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Raman excitation profiles of actinomycin-DNA complex
05~539/82/020219-o3so3.00/0 @ 1982 Pergamon Press
Spectrochimico Acta, Vol. %A, No. 2, pp. 219-221. 1982 Printed in Great Britain.
Ltd.
Raman excitation profiles of actinomycin-DNA complex G. SMULEVICH, L. ANGELONI and M. P. MARZOCCHI
Istituto di Chimica Fisica dell’Universit8,
Via G. Capponi 9, 50121 Firenze, Italy
(Received 1 August 1981) Abstract-The excitation profiles reported. Their analysis allowed electronic state of the corn&x between the nucleic acid and the
of actinomycin and of actinomycin-DNA complex in Hz0 are us to elucidate the vibrational structure of the lowest excited and to obtain some information on the interaction mechanism drug.
INTRODUCTION Resonance Raman spectroscopy, demonstrating the vibrational modes of the chromophore portion of the molecule, has been employed for the study of complex molecules of biological interest. The complex between actinomycin (hereafter AMC) and DNA, owing to the antineoplastic properties of the drug, represents an interesting example for the application of such a spectroscopy to the study of the intermolecular bindings present in many drug-substrate systems. Evidence of the formation of the intercalation complex was found from the absorption (hypochromic and bathochromic effects) and Raman spectra [l, 21. In particular the absorption maxima at 425 and 441 nm of the pure compound which are due to the vibronic structure of a single electronic transition [3] are shifted at 441 and 455 nm, respectively. In addition noticeable and specific intensity changes are observed in the Raman spectra where the strongest band (at 1505 cm-‘) of the pure drug in CH30H disappears. The study of the dependence of the Raman band intensities from the exciting frequencies is a powerful tool for the characterization of the electronic levels and of the corresponding vibronic couplings. We have performed this type of analysis to obtain information on the interaction mechanism of the drug-DNA system. The excitation profiles of AMC in CH,OH have been previously investigated [3]. In the present paper we extend the study to the water solutions of the drug and of the drug-DNA complex. EXPERIMENTAL Actinomycin D and calf thymus DNA were purchased from K&K and Merck, respectively and used without further purification. The complex was obtained by adding small quantities of a dilute solution of AMC in water to a large amount of DNA in NaCl (1 x lo-* M) water solution. The Raman spectra were measured with the aid of a double monochromator Jobin-Yvon model HG-2S and a photon counting system equipped with a thermoelectrically cooled RCA-C31034A photomultiplier. The spectra of the drug and of AMC-DNA were measured in aqueous solution (4x 10m4M) using the lines at 457.9,
476.5, 488.0, 4%.5, 501.7 and 514.5 nm of an Ar’ laser as radiation sources. No photodecomposition effects were found as checked by repeated runs of the Raman and the visible absorption spectra. In order to minimize the reabsorption of the scattered light (90” geometry) the laser beam was kept very close to the edge of the cell. The intensities of some bands were measured by peak heights and by areas with similar results. Peak heights measurements were therefore used throughout. The band at 1058 cm-’ of NOi, added to the solution, was used as an internal standard. The band intensities were corrected for the sensitivity changes of the spectrometer with the wavelength, using a calibration curve established with a standard lamp. Corrections were also made for the v4 dependence. Under these conditions the experimental error was estimated to be less than 15%. The electronic soectra (4 x IO-’ M) were recorded with a Cary 17D spectrbphotometer.
RESULTSANDDISCUSSION The theory of the Raman band intensities involve the sum over all the vibrational sublevels of the excited electronic states [4]. In simplification, approximate methods, based on series expansion have been proposed. In the adiabatic approximation for a multimode model system a very simple expression has been proposed by YOUNG et al.[5]. For a FranckCondon allowed transition the Raman band intensity is given by Ix
a2
1 a=(E,-E,+A-E~)i
(1)
where Er and EC are the energies of the excited and ground states, A is the difference between the adiabatic (E, -EC) and the vertical electronic energy gaps and E,, is the energy of the laser line. The expression is valid for contributing levels for which
4 and l0 being the vibrational energies of the intermediate and ground vibrational states. This expression is obtained by truncating the convergent series at the first non-zero term which is in general the most important. By including the non resonant part of the Raman 219
220 tensor,
G. SMULEVICH et al. expression
(1) becomes
(2) where
Figure 1 shows the visible absorption spectra AMC in CH,OH, AMC in Hz0 and AMC-DNA HZO. Whereas two maxima at 441 and 425 nm occur the visible absorption spectrum of the drug
of in in in
CHsOH, only a broad and smooth band at 441 nm is observed in water. This effect can be
Vk=
E,-E,+A h
and
v. = E,lh.
