moreover, it does not directly reflect the helical content in the studied polynucleotide. By polarography, the reducibility of groups contained in the polynucleotide and the ease of the reduction can be followed. The suppression of the polarographic reducibility in double-stranded polynucleotides as in native DNA, poly(A). poly(U) and poly(I).poly(C) is explained by the inaccessibility of potentially reducible groups for the electrode process. 5,7,sIf changes in availability of potentially reducible groups of a polynucleotide are followed during the helix-coil transition, the curve obtained need not agree perfectly with denaturation curves obtained by other methods (e.g., spectrophotometry). So far relations between the polarographic behavior and the secondary structure of DNA ~,7,8,19,~°,23,~5-4° and the synthetic polyribonucleotides5,6,~ have been studied, but ahnost no measurements have been taken with RNA. 36E. PaleSek, Z. Chem. 2, 260a (1962), Abh. Deut. Akad. Wiss. Berlin, Kl. Med. p. 270 (1964). " E. PaleSek, J. Mol. Biol. 11,839 (1965). 37E. Luk~f~ovfiand F,. Paledek, Biophysik 3, 272 (1966). 38E. Luk~ov~, Biophysik 5, 183 (1968). 39E. Pale~ek, Biochim. Biophys. Acta 145, 410 (1967). 40E. Pale/!ek, Arch. Biochem. Biophys. 125, 142 (1968).

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POLAROGRAPHIC TECHNIQUES

15

The Background Electrolyte. Measurements were performed mostly in buffered 0.3-1.0 M ammonium formate (pH 7), in which single-stranded polynucleotide~ produced well-developed steps, waves, and indentations. Measurements can be carried out even in other background electrolytes with usual polarography responses. If, however, the measurements are performed at neutral or weakly alkaline pH, it is necessary to work at sufficiently high ionic strength and in the presence of ammonium ions [e.g., poly(C) yields a well-developed step in 1 M ammonium formate with Britton-Robinson buffer 4°s (pH 8.5), whereas in the 1 M NaC1 medium with Britton-Robinson buffer it is, at the same pH, already nonreducible]. The effect of ammonium ions is perhaps related to their good ability to screen phosphate groups, and thus decreasing repulsive forces between the polyanionic molecule and the negatively charged electrode. 6,.1 At acidic p H values (where the reduction takes place at more positive potentials), the measurements can be done at lower ionic strength and in the absence of ammonium ions. If D.C. polarography or normal pulse polarography is used, it is advisable to work in a medium such that the studied polynucleotide yields a diffusion-controlled current, especially if the results are to be compared with theory. (The diagnostic tests for differentiation between various polarographic currents can be found in the literature). 1,~ Synthetic Polyribonucleotides In Fig. 5b,c, are given derivative pulse polarograms of the doublestranded complexes of poly(A) poly(U) and poly(I).poly(C) in which the concentrations of poly(A) and poly(C) correspond to the concentrations of the single-stranded polynucleotides given in Fig. 5e, f. The waves produced by complexes are substantially lower and wave summit potentials (Es) are more positive than E, of corresponding single-stranded polynucleotides. Es of poly A differs from E, of poly(A).poly(U) to such an extent that both substances can be determined simultaneously provided that their waves are approximately of the same height. T h e polarographic mixing curves yield in principle the same results as spectrophotometric ones, their shape, however, may be different (Fig. 6). The transition of single-stranded 40~A universal buffer that can be used in the pH range 2-12. It consists of 40 mM acetic acid, 40 mM H3BOs, and 40 mM H,~ 04 plus a certain amount of 0.2 M NaOH necessary for the given pH value -to obtain pH 7.0 buffer, 72.5 ml of 0.2 M NaOH is added to 100 ml of the above-mentioned acids). More details are given in the literature, e.g. : M. Bi~ezina and P. Zuman, "Polarographie in der Medizine, Biochemie und Pharmazie," p. 652. Geest und Portig, Leipzig, 1956. H. T. S. Britton, "Hydrogen Ions," p. 365. Chapman and Hall, London, 1955. 41V. Brabec and E. Paledek, Biophysik 6, 290 (1970).

