Prospects and difficulties in the far- and vacuum-ultraviolet spectroscopy of DNA

Journal of Bzochermcal and Bzophyswal Methods, 5 ( 1981) 319-327

319

Elsevier/North-Holland Biomedical Press

Prospects and difficulties in the far- and vacuum-ultraviolet spectroscopy of DNA I. F61dv~ri ~, A. F e k e t e 2 a n d G. C o r r a d i 1 t Research Laboratory for Crystal Physics, Hungarian Academy of Sciences, P.O. Box 132, 1502 Budapest, and 2 Instztute of Bzophyszcs Semmelwels Medwal Umversltv, P.O. Box 263, 1444 Budapest, Hunga~T

(Received 5 August 1981) (Accepted 27 October 1981)

SUMMARY Typical sources of error in the UV spectroscopy of D N A near and below 200 n m are pointed out. In the case of native D N A a procedure for extending the wavelength range and for analysing the spectra ts proposed. As illustration, spectra for D N A s of various origin recorded in the 164-350 n m range at room temperature and at 110 K are presented. K e y words: vacuum-ultraviolet spectroscopy; D N A thin films.

INTRODUCTION Following the basic work of Preiss and Setlow [1] a number of attempts were made to extend the UV spectroscopy of D N A towards shorter wavelengths. A number of papers [2-8] has dealt with absorption near 200 nm (Y peak). Recently measurements extending to still shorter wavelengths have been carried out [9-13]; a paper reviewing the given field has been published [14]. In addition to studies of the electronic structure and denaturation hyperchromicity the far- and vacuum-UV spectra of D N A were also used to follow the effect of UV- or y-irradiation on the properties of D N A [12,81. In some cases, however, the D N A samples prepared for these experiments were inappropriate for far- or vacuum-UV spectroscopy and, moreover, some methods were extended beyond their limits of validity. A number of spectra and conclusions made on their bases seem therefore to be unreliable. We think that clarifying some basic principles would promote further progress in the field. Earlier, an effective procedure for the preparation of D N A films for vacuum UV absorption measurements was proposed [12]. In the present work we show the feasibility of this method 'for the spectroscopy of D N A films of various origin. 0165-022X/81/0000-0000/$02.50 © 1981 Elsevier/North-Holland Biomedical Press

320 EXPERIMENTAL

Materials Calf thymus DNA was purchased from Calbiochem, salmon sperm DNA from Serva, and chicken erythrocyte DNA from Reanal (Hungary). Phage T7 DNA was prepared following Mandell and Hershey [21] using our earlier resutts for cultivation of T7 phages [22]. Phenol extraction to remove the protein contamination of all the D N A samples was carried out until no appreciable amount of protein could be detected by using the Folin-Ciocalteu reagent [15]. In all experiments the DNAs were first dissolved in aqueous solutions containing 0.05 M NaC]. The quality of D N A was checked by UV absorption spectra (E280/E260 z 0.52, E230/E260 = 0.45) and by melting curves. The DNA thin films were prepared by centrifugal deposition from aqueous solutions. The method of obtaining optically suitable DNA thin films has previously been described in detail [121. The relative humidity (R.h.) of the DNA films was adjusted by exposure over saturated BaC12 (88% R.h.) or drying over P20~ (0% R.h.) for two days-to obtain the native state (B conformation) or the denaturated state (D conformation), respectively. Recording the far- and vacuum- UV spectra The absorption spectra were recorded on a Perkin-Elmer 554 spectrophotometer (190-500 nm) and on a Beaudouin MVR-100 vacuum monochromator (164-300 nm). The latter was complemented by a vacuum-proof measuring cell having Teflon isolation and quartz windows transparent in the far UV. This gave the possibility to investigate DNA films having a thickness of 200-400 nm, maintaining preset relative humidity values. For low-temperature work, an Air Products 'Displex' cryostat or a self-developed cryostat and the same measuring cell were used. The accuracy of wavelength determination was +--0.5 and ~+0.1 nm, and of absorption measurement (in the case l o g / 0 / I = 1)±0.2% and 3% for the Perkin-Elmer 554 spectrophotometer and the vacuum monochromator, respectively. All equipment was checked for reliability, including transparency tests of the cell windows. Correction of DNA spectra for light scattering Despite all efforts in this field, the problem of scattered light in DNA spectroscopy has not yet been resolved. Refraction data permitting the calculation of light scattering have been presented only for dried D N A films in the 15-600 nm region [9]. In the case of native DNA films and solutions, refraction data are available only above 310 nm. Therefore, light scattering corrections for films and solutions are usually made by fitting and extrapolating the Raleigh-Mie formula ( E = k X-n), thus neglecting anomalous behaviour of the refraction index near absorption maxima. Measurements using specially constructed integrating UV photometers, and empirical fitting yielded essentially the same corrections even near absorption maxima (see e~g. Refs. 16, 17). In the case of films, the theoretically expected n = 4 is seldom realized, and even n < 1 may occur due to aggregation, as suggested by measurements of Onari [181 on polypeptides. DNA films prepared by our method showed values of n = 1.5-2.8 [13].