As proposed by MINGARDI and SIEBRAND[6], in the expression (2) the frequency of the absorption maximum of the envelope due to a single progression (vk) is considered. When the absorption maximum is rather far from the exciting frequencies, the equation equals the well known pre-resonance frequency factor [7] being Vk= v., where v. is the frequency of the pure electronic transition. In the near-resonance condition (with the exciting frequencies close to the electronic absorption maximum) the expression (2) fits the exact sum well only for appropriate values of the displacement parameters of the potential wells and of the sublevel widths. Under these conditions large changes of the band intensities occur in function of the exciting frequency. We have shown that equation (2) holds well for near resonance spectrum of pure AMC in CH30H [3]. In this case this equation is not sufficient to describe completely the experimental profiles since the band intensities of the spectra registered with the 457.9 nm (21832 cm-‘) exciting line are lower than the expected ones. However, the analysis of the observed deviations, compared with the previous results obtained for the pure drug allows us to suggest inferences on AMC/H*O and AMC-DNA interactions.
due to hydrogen bonding between the chromophore portion of the drug and the water molecules. The interaction causes a large broadening of the absorption band with loss of the vibrational structure especially due to inhomogeneous distribution of site environments of some vibronic states. The above interpretation is supported by the analysis of the Raman excitation profiles (Fig. 2) where lower intensity enhancements are observed for some Raman bands registered with the 457.9nm laser line. This is well explained if we consider that in the energy denominator of the general expression of the Raman intensity [S] a term appears which accounts for large contribution of homogeneous and site broadening. The visible absorption spectrum of AMC-DNA complex shows the same pattern observed for the pure drug in methanolic solution (Fig. 1). The presence of a similar vibrational structure in both spectra is also supported by the excitation profiles measurements of the complex. In Fig. 3 are shown the resonance Raman spectra of AMC-DNA/ H,O. Figure 4 shows the corresponding excitation profiles, between 19200 and 21000 cm-‘, obtained for some Raman bands. The curves calculated according to the equation (2) with l/vk = 455 nm (AJ and l/vk = 441 nm (A,) fit the experimental points very well relative to the bands at 588 cm-’ (A,) and 1487, 1404, 1263 and 1213cm-’ (A,). The maxima of the excitation
A
20000 Wavenumber,
2loQO
22occJ
cm-’
Fig. 2. Relative intensity of the 1505 (B), 1404 (A), 1263(0) and 1213( x km-’ Raman bands of AMC as a function of Fig. 1. Visible absorption spectra of AMC in CH30H (-.-a-), in Hz0 (--_) and of its complex with DNA in Hz0 (-).
The solution in CHjOH is 2 x lo-’ M. Bars indicate the Raman exciting frequencies.
the excitingfrequency. Because of the closeness of some experimental points, bars include all the corresponding errors. The solid line indicates the calculated excitation profile according to F” with r\k = 425 nm.
Raman excitation profiles of actinomycin-DNA
Fig.3. Ramanspectraof AMC-DNAcomplexinH,Otaken with the 4%.5 (upper), 488.0 (middle) and 476.5 (lower) nm exciting lines. (*) indicates the 1058 cm-’ band of NO;.
profiles exactly follow the shifts of the absorption
bands which occur at 441 and 425 nm for the drug in CH,OH and at 455 and 441 nm for the complex in H,O. On this basis we deduce that, as found for the pure AMC [3], the absorption bands are due to O-l transitions involving the mode at 588 cm-’ and the modes between 1500-1200 cm-‘, respectively.