16

T~.CHNZQU~ FOR STRUCTURAL ANALYSIS

(a)

(b)

[1]

(c)

I I I

,

I

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-I.2I

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(d)

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Potential

FIG. 5. Pulse polarograms of double-stranded and single-stranded polynucleotides. Upper row: double-stranded polynucleotides: (a) Native calf-thymus DNA at a concen-

tration of 470 pg/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/20 (wave II). (b) ] × 10-4 M poly(I) • poly(C) in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/80. (c) 1 X 10-4M poly(A) • poly(U) in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/40. Lower row: single-stranded polynucleotides: (d) Denatured calf-thymus DNA at a concentration of 50 ~,g/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/40 (wave III). (e) 5 × 10-6M poly(C) in 0.3M ammonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/80. (f) 5 × 10-~M poly(A) in 0.3 M anunonium formate with 0.1 M sodium phosphate (pH 7); sensitivity 1/40. Molar concentrations of polynucleotides are based on the phosphorus content. Measurements were performed on a Model A 1700 Southern-Harwell pulse polarograph, Mark II. The potentials were measured against the mercury pool at the bottom of the polarograph/c vessel.

[1]

17

POLAROGRAPHIC TECHNIQUES

"~ c .o

50

.w

.>

o

c-

25

._~ ¢-

o,-

\\

\ C

\

\

i,

i

I

i

~I

l

0

25

50

75

I00

125

Mole % of poly(I)

FIG. 6. Formation of the 1: 1 complex of poly(C) - poly(I) followed by pulse-polarographic method. Homopolymers were mixed in 0.1 M NaC1 with 0.01 M sodium phosphate (pH 7). After 2 hours of incubation at room temperature, the supporting background electrolyte was added. The pulse-polarographic measurements were carried out in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 6.9). The sensitivity of the Model A 1700 Southern-Harwell pulse polarograph was 1/80. Concentration of poly(C) (4 × 10-s M) was held constant in all samples while the amount of poly(I) varied as indicated in the figure. The height of the more positive pulse-polarographic wave of poly(C) was measured. After E. Pale~ek, Pro~. Nucl. Acid. Rea. Mol. Biol. 9, 31 (1969). poly(A) and poly(C) into their protonated double-stranded forms can be also followed b y means of polarographic techniques? ,~ Native and D e n a t u r e d D N A ' s Denatured D N A produces at sufficiently high concentration (about 100-500 ~g/ml) a D.C. polarographic reduction step, which has, under suitable conditions, characteristics of the diffusion-controlled current. 41 On the other hand an equally concentrated solution of native D N A is D.C. polarographically inactive. If native D N A is analyzed under the same conditions b y means of more sensitive pulse-polarographie method (Fig. 5a) a relatively small wave (wave II) can be observed, whose E~ is more positive 4, E. Pale~ek, in preparation.

18

TECHNIQUES FOR STRUCTURALANALYSIS

[1]

than E8 of the wave (wave III) produced by denatured DNA (Fig. 5d). The results so far obtained suggest that for wave II of native DNA, faradaic processes are responsible and that the wave height is influenced by the presence of labilized regions in the DNA double helix (see the next section). It follows from Fig. 5a,d that wave III of denatured calf thymus DNA is more than hundred times higher than wave II of equally concentrated native DNA. The great difference in wave heights (observed in all studied DNA samples isolated from various sources), as well as sufficiently different E8 of both waves, renders it possible to estimate small amounts of denatured DNA in the presence of an excess of native DNA (see below under Pulse-Polarographic Estimation). Polarographic methods can be utilized also in the study of interactions of various substances with DNA. For studying the interaction of a polarographically reducible low-molecular weight substance with DNA, even the simple D.C. polarographic method can be used. Free depolarizer (in a concentration usual in D.C. polarography) produces a step, while the depolarizer bound to DNA is practically polarographically inactive. In this way DNA interactions with daunomycin,43 methylene blue, ~ acridine orange, 45 and inorganic depolarizers 1°,~-48 were followed. DNA Conformational Changes at Premelting Temperatures Polarographic methods can be used for following the denaturation of double-stranded polynucleotides. When following the thermal denaturation of DNA by measurements at room temperature after quick cooling of the sample, the denaturation curve obtained by polarographic methods is very similar to that obtained by observing optical density at 260 m#. ~,85 If, however the measurements are performed at elevated temperatures, gradual changes, preceding the step-increase in polarographic activity, can be observed at temperatures far below the melting temperature (Fig. 7). These changes were demonstrated first in 1962 with the aid of oscillopolarographic method, and they were explained by changes in DNA conformation at premelting temperatures. 36 Shortly afterward, further reports were published showing that, at these temperatures, changes in DNA properties take place that can be demonstrated by viscosimetry,49 circular 48E. Calendi, A. DiMarco, M. Reggiani, B. Scarpinato, and L. Valentini, Biochim. Biophys. Acta 103, 25 (1965). 44M. J. Simons, Trans. Faraday Soc. 64, 724 (1968). 45A. Humlov~, private communication (1969). 4sI. R. Miller and D. Bach, Bioplymers 4, 705 (1966). 4~I. R. Miller and D. Bach, Proc. Int. Congr. Polarography, Prague, 1966, p. 64. 48j. p. Schreiber and M. Daune, C. R. Acad. Sci. Paris Ser. C 264, 1822 (1967). ~9A. M. Freund and G. Bernardi, Nature (London) 200, 1318 (1963).

