Current Polarographic Investigation of Polysaccharides in DNA B. MALFOYAND J. A. REYNAUD


de Biophysique



Orleans Cedex,


Received October 20, 1975; accepted August 29, 1977 Polysaccharides alone or in the presence of DNA are studied by means of alternating current (ac) polarography. When neutral and basic polysaccharides are used, the polarograms recording the quadratic component of the current display one capacitive peak at -1650 mV (SCE). Acid polysaccharides never show this peak and are desorbed from the electrode at more positive potentials. If dextran is used as a reference, this peak allows the determination of the amount of neutral polysaccharides in solution up to 2 PgIml. The height of this peak has no relation to the ionic strength or pH of the solution within the investigated range. Likewise, the concentration and molecular weight of DNA enclosed in the solution exert no influence upon the peak height. On the other hand, the presence of polysaccharides causes DNA peaks to decrease considerably. ac polarography can, therefore, be regarded as a quick, convenient, and sensitive method for carrying out the titration of polysaccharides alone or mixed with DNA.

During our comparative alternating current (ac) polarographic study of DNAs from various sources (l-4), the aberrant behavior of some preparations led us to investigate the problem of DNA contamination by macromolecules. It is a well-known fact that DNA samples can contain some polysaccharides acting as contaminating agents, in addition to RNA and proteins, which can be completely removed by means of enzymatic treatment (Pronase, RNase) and through the agency of organic solvents (chloroform, phenol). The polysaccharides can be detected through a cesium gradient (5). Up to the present time, no efficient method of separation has been defined except for the process of separation on a sucrose gradient (6), but this process can be applied only to small amounts of polysaccharides. More recently a process of separation by affinity chromatography performed on Sepharose with concanavalin A as a substitute has been introduced (7). This procedure solves the problem of removing the starchlike and glycogen-like DNA contaminants. The estimation method used by the authors to determine polysaccharide quantity is based upon the spectrophotometric scattering profile (8). This method is not very accurate 1

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and is only suitable for high polysaccharide concentration. Moreover, the processes of determining the presence and quantity of polysaccharides alone in solution, based exclusively upon calorimetric techniques, are difficult to use, show little sensitivity, and ruin the sample (9). In this paper, we deal with the conditions of a polarographic approach and the dosage of polysaccharides alone or in the presence of DNA. EXPERIMENTAL material. The bacterial DNAs were extracted according to method (10) from cells of various sources: Micrococcus lysodeikticus (ML), lyophilized cells purchased from Miles; Escherichia coli (EC), Proteus vulgaris (PV) hydrated cells obtained from Laboratoire de Chimie Bacterienne, Marseille, France. Calf thymus DNA (CT-DNA) was prepared according to the method of Kay et al. (11) while the extraction of chicken erythrocytes (CE) was performed by means of Bernardi and Sadron’s technique (12). Full deproteinization of DNA following treatment with Pronase was achieved by repeated shaking with phenol. Dextran was purchased from Pharmacia or Sigma (molecular weight: 0.5 x 106). Chondroitine sulfate (from shark cartilage), hyaluronic acid (from human umbilical cord), glycolchitine, polygalacturonic acid, and pectin (from citrus fruits) were purchased from Sigma; deacetyled chitin was purchased from Fluka. Physicochemical methods. The measurements were carried out by means of a SOLEA PRG 3 polarograph including a phase-sensitive ac polarographic unit fitted with a drop time-controlling device. The phase angle was adjustable from -45” to + 135”, allowing for both in-phase and out-of-phase (or the quadratic component) detection of the ac response. A three-electrode system was used. A polarograph cell built in the laboratory allows experiments to be performed with 1 or 2 ml of solution. The capillaries used had the following characteristics: open circuit, 50-cm-high mercury column, 1 M NaCl. Capillary I flow: m = 0.16 mg/sec; drop time t = 23 sec. Capillary II flow: m = 0.17 mg/sec; drop time t = 22 sec. The hanging mercury drop electrode (HMDE) was a P.A.R. Model 9323 with a surface area of 3 x 10e2 cm2. The electrode potentials are referred to the saturated calomel electrode (SCE). The experiments were performed in a buffer solution of 5 x 10e3 M Tris plus 5 x 10e3 M sodium acetate, with NaCl as the supporting electrolyte. The heights of the peaks were measured against the supporting electrolyte. The scan rate used was 1 mV/sec. The ultrasonic DNA degradation was performed with a 19-W MSE ultrasonic disintegrator at a frequency of 20 kC under conditions stipulated by Obrenovitch and Aubel-Sadron (13). The sedimentation coefficient measurements were carried out with a Beckman Type E analytical Biological