321 The development of 'absolute' absorption spectroscopic methods, such as optoacoustic spectroscopy, where luminescence should also be accounted for, could promote the solution of these problems in the near future. For the time being one should accept the above empirical correction methods. In the present experiments the fitting values k and n were calculated from absorption data at 350 and 450 nm. On the basis of a comparison of corrected spectra on the Perkin-Elmer 554, and of spectra on the vacuum monochromator, light-scattering corrections were extended to the vacuum-UV region. In order to diminish the experimental noise, a mean value of four spectra was calculated using the data corrected for light scattering. For data processing a H P desk-top calculator was used.

Low temperature investigations Because of special difficulties low temperature absorption spectra for D N A have not been published yet. The contamination of the windows of the measuring cell by diffusion oil vapour may give rise to pseudoeffects, especially in the region 175-230 nm (e.g. Ref. 19). To avoid this problem two methods were tested. In one, the reference and measuring cells were cooled simultaneously in a common vacuum chamber, expecting the vapour condensation to be practically the same. This method is proposed only for non-automatic spectrophotometers where the light intensity in the reference path can be controlled. Alternatively, the sample was kept at low temperature for hours and the spectrum was measured from time to time to follow the kinetics of contamination. In this way the absorption of the condensed vapour could be extrapolated for the time during and immediately after cooling. The spectra obtained by both methods were similar to each other within the experimental error.

RESULTS A N D D I S C U S S I O N

The wavelength limit for far- and vacuum-UV absorption measurements of DNA Analysing the validity of the L a m b e r t - B e e r law for dilute D N A solutions, Voet et al. [7] determined the short-wavelength limit for measurements on such systems. They used nitrogen flushing to remove oxygen from the light paths, applied cuvettes 1-2 m m wide and obtained reliable results down to 190 nm. Falk [6] extended this limit to 185 nm employing 2H20solvent and a cuvette width of 1 cm. No substantial extension further can be expected even by using very thin cuvettes because of high absorption of the solvent. In fact, for shorter wavelengths the absorption of the solvent ( a n d / o r the ambient atmosphere) becomes higher, i.e., the light intensity in both light paths (reference and sample) becomes lower than the limit necessary for the detector system to work reliably. As a result, a steep decrease of 'absorption' is recorded if the usual double-beam automatic spectrophotometers are used. In the case of water as a solvent, a cuvette width of 1 cm and air as an ambient, this pseudo-effect occurs already at 200 nm. This was checked by UV absorption measurements where the D N A and the solvents were separated. A film of calf thymus D N A contained by a cassette and a separate cuvette (which was identical with the one in the reference path) was placed in the measuring path, putting various

322 solvents in both cuvettes. The spectra for the solvents 0.1 M NaC1 and standard saline citrate solution are shown in Fig. 1 (dashed and dotted lines, respectively) together with one for no solvent at all, with the cuvettes empty (solid line). Thus we reproduced the characteristic steep decrease of 'absorption' at the short wavelength side of the 'Y peak' presented by Basu [21, Basu and Dasgupta [3,4] and Lystov et al. [5], and showed that its origin has nothing to do with interaction between the D N A and the buffer as stated by Attri and Mookerjee [8]. In the case of lyophylized D N A films the spectroscopic limit is determined by (1) the sensitivity of the equipment, and (2) the absorption of the substrate. For reflexion spectroscopy the limit so far reported is 15 nm [9], and for absorption in the case of self-supporting films 50 nm !10]. For native wet D N A films a short-wavelength reliability limit arises at about 165 nm due to structural water and NaC1 necessary for stabilization of the native structure. In the absence of structural water and NaC1, native D N A cannot be stable, therefore the 165 nm value should be considered as the short-wavelength limit for native D N A spectroscopy [13].