complex
221
Accordingly the smaller spacing between the two absorption maxima of the complex (600 cm-’ compared to 850cm-’ for the pure drug) is explained in terms of the missing contribution of the mode at the highest frequency (1505 cm-‘) which is absent in the Raman spectrum of the complex. In Fig. 4 the data relative to the spectra registered with the 457.9 nm (21832 cm-‘) exciting line are missed. Actually the experimental intensities observed in this spectrum are much lower than the calculated values using equation (2). This is not unexpected since the laser frequency is very close to the exact resonance and therefore the above expression is not valid in this region. All the results show that the Raman excitation profiles taken with the exciting frequencies close to the absorption maxima are very useful in elucidating the vibrational structure of the excited electronic states and the interaction mechanism between biomolecules and large chromophores. The expression (2) can be used in the nearresonance cases when the absorptions are mainly due to single progressions of so sharp levels that damping factors can be neglected. The broadening of the excited vibronic states of the drug in water and the restoration of the vibrational structure for the complex suggest the occurrence of two different kinds of interaction for AMC/H*O and AMC-DNA/H,0 systems. This probably means that hydrogen bonding plays an important role in the intercalation process and that Hz0 molecules linked to the chromophore portion of the drug are removed by addition of DNA. Acknowledgement-This work was supported by the Italian Consiglio Nazionale delle Ricerche. G. S. wishes to thank the “Cassa di Risparmio di Firenze” for supporting a research fellowship.
REFERENCES
PI W. A. REMERS,The Chemistry of Antitumor Anti-
Wovenumber,
cm-’
Fig. 4. Excitation profiles of the 588 (0), 1213(U), 1263(x), 1404 (0) and 1487 (A) cm-’ Raman bands of AMC-DNA complex in H20. Because of the closeness of some experimental points, bars include all the corresponding errors. Solid lines indicate the calculated profiles according to F*‘ with At = 441 nm (AZ) and 455 nm (A,).
SAAVol.3s. No.2-G
biotics, Vol. 1. John Wiley, and references therein (1979). PI G. DE VOE and I. TINOCOJR.. J. Mol. Biol. 4, 518 (1%2). L. ANCELONIand M. P. MARZOCCHI, r31 G. SMULEVICH, Biochim. Biophvs. Acta 610, 384 (1980). Rim&Spectroscopy, [41J. TANGand ‘A.-C. ALBRECHT, Vol. 2 p. 33, (Edited by H. A. SZYMASKY).Plenum Press, New York (1970). 151E. S. YEUNG, M. HEILINGand G. I. SMALL,Spectrochimica Acta 31A, 1921 (1975). and W. SIEBRAND,.I. Chem. Phys. 62, KJIM. MINGARDI 1074 (1975). [7] A. C. ALBRECHT and M. C. HUTLEY,J. Chem. Phys. 55.4438 (1971). [8] P. M. CHAMPIONand A. C. ALBRECHT,J. Chem. Phys. 71, 1110 (1979).
Spectrochimico Acta, Vol. %A, No. 2, pp. 219-221. 1982 Printed in Great Britain.
Ltd.
Raman excitation profiles of actinomycin-DNA complex G. SMULEVICH, L. ANGELONI and M. P. MARZOCCHI
Istituto di Chimica Fisica dell’Universit8,
Via G. Capponi 9, 50121 Firenze, Italy
(Received 1 August 1981) Abstract-The excitation profiles reported. Their analysis allowed electronic state of the corn&x between the nucleic acid and the
of actinomycin and of actinomycin-DNA complex in Hz0 are us to elucidate the vibrational structure of the lowest excited and to obtain some information on the interaction mechanism drug.
INTRODUCTION Resonance Raman spectroscopy, demonstrating the vibrational modes of the chromophore portion of the molecule, has been employed for the study of complex molecules of biological interest. The complex between actinomycin (hereafter AMC) and DNA, owing to the antineoplastic properties of the drug, represents an interesting example for the application of such a spectroscopy to the study of the intermolecular bindings present in many drug-substrate systems. Evidence of the formation of the intercalation complex was found from the absorption (hypochromic and bathochromic effects) and Raman spectra [l, 21. In particular the absorption maxima at 425 and 441 nm of the pure compound which are due to the vibronic structure of a single electronic transition [3] are shifted at 441 and 455 nm, respectively. In addition noticeable and specific intensity changes are observed in the Raman spectra where the strongest band (at 1505 cm-‘) of the pure drug in CH30H disappears. The study of the dependence of the Raman band intensities from the exciting frequencies is a powerful tool for the characterization of the electronic levels and of the corresponding vibronic couplings. We have performed this type of analysis to obtain information on the interaction mechanism of the drug-DNA system. The excitation profiles of AMC in CH,OH have been previously investigated [3]. In the present paper we extend the study to the water solutions of the drug and of the drug-DNA complex. EXPERIMENTAL Actinomycin D and calf thymus DNA were purchased from K&K and Merck, respectively and used without further purification. The complex was obtained by adding small quantities of a dilute solution of AMC in water to a large amount of DNA in NaCl (1 x lo-* M) water solution. The Raman spectra were measured with the aid of a double monochromator Jobin-Yvon model HG-2S and a photon counting system equipped with a thermoelectrically cooled RCA-C31034A photomultiplier. The spectra of the drug and of AMC-DNA were measured in aqueous solution (4x 10m4M) using the lines at 457.9,
476.5, 488.0, 4%.5, 501.7 and 514.5 nm of an Ar’ laser as radiation sources. No photodecomposition effects were found as checked by repeated runs of the Raman and the visible absorption spectra. In order to minimize the reabsorption of the scattered light (90” geometry) the laser beam was kept very close to the edge of the cell. The intensities of some bands were measured by peak heights and by areas with similar results. Peak heights measurements were therefore used throughout. The band at 1058 cm-’ of NOi, added to the solution, was used as an internal standard. The band intensities were corrected for the sensitivity changes of the spectrometer with the wavelength, using a calibration curve established with a standard lamp. Corrections were also made for the v4 dependence. Under these conditions the experimental error was estimated to be less than 15%. The electronic soectra (4 x IO-’ M) were recorded with a Cary 17D spectrbphotometer.
