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Changes in oscillopolarographic behaviour of deoxyribonucleic acids at temperatures below denaturation temperature
J. Mol. BioI. (1965) 11, 839-841
LETTERS TO THE EDITOR
Changes in Oscillopolarographic Behaviour of Deoxyribonucleic Acids at Temperatures below Denaturation Temperature When following the course of heat denaturation of DNA with the aid of alternating current oscillographic polarography (Heyrovsky & Kalvoda, 1960) so that measurements are carried out at room temperature after quick cooling of the sample, the denaturation curve obtained is very similar to that of the dependence of optical density at 260 mJL on temperature (Palecek, 1964,1965). If, however, measurements are performed at elevated temperature, changes can already be observed at temperatures lower than denaturation temperature (Palecek, 1962,1964). This result suggests that the oscillopolarographic behaviour below the melting temperature is due to a change in configuration ofthe DNA molecule. A presumed conformational change has also been observed in DNA at temperatures below the denaturation temperature by studies of viscosity (Freund & Bernardi, 1963), circular dichroism (Brahms & Mommaerts, 1964) and low-angle X-ray scattering (Luzzati, Mathis, Masson & Witz, 1964). The oscillogram dE Idt against E of denatured DNA (Fig. 1) shows an indentation in the anodic part caused by the residues of deoxyguanylic acid (Palecek, 1961) and another indentation in the cathodic part which is not present in the oscillogram of native DNA (Palecek, 1964). We followed the appearance of the cathodic indentation dE
dt
Denatured DNA
Native DNA
/
E FIG. 1. Oacillograms of native and denatured DNA in ammonium formate medium. C, oathodic part; A, anodic part. In this paper only the depth of the cathodic indentation (CJ) W8Il followed.
and its increase with temperature. DNA samples isolated from different bacteriophages, bacteria and mammalian organs were analysed. Measurements were carried out at elevated temperature in an ammonium formate medium in concentrations of 0·1 M, 0·25 M, 0·3 M and 0·5 M with 0·02 M, 0·025 M, 0·05 M and 0·1 M-sodium phosphate (pH 7,0). The apparatus used was a Polaroscope P 524 (Kfizik, Praha), with dropping mercury electrode. The mercury dropping electrode was polarized with alternating current of frequency 50 cIs. In all cases the appearance of the cathodic indentation at temperatures far below denaturation temperature was registered. 839
E. PALECEK
840
The changes observed at low temperatures could be explained by the idea that unchanged DNA was present in the bulk of the solution, but that its structure was changed only secondarily on the electrode due to the repeated cycles of the alternating current. Therefore, we used the so-called "first curve technique" (Heyrovsky & Kalvoda, 1960; Palecek, 1965), which makes it possible for us to distinguish primary and secondary oscillopolarographic processes (so-called oscillopolarographic artifacts). In principle this involves the polarization of the dropping mercury electrode with a single cycle of alternating current for each drop of mercury dripping from the electrode. An indentation of secondary character does not appear on the "first curve"; however, it does appear on the following curves. The experimental arrangement was similar to that employed in our previous studies (Pale~ek, 1965). By the "first curve technique" we followed the course of denaturation of Escherichia coli DNA and recorded changes at as Iowa temperature as about 50°0. The same results were obtained if every drop from the mercury electrode had been polarized through 100r----------------.ii-"'"
~•
80
60
40
20
o
i
x
40
60 Temperature
80
100 (Oe)
FIG. 2. Denaturation curves of E. coli DNA in 0·1 M·ammonium formate with 0·02 M-sodium phosphate (pH 7·0). -e--e-, oscillopolarographic measurements: depth of cathodic indent.ation at polarization of one mercury drop through multiple cycles of alternating current (50 cIs); DNA concentration 60 p.g/mI. -0--0-, oscillopolarographic measurements: depth of cathodio indentation of polarization of one mercury drop through a single cycle of alternating current (500/s), i.e, first curve technique, DNA concentration 60p.g/mI. - X - - X - , spectrophotometric measurements at 260 mp.; DNA concentration 40 p.g/ml. All measurements were carried out at temperatures given on the graph. The measured maximum absorbance at 260 mp. and the maximum depth of indentation were taken as 100%. As zero value, absorbance at 20°C'was used; zero value in oscillopolarographic measurements means that the indentation followed was not present in the oscillogram.
