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Fluorescence lifetime, precision calorimetry, and fluorescence energy transfer measurements in the study of normal and tumoral chromatin structure
Journal
of
MOLECULAR STRUCTURE Journal of Molecular Structure 4081409 ( 1997) I9 I- 194
Fluorescence lifetime, precision calorimetry, and fluorescence energy transfer measurements in the study of normal and tumoral chromatin structure Liliana Raduav*, Vasile Preoteasaa, Irina Radulescu”, Serban Radub “Department of Molecular Genetics, Victor Babes Institute, Spl. Independentei 99-101, Bucharest 76201, Romania bDepartment of Electrical Engineering, Polytechnic University. Spl. lndependentei 313, Bucharest 77703, Romania
Received 30 August 1996; revised 19 November
1996; accepted 22 November
1996
Abstract Chromatin is a complex of deoxyribonucleic acid (DNA) with proteins, that exists in the nuclei of eukaryotic cells. Three methods have been used to study protein-DNA interactions in chromatin and to compare the chromatin from normal tissue with that from tumoral tissue: determination of the fluorescence lifetimes and measurement of the heats of reaction of complexation of the hgand ethidium bromide with chromatin, and evaluation of the fluorescence energy transfer between two ligands dansyl chloride and acridine orange when coupled with chromatin. 0 1997 Elsevier Science B.V.
Keywords: Calorimetry;
Chromatin;
Energy transfer; Fluorescence;
1. Introduction Within
the
nucleus
of eukaryotic
cells,
nuclear
(DNA) is complexed with basic proteins (histones) and a variety of nonhistone proteins, to form chromatin, which is further organized into higher-order structures and tightly packed inside the nucleus [l] In last twenty years, important progress has been made in the elucidation of the structure of chromatin, in DNA-basic proteins (histones) especially complexes [2,3]. Despite great progress in the determination of chromatin substructures (the nucleosoms-complexes of DNA with five histones deoxyribonucleic
* Corresponding 0022-2860/97/$17.00
acid
author. 0 1997 Elsevier
PIISOO22-2860(96)0973
I- 1
Science
Lifetime
[4]), some aspects of the structure of chromatin remain unknown. Our previous studies were oriented toward analysis of the structure of chromatin by absorption and emission spectroscopy of complexes of chromatin with specific DNA ligands [5,6] or by isotope uptake and ‘H NMR spectroscopic methods [7]. In this paper we analyze the structure of chromatin from normal tissue-the liver of Wistar rats-and that from tumoral tissue-Walker carcinosarcoma maintained on Wistar rats. The methods used for this purpose were determination of fluorescence lifetimes and precision calorimetry for the binding of the ligand ethidium bromide to normal and tumoral chromatin, and fluorescence energy transfer measurements between a pair of fluorescent ligands, dansyl chloride and acridine orange, coupled to chromatin.
B.V. All rights reserved
192
L. Radu et al./Journal of Molecular Siructure 408/409 (1997) 191 -I 94
2. Experimental
2 ,
The chromatin was extracted from the livers of Wistar rats and from the Walker carcinosarcoma, maintained on Wistar rats, by Lewin’s method[8]; purity was verified by an absorption test [9]. Ethidium bromide (c = lo-’ M) was complexed with chromatin samples (chromatin DNA concentration 2.5 x lOA M). In the experiments of double fluorescent labeling, the chromatin proteins were covalently bonded with dansyl chloride and the chromatin DNA was uncovalently coupled with acridine orange. For dansyl chloride hexcitation= 323 nm and Xemission= 505 nm and for acridine orange Xex,-itation= 505 nm and Xemission= 530 nm. The transfer efficiency between the two ligands is [6]:
220
240
260
280
300
Wavelength (nm) Fig. 1. The spectra of chromatim from liver (1) and Walker tumor (2).
E=($.($) where IA, Zg are the relative fluorescence intensities of the acceptor, in the absence and the presence, respectively, of the donor, and eA and eo are the molar extinction coefficients of the acceptor and donor, respectively, at the wavelength of excitation. The transfer efficiency is related to the distance, r, between the donor and the acceptor by the relationship: E=
re6 r %R$ 1
where R. = (Jk2.Qo~n”) i; x 9.79 x 103, J is the overlap integral, k* is the orientation factor for dipoledipole transfer, n the refractive index of the medium, and Q0 the quantum yield of the donor in the absence of transfer. A Pharmacia LKB Ultrospec III spectrophotometer, an Aminco Bowman spectrofluorimeter, a time-resolved fluorimeter FL 900 CD and a LKB 8700 precision calorimeter were used.