ultracentrifuge. The molecular weights were calculated by using Studier’s formulas (14). The dichroism spectra measurements were carried out with a JOUAN dichrograph. RESULTS I. ac Polarography

of Native


Adsorption of native DNA onto a mercury electrode occurs within a wide range of potentials until about -1 V (2-4,15-17). At the most negative potentials several peaks appear. The capacitive nature of peak 1 (Fig. 1) corresponds to some phenomenon of rearrangement which takes place at the level of the adsorbed bases (3). Peak 2 does not appear on the polarograms which record the quadratic component of the current, but does appear when DNAs of low molecular weight are used. In an acid medium, peak 2 indicates the reduction of the DNA bases in the adsorbed state while no desorption of the reduced product occurs (1,2). In a basic medium, peak 2 corresponds to the desorption of the macromolecules (3). If there is some mixed denatured DNA in a solution of native DNA, a new peak 3 appears on the polarograms. 2. Polarographic


of Polysaccharides

Until now there have been only a few polarographic studies of polysaccharide derivates. Brabec and Palecek (15), after studying a number of polysaccharide polarograms obtained by ac polarography, came to the conclusion that these substances are adsorbed at the electrode around the electrocapillary maximum and that most of them yield a desorption peak around the potential of - 1 V. Flemming (18) performed a study of the dextran sulfate derivatives as a DNA model, using ac polarography at hanging mercury drop electrode. The presence of two


- 0,5


FIG. 1. ac polarograms of native DNA. CT-DNA: pH 5.6, phase angle: W, frequency: 78 Hz, drop



400 pg/ml, M,: 0.5 x 106, NaCI: time: 7.6 sec. Capillary I.

0.5 M,




FIG. 2. ac polarograms of dextran. Dextran: 170 pg/ml, NaCI: 0.5 90”, frequency: 78 Hz, drop time: 7.6 sec. Capillary I.


pH 5.6, phase angle:

peaks is indicated, the first being located at about -650 mV, the second, about - 1650 mV. We investigated the conditions of polysaccharide adsorption at the mercury electrode by using ac polarography with phase-sensitive demodulation. When dextran is used, the polarograms registering the quadratic component of the current display two peaks (Fig. 2): One is weakly developed around -900 mV (peak I); the other is largely spread out around -1650 mV (peak II). Peak I at -900 mV is characterized by a phase angle approaching 90” (Fig. 3A). At 0 and 90”, the resistive and capacitive components go through a linear variation in terms of the superimposed voltage (Fig. 3B). This peak is linked with a change occurring under the conditions of macromolecule adsorption at the electrode. This change, however, does not give rise to any desorption (19,20). Peak II at - 1650 mV is characterized by a phase angle of 90” and goes through a linear variation in terms of the frequency (Fig. 3A). This variation means, therefore, that the phenomenon we are dealing with is of a capacitive nature, such as some fast rearrangement at the electrode (19,20). While peak height does not depend upon ionic strength, the potential of the peak shifts toward positive values when the ionic strength increases. Such behavior shows the influence of the supporting electrolyte upon the thickness of the double layer and the electrokinetic potential (21). Moreover, peak II appears with low concentrations of dextran (2 pg/ml) and allows, therefore, determination of the presence of a small



Ip nA


FIG. 3. Dextran’s peaks I and II. Dextran: 100 &ml, NaCI: 0.5 M, pH 5.6. Capillary I. (A) Phase angle dependence; frequency; 78 Hz: (0 0) peak I, (M n ) peak II. (B) Frequency dependence: (0 l ) peak I, r$ = 90”; (0 0) peak II, 4 = 0”; (W W) peak II, d=W.