LETTERS TO THE EDITOR
841
the repeated cycles of the alternating current. On the other hand, the curve for the dependence of optical density at 260 mp. on temperature, in the same medium, did not show any increase below the region of denaturation temperature (Fig. 2). The changes registered oscillopolarographically at a temperature lower than that of denaturation were reversible and the cathodic indentation did not show any deepening on the "second" and "third" curves. From the results obtained we can conclude that the changes observed at temperatures lower than the denaturation temperature are not caused by disturbance of the DNA structure due to repeated polarization of the dropping mercury electrode through several cycles of the alternating current. Should the changes in the DNA structure occur on the electrode, they would have to set in at a time interval shorter than 10- 2 second at the beginning of the first cycle and then not continue in the following cycles. We consider it more probable that the changes set in already in the solution due to higher temperatures, but are not detectable by means of the usual methods permitting measurements at elevated temperatures such as optical density and optical rotation measurements. If this is correct, we can imagine that thermal denaturation of DNA is preceded by slow "opening" of the double helical structure over 'a rather wide temperature range. Evidence has been obtained that there exists a certain relationship between nucleotide sequence in DNA and the extent of changes observed at temperatures lower than denaturation temperature (Palecek, unpublished experiments). Institute of Biophysics Czechoslovak Academy of Sciences Brno, Czechoslovakia Received 14 December 1964
E.
PALECEK
REFERENCES Brahms, J. & Mommaerts, W. F. H. M. (1964). J. Mol. Biol. 10, 73. Freund, A. M. & Bernardi, G. (1963). Nature, 200, 1318. Heyrovsky, J. & Kalvoda R. (1960). Oszillographische Polarographie. Berlin: Akademie Verlag. Luzzati, V., Mathis, A., Masson, F. & Witz, J. (1964). J. Mol. Biol. 10, 28. Palecek, E. (1961). Biochim. biophys. Acta, 51, 1. Palecek, E. (1962). Z. Ohem, 2, 260a. Palecek, E. (1964). Abh. deutechen, Akad. Wiss. 270. Palecek, E. (1965). Biochim. biophys. Acta, 94, 293.
LETTERS TO THE EDITOR
Changes in Oscillopolarographic Behaviour of Deoxyribonucleic Acids at Temperatures below Denaturation Temperature When following the course of heat denaturation of DNA with the aid of alternating current oscillographic polarography (Heyrovsky & Kalvoda, 1960) so that measurements are carried out at room temperature after quick cooling of the sample, the denaturation curve obtained is very similar to that of the dependence of optical density at 260 mJL on temperature (Palecek, 1964,1965). If, however, measurements are performed at elevated temperature, changes can already be observed at temperatures lower than denaturation temperature (Palecek, 1962,1964). This result suggests that the oscillopolarographic behaviour below the melting temperature is due to a change in configuration ofthe DNA molecule. A presumed conformational change has also been observed in DNA at temperatures below the denaturation temperature by studies of viscosity (Freund & Bernardi, 1963), circular dichroism (Brahms & Mommaerts, 1964) and low-angle X-ray scattering (Luzzati, Mathis, Masson & Witz, 1964). The oscillogram dE Idt against E of denatured DNA (Fig. 1) shows an indentation in the anodic part caused by the residues of deoxyguanylic acid (Palecek, 1961) and another indentation in the cathodic part which is not present in the oscillogram of native DNA (Palecek, 1964). We followed the appearance of the cathodic indentation dE
dt
Denatured DNA
Native DNA
/
E FIG. 1. Oacillograms of native and denatured DNA in ammonium formate medium. C, oathodic part; A, anodic part. In this paper only the depth of the cathodic indentation (CJ) W8Il followed.