[1]

POLAROGRAPHIC TECHNIQUES

19

1500

/J ,,'/]

1.3

g .>_ "0 v

1000

J [

0

X

t--

8

/

¢.-

//

500

•

x/ Y -

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~-A--A--~--~-..~--~..T-~--~--~ 40

60

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FIo. 7. ThermaltransitionofBacillussubtilisDNAtreatedwithdeoxyribonucleaseI. A sample of 375 ~g of B. subtilis DNA per milliliter in 10 mM MgSO4 with 10 mM sodium phosphate (pH 7) was incubated with 9 X 10-4 ~g of DNase I per milliliter at 27 °. The reaction was stopped by adding 0.1 volume of 0.15 M sodium citrate, and samples were withdrawn immediately after the addition of the enzyme and after 30 minutes of incubation. Enzyme-treated DNA: X, pulse polarography; A, optical density at 260 m~. Controh Q, pulse polarography; O, optical density at 260 m~. Pulse-polarographic measurements were carried out at a DNA concentration of 50 ~g/ml in 0.3 M ammonium formate with 0.1 M sodium phosphate. For optical density measurements, the same medium was used. The sensitivity of the pulse polarograph was 1/5 or lower, and the number of divisions was calculated for a sensitivity of 1/5. After E. PaleSek, Arch. Biochem. Biophys. 125, 142 (1968). dichroism, 6° m i c r o c o c c a l nuclease, ~1,52h y d r o g e n e x c h a n g e a n a l y s i s y t h r o u g h following cross-link f o r m a t i o n in D N A b y U V light, 54 etc. I t h a s b e e n shown that polarographic methods, namely, pulse polarography, are very s u i t a b l e for t h e s t u d y of p r e m e l t i n g c h a n g e s in D N A . E , of t h e p u l s e polarographic wave produced by native DNA at premelting temperatures is m o r e p o s i t i v e t h a n E , of d e n a t u r e d D N A , a n d b o t h w a v e s can b e ob50j. Brahms and W. H. F. M. Mommaerts, J. Mol. Biol. 10, 73 (1964). 51 p. H. von Hippel and G. Felsenfeld, Biochemistry 3, 27 (1964). ~ L. Ungert and P. H. von Hippel, Biochim. Biophys. Acta 157, 114 (1968). 53 p. H. von Hippel and M. P. Printz, Fed. Proc. Fed. Amer. Soc. Exp. Biol. 24, 1458 (1965). 54V. R. Gli~in and P. Doty, Biochim. Biophys. Acta 142, 314 (1967).

20

TECHNIQUES FOR STRUCTURAL ANALYSIS

[1]

served simultaneously provided their heights are not too different.~° The wave of native DNA appearing at premelting temperature is probably identical with wave II observed with concentrated DNA solutions at room temperature (Fig. 5a). The thermal denaturation of the DNA samples gently modified by various agents, such as deoxyribonuclease I, gamma rays (10(O4000 rads),4° cross-linking agents, monofunctional alkylating agents, UV light, ultrasound, shearing, 56,~ was followed by means of polarographic and spectrophotometric methods. While no changes in spectrophotometric curves were observed, polarographic curves of the modified DNA samples in most cases differed in premelting temperature regions from the untreated controls. It was presumed that changes in DNA conformation, which occur at premelting temperatures, take place in labilized regions of the double helix, i.e., in sequences rich in adenine and thymine on one hand, and on the other hand, in regions where bases loop out from the double-helical structure, where phosphodiester bonds are broken, etc. m° These changes are characterized by a local opening of the double helix that might include changes in angles and distances between the adjacent bases, the rupture of the hydrogen bonds, and changes in hydration; the bases, however, remain vertically stacked. Pulse-Polarographic Estimation of Denatured D N A in the Presence of Native D N A

Method Principle. Denatured DNA produces a pulse-polarographic wave, and from the height of this wave, DNA concentration is estimated. 67 The presence of native DNA practically does not interfere with the estimation.