Vacuum- UV absorption spectra of various DNA films Vacuum-UV spectra of native D N A films of various origin are shown on Fig. 2. The position and the shape of the X peak (near 260 nm) show little variation in the cases investigated and correspond to spectroscopic features of native D N A solutions. Contrary to previous assumptions, the shape of the Y peak (near 200 nm) and its intensity relative to the X peak is found to depend on the origin of DNA. The fine structure of the long wavelength side of the Y peak was first described by Falk [6]. The analysis of the spectra was extended in previous works of our team to the short wavelength side of the Y peak and to the long wavelength tail of the Z peak (near t50 nm) [12,t3]. The spectra are rather compact, the band components of the fine structure showing up in most cases only as shoulders or badly resolved inflexions. These features can be readity reproduced; the absolute values of the parameters derived, however, should not be relied upon too seriously. Nevertheless, external effects (e.g. variation of NaC1 concentration or UV irradiation) can be clearty observed as shifts of band components and as changes in their relative intensities. As shown by Fig. 2 and Table 1 for D N A s of various origin there is a variation in the fine structure of the Y peak. Taking into account the careful preparation procedures, these variations cannot be attributed to changes of any protein impurity content. There is a difference, however, in the G + C base content and the base sequence for various samples. For the calf thymus, salmon sperm and chicken erythrocyte DNAs, the G + C content was similar and differed from that of T7 DNA. As the spectra of the calf thymus, salmon sperm and chicken erythrocyte D N A s are also different, the effect may be due to the various base sequence of the samples. A similar conclusion was reached by Lewis and Johnson [20] on the basis of their measurements of vacuum-UV circular dichroism. The effect of denaturation by drying has been also detected by observing changes of both the positions and relative intensities of the band components in the fine structure of the ¥ peak (Fig. 3).

323

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60'000

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Fig. 1. Absorbance of a DNA thin film using various solvents in 1 cm cells placed separately from the DNA film in both reference and measuringpaths.

Fig. 2. Absorption spectra of various native DNA thin films (R.h. 88%)

Along with the band parameters, values of the observed hyperchromicity are included in Table 2. It should be pointed out that hyperchromicity values at 261 nm for various D N A s are essentially the same (20-25%), taking into account the experimental error for parallel measurements. As shown in Table 2, the wavelength dependence of the hyperchromicity is different for various DNAs.

Low temperature spectra Fig. 4 shows the absorption spectrum of calf thymus D N A at 110 K as compared to the spectrum at room-temperature of the native films. The low temperature band parameters are collected in Table 1. During cooling of the sample some structural changes are expected due to freezing of the electrolyte environment of the D N A . According to our findings the native state of D N A is restored after raising the temperature up to room temperature. In the 110 K spectrum a hypsochromic shift of the X band appeared without a resolved fine structure. The separation of the band components of the Y band was more pronounced than in the spectrum at room temperature. At the same time the shift of the bands and intensity changes were also significant. This clear separation proved the reliability of the fine structure of the Y peak in the room temperature spectra of D N A .

48%

42%

43 %

43 %

T7

Calf thymus

Chicken erythrocyte

Salmon sperm

Calf thymus at 110 K

G+C content

Origin

(209) (1.04) (212) (1.25) (210) (1.28) (206) (t.43) (209) (1.24)

200 (1.78) 197 1.65 (202) ( t .54) (198) (t.59) 198 1.72 195 1.66 188 1.78 -

192 1.78 -

1.77 187 1.76 182 1.80 183 2.17

188

187 1.78

4

179

179 1.88 1.80 ( 181) ( l 66) (176) (1 69) 175 2.28

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2.17 172 1.72 (166) (2.08) (166) (2.46)

168

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Y peak

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7

The first lines show the positions in nm, the second hnes mdmate the intensities relative to the 261 n m band (in the case of the 110 K spectrum relative to the 261 n m band of the room-temperature spectrum).

ABSORPTION B A N D P A R A M E T E R S OF PEAKS, S H O U L D E R S OR INFLEX1ONS (IN PARENTHESIS) IN T H E V A C U U M - U V SPECTRA OF VARIOUS D N A T H I N FILMS

TABLE 1

325 200 i

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Fig. 3. Absorption spectra of varxous denatured D N A thin films (R.h. 0%)

Conclusion

The vacuum-UV absorption spectra obtained illustrate the feasibility of the techniques proposed in this paper for studying the DNA structure. Significant 200 L

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Fig. 4. Absorption spectra of r~ative calf thymus D N A thin film at room temperature and at 110 K.

Salmon sperm

Chicken erythrocyte

Calf thymus

T7

Origin

(209) (1.041 (23) (213) (1.25) (23) (207) ('1.27) (21) (210) (1,20) (15) (199) (1.481 (17)

200 1.78 21 (200) (1.62) (2(/) 194 1,67 23 t 89 1.59 16

194 1.82 21 -

188 1 80 21 194 1 66 15 186 1.77 13 181 1.63 16

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(1o)

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7

The first lines show the band poslhons in nm, the second hnes m&cate the intensity values relat:ve to the 261 n m band, and the percent values of denaturation hyperchromic:ty are shown in the thtrd lines.