and its increase with temperature. DNA samples isolated from different bacteriophages, bacteria and mammalian organs were analysed. Measurements were carried out at elevated temperature in an ammonium formate medium in concentrations of 0·1 M, 0·25 M, 0·3 M and 0·5 M with 0·02 M, 0·025 M, 0·05 M and 0·1 M-sodium phosphate (pH 7,0). The apparatus used was a Polaroscope P 524 (Kfizik, Praha), with dropping mercury electrode. The mercury dropping electrode was polarized with alternating current of frequency 50 cIs. In all cases the appearance of the cathodic indentation at temperatures far below denaturation temperature was registered. 839
E. PALECEK
840
The changes observed at low temperatures could be explained by the idea that unchanged DNA was present in the bulk of the solution, but that its structure was changed only secondarily on the electrode due to the repeated cycles of the alternating current. Therefore, we used the so-called "first curve technique" (Heyrovsky & Kalvoda, 1960; Palecek, 1965), which makes it possible for us to distinguish primary and secondary oscillopolarographic processes (so-called oscillopolarographic artifacts). In principle this involves the polarization of the dropping mercury electrode with a single cycle of alternating current for each drop of mercury dripping from the electrode. An indentation of secondary character does not appear on the "first curve"; however, it does appear on the following curves. The experimental arrangement was similar to that employed in our previous studies (Pale~ek, 1965). By the "first curve technique" we followed the course of denaturation of Escherichia coli DNA and recorded changes at as Iowa temperature as about 50°0. The same results were obtained if every drop from the mercury electrode had been polarized through 100r----------------.ii-"'"
~•
80
60
40
20
o
i
x
40
60 Temperature
80
100 (Oe)
FIG. 2. Denaturation curves of E. coli DNA in 0·1 M·ammonium formate with 0·02 M-sodium phosphate (pH 7·0). -e--e-, oscillopolarographic measurements: depth of cathodic indent.ation at polarization of one mercury drop through multiple cycles of alternating current (50 cIs); DNA concentration 60 p.g/mI. -0--0-, oscillopolarographic measurements: depth of cathodio indentation of polarization of one mercury drop through a single cycle of alternating current (500/s), i.e, first curve technique, DNA concentration 60p.g/mI. - X - - X - , spectrophotometric measurements at 260 mp.; DNA concentration 40 p.g/ml. All measurements were carried out at temperatures given on the graph. The measured maximum absorbance at 260 mp. and the maximum depth of indentation were taken as 100%. As zero value, absorbance at 20°C'was used; zero value in oscillopolarographic measurements means that the indentation followed was not present in the oscillogram.
LETTERS TO THE EDITOR
841
the repeated cycles of the alternating current. On the other hand, the curve for the dependence of optical density at 260 mp. on temperature, in the same medium, did not show any increase below the region of denaturation temperature (Fig. 2). The changes registered oscillopolarographically at a temperature lower than that of denaturation were reversible and the cathodic indentation did not show any deepening on the "second" and "third" curves. From the results obtained we can conclude that the changes observed at temperatures lower than the denaturation temperature are not caused by disturbance of the DNA structure due to repeated polarization of the dropping mercury electrode through several cycles of the alternating current. Should the changes in the DNA structure occur on the electrode, they would have to set in at a time interval shorter than 10- 2 second at the beginning of the first cycle and then not continue in the following cycles. We consider it more probable that the changes set in already in the solution due to higher temperatures, but are not detectable by means of the usual methods permitting measurements at elevated temperatures such as optical density and optical rotation measurements. If this is correct, we can imagine that thermal denaturation of DNA is preceded by slow "opening" of the double helical structure over 'a rather wide temperature range. Evidence has been obtained that there exists a certain relationship between nucleotide sequence in DNA and the extent of changes observed at temperatures lower than denaturation temperature (Palecek, unpublished experiments). Institute of Biophysics Czechoslovak Academy of Sciences Brno, Czechoslovakia Received 14 December 1964
E.
PALECEK
REFERENCES Brahms, J. & Mommaerts, W. F. H. M. (1964). J. Mol. Biol. 10, 73. Freund, A. M. & Bernardi, G. (1963). Nature, 200, 1318. Heyrovsky, J. & Kalvoda R. (1960). Oszillographische Polarographie. Berlin: Akademie Verlag. Luzzati, V., Mathis, A., Masson, F. & Witz, J. (1964). J. Mol. Biol. 10, 28. Palecek, E. (1961). Biochim. biophys. Acta, 51, 1. Palecek, E. (1962). Z. Ohem, 2, 260a. Palecek, E. (1964). Abh. deutechen, Akad. Wiss. 270. Palecek, E. (1965). Biochim. biophys. Acta, 94, 293.