Reagents Background electrolyte containing ammonium formate, 0.3 M, and sodium phosphate, 0.1 M, pH 7.0 Mercury Oxygen-free nitrogen or argon

Apparatus. Pulse polarograph Model A 1700 or A 3100 (Southern Analytical Ltd.) with a dropping mercury electrode. Procedure. Reference DNA is prepared by denaturing of a small part of the sample in which denatured DNA is to be estimated. The denaturation 66 E. Luk~tov~ and E. Pale~!ek, in preparation. 6, M. Vorlidkov~ and E. Paledek, FEBS Lett. 7, 38 (1970). b7 E. PaleSek and B. D. Frary, Arch. Biochem. Biophys. 115, 431 (1906).

[1]

21

POLAROGRAPHIC TECHNIQUES

is usually carried out b y heating D N A in a concentration of 15-30 #g/ml in 0.015 M NaC1 with 0.0015 M sodium citrate (pH 7) at 100 ° for 10 minutes and a subsequent quick cooling in an ice-bath. Denatured reference DNA, 3-5 ml, in a known concentration (usually 10-30 #g/ml) in 0.3 M ammonium formate with 0.1 M sodium phosphate (pH 7.0) is placed into the conventional pulse-polarographic vessel (with a pool of mercury on the b o t t o m serving as a reference electrode). The volume of the D N A sample can be reduced to about 0.5 ml if a special vessel is available (see above in section on pulse polarography). T h e vessel is then placed into the polarographic stand, and the solution is bubbled with oxygen-free nitrogen or argon for about 5 minutes (altogether 4 samples can be simultaneously bubbled). Only short bubbling (about 1 minute) is necessary for a prebubbled sample after the dropping mercury electrode is dipped into the solution. T h e pulse-polarographic curve is recorded starting from - 1 . 2 V.

oo I

/

200

~'

,oo~-

=p-

0

0

I

I

I

I

20

40

60

80

DNA concentration (/~g/rnl)

FiG. 8. The dependence of the height of pulse-polarographic wave III on the concentration of denatured DNA. O, Denatured DNA only; O, 1 mg of native DNA/ml plus denatured DNA in the concentration given in the graph. Measurements were carried out in 0.3 M ammonium formate with 50 mM sodium phosphate (pH 7). The sensitivity of the Southern-Harwell pulse polarograph was 1/5 or lower, and the number of divisions was calculated for the sensitivity 1/5. After E. Pale~ek and B. D. Frary, Arch. Biochem. Biophys. llS, 431 (1966).

22

TECHNIQUES FOR STRUCTURAL ANALYSIS

[1]

Usually, the following instrument setting is used: 1 V in 7.5 minutes; derivative 35 mV; integration 3; recorder sensitivity 1/5; amplifier sensitivity within 1/1-1/16 depending on the concentration of denatured DNA; autotrigger 2 seconds. Registration of one curve takes less than 5 minutes. Denatured DNA at a concentration of 15 ~g/ml can be estimated with a standard error -4-2%. The height of wave III is linearly dependent on concentration of denatured DNA, and the influence of the presence of native DNA on height of wave III of denatured DNA is very small (Figs. 8 and 9). If, however, the analysis of small amounts of denatured DNA is performed in concentrated, very viscous solutions of native DNA, larger decrease in the height of wave I l I may occur. In such a case, it is advisable to compare the obtained wave height with height of wave III of a model mixture of native and denatured DNA's having the same optical density (at 260 m~) and similar height of wave III as the analyzed sample, or first to record the polarogram of an exactly known volume of the unknown sample and

Ill

A

J d

c

b

o

FIG. 9. Derivative pulse polarograms of calf thymus DNA. (a) 15 ~g of denatured D N A per milliliter; (b) 15 ~g of denatured D N A plus 7.5 ~g of native DNApermilliliter; (c) 15 ~g of denatured D N A plus 75 ~g of native D N A per milliliter; (d) 15 ~g of denatured D N A plus 212 ~,g of native D N A per milliliter. The measurements were carried out in 0.3 M ammonium formate with 50 m M sodium phosphate, pH 7.0, with the Model A 3100 pulse polarograph. Instrument settings were: 1 V in 15 minutes; derivative 50 mV; integration 3, from - 1 . 3 V; recorder sensitivity, 1/5; amplifier sensitivity, 1/8; autotrigger, 2 seconds. In the figure, two possible ways of the measurements of wave height (A, B) are demonstrated.