amount of dextran in solution. With increasing concentration of polysaccharides, the peak rises, then comes to a limit when approaching values above 150 &ml (Fig. 4A). Its height has no relation to pH, which means that the behavior of the neutral macromolecule of dextran at the electrode has no connection with the pH within the investigated range (5 < pH < 8). We have compared the ac polarographic results with those obtained by the classical method of neutral sugar dosage: degradation by concentrated sulfuric acid in the presence of phenol and optical measurements of the concentration (22). The detection limit of this method is similar to that of the polarographic method, but the reproducibility and accuracy of the classical method are inferior at low concentration. When a test solution of 10 &ml of dextran is used, the margin of error for the polarographic method is about 2.5% (30 -+ 0.7 nA), and with the optical method the margin of error is 7% (0.07 -+ 0.005 OD,,,). The polarographic method also introduces the advantages of not being destructive and of being usable in various media. With dextran sulfate, the polarograms recording the quadratic component of the current display only one peak at -6.50 mV (Fig. 5). Its height and potential have no relation with the pH over the range investigated. Beyond this peak, the polarogram of the supporting electro-




150 pg/ml


50 Dextmn


150 pg/ml


FIG. 4. (A) Variation of peak II of dextran with the concentration. NaCl: 0.5 M, phase angle: 90”, frequency: 78 Hz, drop time: 7.6 sec. (0 0) pH 5.6, (0 0) pH 7.8. (B) Variation of dextran’s peak II with the concentration in a 400~pg/ml solution of DNA. NaCl: 0.5 M, phase angle: W, frequency: 78 Hz, drop time: 7.6 sec. (0 0) DNA PV, M, = 0.5 x 106, pH 5.6; (0 0) DNA PV, Mw = 0.5 x 108, pH 7.8; (0 0) DNA PV, Mw = 9.6 x 106, pH 5.6. Capillary 1.

lyte joins that of the polysaccharide. The electrochemical properties of the peak (phase angle between 45 and 90”, with a shift when the frequency is increased) allow us to conclude that the properties are connected with the desorption of the negatively charged macromolecules when the electrode becomes negative. Similar behavior is observed with polyphosphate (23). The limit of detection is 5 pg/ml. Using ac polarography at HMDE, we were not able to record any peak at -1650 mV as Flemming did (18). Whatever the concentration, pH, or ionic strength may

FIG. 5. ac polarograms of dextran sulfate (200 &ml). frequency: 78 Hz, drop time: 7.6 sec. Capillary II.

NaCl: 0.5 M, pH 5.6, phase angle: 90”,




be, the peak stays at about -600 mV. Meanwhile, at HMDE as DME, this peak disappears in a few hours, and badly resolved peaks become evident at the more negative potentials. These kinds of peaks are also observable when solutions are used in which the macromolecules are not completely dissolved. This behavior can be connected with hydrolysis and/or the aggregation of dextran sulfate in solution. With amino- or acetamidopolysaccharides, the behavior of the macromolecules at the electrode is more complex. Chondroitine sulfate which carries two charges in repeating unit (carboxyl and sulfate) presents only one desorption peak at -850 mV. Hyaluronic acid (one carboxyl group by sugar) presents one desorption peak at - 1200 mV. On the other hand, polysaccharides without the acidic function (deacetylchitin, glycolchitin) present a peak at - 1650 mV. One can conclude from these experiments that, when a polysaccharide does not carry negative charges, it is characterized by one peak largely spread out around -1650 mV. If the polysaccharide carries negative charges, the macromolecule is desorbed in the area of the electrocapillary maximum. The desorption potential is largely dependent on the nature of the other sugar substituents. A typical result of this behavior, for example, is that polygalacturonic acid (Fig. 6) is desorbed at -700 mV. But when the acidic function is esterified, this peak disappears and is replaced by a peak at -1650 mV. 3. Neutral Polysaccharides in the Presence of DNA