RESULTSANDDISCUSSION The theory of the Raman band intensities involve the sum over all the vibrational sublevels of the excited electronic states [4]. In simplification, approximate methods, based on series expansion have been proposed. In the adiabatic approximation for a multimode model system a very simple expression has been proposed by YOUNG et al.[5]. For a FranckCondon allowed transition the Raman band intensity is given by Ix
a2
1 a=(E,-E,+A-E~)i
(1)
where Er and EC are the energies of the excited and ground states, A is the difference between the adiabatic (E, -EC) and the vertical electronic energy gaps and E,, is the energy of the laser line. The expression is valid for contributing levels for which
4 and l0 being the vibrational energies of the intermediate and ground vibrational states. This expression is obtained by truncating the convergent series at the first non-zero term which is in general the most important. By including the non resonant part of the Raman 219
220 tensor,
G. SMULEVICH et al. expression
(1) becomes
(2) where
Figure 1 shows the visible absorption spectra AMC in CH,OH, AMC in Hz0 and AMC-DNA HZO. Whereas two maxima at 441 and 425 nm occur the visible absorption spectrum of the drug
of in in in
CHsOH, only a broad and smooth band at 441 nm is observed in water. This effect can be
Vk=
E,-E,+A h
and
v. = E,lh.
As proposed by MINGARDI and SIEBRAND[6], in the expression (2) the frequency of the absorption maximum of the envelope due to a single progression (vk) is considered. When the absorption maximum is rather far from the exciting frequencies, the equation equals the well known pre-resonance frequency factor [7] being Vk= v., where v. is the frequency of the pure electronic transition. In the near-resonance condition (with the exciting frequencies close to the electronic absorption maximum) the expression (2) fits the exact sum well only for appropriate values of the displacement parameters of the potential wells and of the sublevel widths. Under these conditions large changes of the band intensities occur in function of the exciting frequency. We have shown that equation (2) holds well for near resonance spectrum of pure AMC in CH30H [3]. In this case this equation is not sufficient to describe completely the experimental profiles since the band intensities of the spectra registered with the 457.9 nm (21832 cm-‘) exciting line are lower than the expected ones. However, the analysis of the observed deviations, compared with the previous results obtained for the pure drug allows us to suggest inferences on AMC/H*O and AMC-DNA interactions.
due to hydrogen bonding between the chromophore portion of the drug and the water molecules. The interaction causes a large broadening of the absorption band with loss of the vibrational structure especially due to inhomogeneous distribution of site environments of some vibronic states. The above interpretation is supported by the analysis of the Raman excitation profiles (Fig. 2) where lower intensity enhancements are observed for some Raman bands registered with the 457.9nm laser line. This is well explained if we consider that in the energy denominator of the general expression of the Raman intensity [S] a term appears which accounts for large contribution of homogeneous and site broadening. The visible absorption spectrum of AMC-DNA complex shows the same pattern observed for the pure drug in methanolic solution (Fig. 1). The presence of a similar vibrational structure in both spectra is also supported by the excitation profiles measurements of the complex. In Fig. 3 are shown the resonance Raman spectra of AMC-DNA/ H,O. Figure 4 shows the corresponding excitation profiles, between 19200 and 21000 cm-‘, obtained for some Raman bands. The curves calculated according to the equation (2) with l/vk = 455 nm (AJ and l/vk = 441 nm (A,) fit the experimental points very well relative to the bands at 588 cm-’ (A,) and 1487, 1404, 1263 and 1213cm-’ (A,). The maxima of the excitation
A
20000 Wavenumber,
2loQO
22occJ
cm-’
Fig. 2. Relative intensity of the 1505 (B), 1404 (A), 1263(0) and 1213( x km-’ Raman bands of AMC as a function of Fig. 1. Visible absorption spectra of AMC in CH30H (-.-a-), in Hz0 (--_) and of its complex with DNA in Hz0 (-).