[1]

POLAROGRAPHIC TECHNIQUES

23

then add a known volume of the reference DNA ("standard addition method," p. 398 of Meitesl).

The Influence of Molecular Weight and Aggregation of Denatured DATA The estimation of denatured DNA is based on he presumption that the molecular weight and the degree of aggregation of the analyzed DNA do not differ from those of the reference DNA. Small changes in molecular weight of the denatured DNA do not substantially influence the height of wave III (the decrease of the molecular weight of calf thymus DNA by one third, caused by shearing of native DNA of molecular weight about 1 X 107, manifested itself after denaturation by 8% increase in the wave III). When large differences between the molecular weight of the analyzed DNA's and reference DNA are expected, it is advisable to correct the wave heights obtained or to use a more suitable reference DNA. Aggregation of the denatured DNA decreases the height of wave III. If, for some reason, larger aggregation is expected in the analyzed sample than in the reference DNA, at least partial disaggregation can be achieved by heating the samples at low ionic strength (e.g., in 0.015 M NaC1 with 0.0015 M sodium citrate pH 7 for 15 minutes at 65 °) and subsequent quick cooling.

Influence of Impurities Substances producing pulse-polarographic waves at potentials close to the potential of wave III of denatured DNA [e.g., poly(C)] can interfere with the estimation. The estimation can be also influenced by substances causing t h e shift of the background electrolyte discharge to more positive potentials (this is because wave III appears in the vicinity of the potential of the background discharge). If the measurements are carried out at top sensitivities of the instrument, the polarogram may be deformed also by polarographically nonreducible substances adsorbing on the electrode. The pulse-polarographic method cannot be used for the estimation of denatured DNA in a nucleoprotein. The presence of proteins causes the shift of the background electrolyte discharge to more positive potentials and decrease of the wave III. Larger amounts of RNA in the analyzed sample should also be avoided. RNA may deform wave III and increase its height. In comparison with proteins and RNA the presence of polysaccharides is less critical (the influence of starch, agar, and polygalacturonic acid was tested). Amounts of proteins and RNA varying around the values usual in purified DNA samples do not interfere with the estimation. Closing Remarks Methods of electrochemical analysis have been introduced in nucleic acid research much later than most of other physical chemical methods (e.g., optical methods). In spite of this delay it appears that application of

24

TECHNIQUES

FOR STRUCTURAL

ANALYSIS

[2]

polarographic techniques may still yield useful results and that these techniques should be further developed. In the experiments where the polarographic method is used for the detection or estimation of a polynucleotide with a known secondary structure (e.g., for the estimation of denatured DNA in the presence of native DNA), the method can be used by the worker who is not interested in electrochemistry. If, however, from the polarographic behavior of a polynucleotide (e.g., from a comparison of heights and EI/~ of two steps) conclusions on polynucleotide secondary structure are to be made, it is necessary to have a certain knowledge of electrochemistry, discussion of which has not been included in this article, as well as knowledge of structural polymer chemistry. Some results not described in this paper have been obtained also with the aid of methods following adsorption of nucleic acids in a polarized water/mercury interface? ,8,~s-6° Further development of the study of adsorption of nucleic acids in electrically charged surfaces may yield information basic to a better understanding of the interaction of nucleic acids with cell membranes and particles as well as with various macromolecules. ~8I. R. Miller, J. Mol. Biol. 3, 229 (1961). ~gH. Berg, H. Biir, and F. A. Gollmick, Biopolymers 5, 61 (1967). J. Flemming, Biopolymers 6, 1697 (1968).

[2] L u m i n e s c e n c e S p e c t r o s c o p y of N u c l e i c A c i d s

By J. EISINGER and A. A. LAMOLA I. Introduction A. Emission Spectroscopy in Biochemistry

Absorption spectroscopy is probably the most widespread physical technique employed in biochemistry. Its uses include the spectroscopic identification of compounds, the determination of concentrations of solutes in solution, and the monitoring of reactions or conformational changes. The inverse process to light absorption, the emission of photons from excited molecules, has on the other hand found only relatively specialized applications in molecular biology. The reasons for this are manyfold: Emission spectroscopy requires more elaborate instrumentation than does absorption spectroscopy; it is not universally applicable (some molecules do not emit light); it requires greater care (impurities produce artifacts more commonly than they do in absorption); and emission spectra are generally more sensitive to the temperature, pH, concentration, and other conditions