With some DNAs, we observe, on the polarograms registering the quadratic or out-of-phase component of the current, a peak, round shaped and

FIG.6. ac polarograms. NaCl: 0.5 M, pH 5.6, phase angle: 90”, frequency: 78 Hz, drop time: 7.60 sec. Capillary II. (0) Polygalacturonic acid, 200 fig/ml; (----) pectin, 300 pg/ml; (- - - -) supporting electrolyte.





widely spread out, situated at - 1650 mV (Fig. 7B,), and with some of the samples this peak completely screens off DNA peaks 1 and 2 (Fig. 7C,). At the same time, the in-phase component of DNA peak 2 current decreases whereas the molecular weights of the DNA investigated are identical (Figs. 7Az, Bz, C,). This round-shaped peak cannot be removed by dialysis or by precipitation of DNA with ethanol. The addition of RNA and protein has no direct influence upon it. In terms of the frequency, this peak goes through a linear variation, and it reaches its maximum value at 90” (the presence of DNA prevents an accurate determination of the peak’s height for the lowest values of phase angle). If dextran is added to a DNA solution which already registers this peak, intensity increases. All these characteristics suggest that the peak displayed at - 1650 mV (peak II) on DNA polarograms is due to the presence of neutral polysaccharides in the solution. Interaction between polysaccharides and DNA. The presence of neutral polysaccharides in a solution of DNA makes DNA peaks 1 and 2 collapse. IpnA


Ip nA Bl



Ip nA 30

- 0,7







-I,3 -I,4


-I,6 v


FIG. 7. ac polarograms of native DNAs containing neutral polysaccharides. DNA: 400 pg/ml, Mw = 0.5 x 106, NaCI: 0.5 M, pH 5.6. Capillary I. (-) A, B, and C: EC-DNA from various preparations; (- - - -) supporting electrolyte. (1) Phase angle: 90”. frequency: 78 Hz, drop time: 7.6 sec. (2) Phase angle: 0”. frequency: 32 Hz, drop time: 3 sec.




This effect is much more pronounced in a basic than in an acidic medium. In an acidic medium, peak 1 slightly decreases at first and remains constant afterward, while in a basic medium, it decreases continually (Fig. 8A). In an acidic medium, peak 2 decreases when dextran is added to the solution (Fig. 8B), and the slope of the curve shows a discontinuity; whereas in a basic medium, peak 2 decreases regularly and disappears completely when the concentrations are greater than 50 &ml (Fig. 8B). On the other hand, under our experimental conditions, the presence of DNA has no influence upon the height of the neutral polysaccharides’ peak II. The height ofthe peak, within the margin of experimental error, does not depend on the concentration and molecular weight of DNA; for any amount of dextran in solution, with or without an added 400 &ml of DNA, the heights are identical (Fig. 4). These conclusions are in agreement with recent studies concerning the adsorption of DNA (24-26), which show that whatever the pH may be the nonreduced DNA is no longer adsorbed at potentials more negative than - 1600 mV. Therefore, the competition of adsorption which takes place at the electrode between DNA and neutral polysaccharides is favorable to the latter in the potential region of this peak II, and thus the highly changeable amount of polysaccharides enclosed in bacterial DNA and the strong influence their amount exerts upon the peaks of DNA in ac polarography make it quite difficult to carry out a comparative study of the polarographic signals of native DNAs of various origins. The dichroism spectra of DNA and of DNA plus dextran are identical; this means that there is no direct interaction between dextran and DNA in solution.



25 FIG. DNA: lary I. angle:


75 pg/ml



75 w/ml

8. Variation of native DNA’s peaks with the concentration of dextran in solution. PV0) pH 7.8, (0 0) pH 5.6. Capil4OO&ml, MW: 0.5 x 106, NaCI: 0.5 M, (0 __ (A) Peak 1: phase angle: 90”, frequency: 78 Hz, drop time; 7.6 sec. (B) Peak 2: Phase 0”, frequency: 32 Hz, drop time: 3 sec.