ABSORPTION PARAMETERS OF PEAKS, S H O U L D E R S OR I N F L E X I O N S (IN PARENTHESIS) IN T H E V A C U U M - U V SPECTRA OF V A R I O U S D E N A T U R E D D N A T H I N FILMS (R.h. 0%)

TABLE 2

CJ~

327 d i f f e r e n c e s w e r e s h o w n a m o n g t h e v a c u u m - U V s p e c t r a of v a r i o u s D N A s . N e v e r t h e less, the i n c r e a s e of r e l i a b i l i t y of the m e a s u r e m e n t s , t h e t h e o r e t i c a l i n t e r p r e t a t i o n a n d t h e p r a c t i c a l a p p l i c a t i o n of the s p e c t r a d e m a n d f u r t h e r efforts.

SIMPLIFIED

DESCRIPTION

OF THE METHOD

AND

ITS APPLICATIONS

The method described is an improvement of tile thin fihn techmque in DNA absorption spectroscopy. The optical quality of the films obtained by centrifugal deposition permits to follow subtle differences m the DNA spectra. The vacuum-tight measuring cell maintains the adjusted relative humidity even m a vacuum monochromator. This way the absorption spectra of native DNA can be extended down to 165 nm. This limit is determined by the absorption of the structural and environmental water of the native DNA films. The extended wavelength range may be used to study: (1) the fine structure of electronic transitions and its dependence on the DNA secondary structure: (2) the wavelength dependence of denaturation hyperchromlclty: (3) the &fference in the electronic structure of DNA of various origin: (4) the UV-mduced photoreactions in DNA thin films; and (5) any other effects modifying the DNA electron structure.

ACKNOWLEDGEMENTS T h e a u t h o r s w o u l d like to e x p r e s s t h e i r g r a t i t u d e to P r o f e s s o r s Gy. R o n t 6 a n d R. V o s z k a for h e l p f u l discussions, to Mrs. K. P a t a k i for p r e p a r a t i o n o f T7 p h a g e , a n d to Mrs. I. P e r c z e l a n d M i s s M. D r a b a n t for t e c h n i c a l help. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Preiss, J.W. and Setlow, R. (1956) J. Chem. Phys. 25, 138-141 Basu, S. (1967) Biopolymers 5, 876-878 Basu, S. and Dasgupta, N.N. (1967) Biochlm. Biophys. Acta 145, 391-397 Basu, S. and Dasgupta, N.N. (1969) Biochim. Blophys. Acta 174, 174-182 Lystov, V.N., Sukhorukov, B.I., Bliumenfeld, L A., Moshkovskxi. Yu.Sh. and Petukhov, V.A. (1962) Biofizika 7, 662-663 Falk, M. (1964) J. Am. Chem. Soc. 86, 1226-1228 Voet, D., Gratzer, W.B., Cos, R.A. and Doty, P. (1963) Blopolymers 1, 193-208 Attri, A.K. and Mookerjee, A. (1981) Radiat. Environ. Biophys. 19, 51-56 Inagakl, T., Hamm, R.N., Arakawa, E.T. and Painter, L.R (1974) J. Chem. Phys 61, 4246-4250 Sontag, W. and Weibezahn, K.F. (1975) Radiat. Environm. Blophys 12, 169-174 Kiseleva, M.N., Zarothentseva, E.P. and Dodonova, N.Ya. (1975) Blofizika 20, 561-565 Fekete, A. and FOldvfiri, I. (1978) Studia Blophys. 73, 47-53 Fekete, A., F61dv/tri, I. and P+ter, A. (1979) Studia Blophys. 77, 133-139 Fekete, A. (1980) Biok6mia 4, 23-28 (in Hungarian) Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Gratton, E. (1971) Biopolymers 10, 2629-2634 Tikchonenko, T.I., Kislina, O.S. and Dobrov, E.N. (1974) Arch. Biochem Biophys. 160, 1-13 Onari, S. (1971) J. Phys. Soc. Japan 30, 811-818 Sowers, B.L., Williams, M.W., Harem, R.N. and Arakawa, E.T. (1971) J. Appl. Phys. 42, 4252-4257 Lewis, D.G. and Johnson, W.C. (1974) J. Mol. Biol. 86, 91-96 Mandell, J.D. and Hershey, A.D. (1969) Anal. Biochem. 1.66-77 G~spgr, S., Ront6, Cry and Mtiller, G. (1979) Z. Allgem. Mikroblol. 19, 163-169