The solution in CHjOH is 2 x lo-’ M. Bars indicate the Raman exciting frequencies.
the excitingfrequency. Because of the closeness of some experimental points, bars include all the corresponding errors. The solid line indicates the calculated excitation profile according to F” with r\k = 425 nm.
Raman excitation profiles of actinomycin-DNA
Fig.3. Ramanspectraof AMC-DNAcomplexinH,Otaken with the 4%.5 (upper), 488.0 (middle) and 476.5 (lower) nm exciting lines. (*) indicates the 1058 cm-’ band of NO;.
profiles exactly follow the shifts of the absorption
bands which occur at 441 and 425 nm for the drug in CH,OH and at 455 and 441 nm for the complex in H,O. On this basis we deduce that, as found for the pure AMC [3], the absorption bands are due to O-l transitions involving the mode at 588 cm-’ and the modes between 1500-1200 cm-‘, respectively.
complex
221
Accordingly the smaller spacing between the two absorption maxima of the complex (600 cm-’ compared to 850cm-’ for the pure drug) is explained in terms of the missing contribution of the mode at the highest frequency (1505 cm-‘) which is absent in the Raman spectrum of the complex. In Fig. 4 the data relative to the spectra registered with the 457.9 nm (21832 cm-‘) exciting line are missed. Actually the experimental intensities observed in this spectrum are much lower than the calculated values using equation (2). This is not unexpected since the laser frequency is very close to the exact resonance and therefore the above expression is not valid in this region. All the results show that the Raman excitation profiles taken with the exciting frequencies close to the absorption maxima are very useful in elucidating the vibrational structure of the excited electronic states and the interaction mechanism between biomolecules and large chromophores. The expression (2) can be used in the nearresonance cases when the absorptions are mainly due to single progressions of so sharp levels that damping factors can be neglected. The broadening of the excited vibronic states of the drug in water and the restoration of the vibrational structure for the complex suggest the occurrence of two different kinds of interaction for AMC/H*O and AMC-DNA/H,0 systems. This probably means that hydrogen bonding plays an important role in the intercalation process and that Hz0 molecules linked to the chromophore portion of the drug are removed by addition of DNA. Acknowledgement-This work was supported by the Italian Consiglio Nazionale delle Ricerche. G. S. wishes to thank the “Cassa di Risparmio di Firenze” for supporting a research fellowship.
REFERENCES
PI W. A. REMERS,The Chemistry of Antitumor Anti-
Wovenumber,
cm-’
Fig. 4. Excitation profiles of the 588 (0), 1213(U), 1263(x), 1404 (0) and 1487 (A) cm-’ Raman bands of AMC-DNA complex in H20. Because of the closeness of some experimental points, bars include all the corresponding errors. Solid lines indicate the calculated profiles according to F*‘ with At = 441 nm (AZ) and 455 nm (A,).
SAAVol.3s. No.2-G
biotics, Vol. 1. John Wiley, and references therein (1979). PI G. DE VOE and I. TINOCOJR.. J. Mol. Biol. 4, 518 (1%2). L. ANCELONIand M. P. MARZOCCHI, r31 G. SMULEVICH, Biochim. Biophvs. Acta 610, 384 (1980). Rim&Spectroscopy, [41J. TANGand ‘A.-C. ALBRECHT, Vol. 2 p. 33, (Edited by H. A. SZYMASKY).Plenum Press, New York (1970). 151E. S. YEUNG, M. HEILINGand G. I. SMALL,Spectrochimica Acta 31A, 1921 (1975). and W. SIEBRAND,.I. Chem. Phys. 62, KJIM. MINGARDI 1074 (1975). [7] A. C. ALBRECHT and M. C. HUTLEY,J. Chem. Phys. 55.4438 (1971). [8] P. M. CHAMPIONand A. C. ALBRECHT,J. Chem. Phys. 71, 1110 (1979).