4. Titration Conditions of Neutral Polysaccharides in DNA

The classical calorimetric method cannot be used when one wants to determine neutral polysaccharides in the presence of nucleic acid, due to the reactivity of the ribose moieties of the DNA. The use of the ac polarographic technique allows determination of the presence of a small amount of neutral polysaccharides in solution when alone or mixed with DNA. The height of peak II is not related to the pH or the ionic strength, nor to the concentration or molecular weight ofthe DNA possibly present in solution. When investigating with a superimposed voltage of 10 mV and at a frequency of 78 Hz, it is possible to detect the presence of a small amount of polysaccharides: 2 kg/ml. The quantity of polysaccharides is formulated in an equivalent amount of dextran by referring to a standard curve. We have determined the amount of neutral polysaccharides present in various preparations of DNA by referring to the standard curve (Fig. 4). This amount varies in a manner which cannot be accounted for by the difference in the conditions of DNA preparation. With six different samples of DNA from E. coli, the variation was between 2 and 160 pg/ml. One sample of DNA from Micrococcus fysodeikticus was without polysaccharides; three others contained between 20 and 40 pg/ml. The sample ofProteus vufgaris DNA used in our experiments and all the tested preparations of calf thymus DNA did not contain any neutral polysaccharides. The ac polarographic method appears to be more accurate than the classical calorimetric method. Moreover, it is not a “destructive” method and can be applied, without correction, under various experimental conditions. This method is unrivaled for the low-quantity dosage of neutral polysaccharides in the presence of a large excess of DNA. ACKNOWLEDGMENT The authors are indebted to Mr. Gremy for his skillful technical assistance.

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10. Marmur, J. (1961)J. Mol. Biol. 3, 208-218. 11. Kay, E. R. M., Simmons, N. S., and Dounce, A. L. (1952) J. Amer. Chem. Sot. 74, 1274- 1276. 12. Bernardi, G., and Sadron, Ch. (1964) Biochemistry 3, 1411-1418. 13. Obrenovitch, A., and Aubel-Sadron, G. (1971)5. Chem. Phys. 68, 521-526. 14. Studier, F. W. (1%5)J. Mol. Biol. 11, 373-390. 15. Brabec, V., and Palecek, E. (1972) Biopolymers 11, 2566-2589. 16. Berg, H., Tresselt, D., Flemming, G. J., Bar, H., and Horn, G. (1969)J. Electroanal. Chem. 21, 181-186. 17. VaJenta, P., and Niimberg, M. W. (1974) J. Electroanal. Chem. 49, 55-75. 18. Flemming, J. (1973) J. Electroanaf. Chem. 47, 127-145. 19. Frumkin, A., and Damaskin, B. B. (1964) Modem Aspects of Electrochemistry (Bockris, J. O., and Conway, B. E., eds.), Vol. 3, p. 149, Butterworth, London. 20. Frumkin, A. N., and Melik-Gaikazyan, V. I. (1963) Zh. Friz. Khim. 26, 1184-1188. 21. Grigoriev, M. B., and Damaskin, B. B. (1968) Advances in the Electrochemistry of Organic Compounds, p. 68 IZD Nauka, Moscow. 22. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956) Anal. Chem. 28, 350. 23. Vetterl, V., and Bohacek, J. (1968) J. Electroanal. Chem. 16, 313. 24. Malfoy, B., Sequaris, J. M., Niimberg, M. W., and Valenta, P., (1976) Bioelecfrochem. Bioenerg. 3, 440-460. 25. Sequaris, J. M., Malfoy, B., Niimberg, M. W., and Valenta, P. (1976) Bioeleclrochem. Bioenerg. 3, 461-473. 26. Malfoy, B., Sequaris, J. M., Valenta, P., and Numberg, H. W. (1977) J. Electroanal. Chem. 75. 455-469.

Alternating current polarographic investigation of polysaccharides in DNA.

ANALYTICAL BIOCHEMISTRY Alternating 84, I- 11 (1978) Current Polarographic Investigation of Polysaccharides in DNA B. MALFOYAND J. A. REYNAUD...
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