E260nm
-=1.7and E 280nm
E2mm
spectrum
-= E 240nm
1
2 cor,*x
lo4
M
Fig. 2. The relative fluorescence intensities of the complexes of ethidium bromide with liver (1) and Walker tumor (2) chromatin, versus chromatin DNA concentration.
while for Walker tumor chromatin these ratios are 1.75 and 1.45, respectively. Fig. 2 shows the relative fluorescence intensities for complexes of normal chromatin and of tumoral chromatin with ethidium bromide. The values obtained indicate a greater availability to ligand binding in tumoral chromatin. The fluorescence decay curves have three components, with half-lives (7) of 2 ns for unbound ethidium bromide, 8 ns for ethidium bromide loosely bound to chromatin DNA, and 24 ns for intercalated ethidium bromide in chromatin DNA (Table 1). Table 1 The lifetimes (7) and the percentages of lifetimes v) of the chromatin-ethidium bromide complexes, for normal chromatin (NC) and for tumoral chromatin (TC)
3. Results and discussion The liver chromatin characteristics:
0
14
’
(Fig. 1) presents the
NC TC
7 (/ns) f US) r(/ns) f U%)
2.24 2 0.06 12.10 2.11 t 0.07 11.82
8.63 + 0.05 42.73 8.14 + 0.02 38.15
24.11 + 0.08 45.17 24.72 + 0.02 50.03
193
L. Radu et al./Joumal of Molecular Structure 4OW409 (1997) 191-194
Table 2 The parameters
deduced from calorimetric
Sample
CDNA
DNA DNA DNA NC TC
25.00 10.25 4.75 10.11 4.40
x
lo4
determinations n x
CM)
6.20 2.53 1.18 2.60 2.57
These three similar components were observed both in liver chromatin and in the Walker tumor chromatin, but with different relative contributions (f). In tumoral chromatin, a 9% increase of the 24 ns component was observed, indicative of a less rigid chromatin structure. The heat of interaction (Q) of the ethidium bromide with chromatin is dependent on the concentration of DNA (Table 2, in which n is the number of moles of ligand. The mean value obtained for the enthalpy (AH) of complexation of ethidium bromide with DNA is -6.54 kcal mall’. Table 2 also includes values for Q and AH for normal chromatin (NC) from liver and for tumoral chromatin (TC) from Walker carcinosarcoma. The values obtained for the enthalpy of complexation (AH) indicate also the greater possibility of coupling in tumoral chromatin. The emission spectrum of dansyl chloride can be perfectly superimposed on the excitation spectrum of acridine orange (Fig. 3), indicating that energy transfer from dansyl chloride to acridine orange is taking place. For example, at 2.5 x 10” M liver chromatin
AH (kcal mol-‘)
Q (Cal)
1O-5 (M)
0.39945 0.17477 0.07486 0.08242 0.09465
-
6.44 6.90 6.30 3.17 3.68
DNA concentration, the relative fluorescence intensity of the donor was lo = 1.93, of the acceptor IA = 4.12, of the donor in the presence of the acceptor Zk = 1.4 1, and of the acceptor in the presence of the donor Zi = 5.95 (Fig. 4). The values obtained for the transfer efficiency between the two fluorescent ligands and for the mean donor-acceptor distance for normal chromatin (NC) and for tumoral chromatin (TC) are listed in Table 3. The lower efficiency and the greater donor-acceptor distance in the tumoral chromatin, compared with normal chromatin, is an indication of a less rigid structure in tumor chromatin. 4. Conclusions The fluorescence lifetime and precision calorimetry determinations for DNA stain-chromatin complexes
-. 1.8
1
0' 465
490
540
Wavelength (nm) Fig. 3. The emission spectrum of dansyl chloride (1) and the excitation spectrum of acridine orange (2).
510
Wavelength (nm) Fig. 4. The relative fluorescence intensities of the donor and of the acceptor, either alone or in the presence of the other ligand: I, I,; 2, I$ 3, IA; 4, I:.
194
L. Radu et al./Journal of Molecular Structure 408/409 (1997) 191-194
Table 3 The transfer efficiency (E) between dansyl chloride and acridine orange and the mean donor-acceptor distance (r) for normal chromatin (NC) and for tumoral chromatin (TC) Sample
E
r (/A,
NC TC
0.274 i- 0.004 0.187 ? 0.006
49.98 ? 0.012 57.14 ? 0.015
and also the energy transfer measurements are useful in the study of the protein-nucleic acid interactions in chromatin. The results indicate a higher proportion of euchromatic regions in tumor cells, a conclusion important for the understanding of tumoral development mechanisms. The greater distance between the two ligands in tumoral chromatin compared with normal chromatin
denotes a less rigid structure of tumoral chromatin intense genie activity.
and
References [I] [2] [3] [4] [5] [6] [7]
[8] [9]
S. Weibrod, Nature 297 (1982) 289. R.D. Komberg, Ann. Rev. B&hem. 46 (1977) 931. G. Felsenfeld, J.D. McGhee, Cell 44 (1986) 375. G.W. Braddock, J.P. Baldwin, E.M. Bradbury, Biopolymers 20 (1981) 327. L. Radu, V. Preoteasa, Z. Lenghel, J. Mol. Struct. 348 (1995) 29. L. Radu, I, Mihailescu, B. Constantinescu, J. Int. Sot. Opt. Eng. 4 ( 1994) 405. L. Radu, 0. Horer, B. Constantinescu, V. Preoteasa, in: Spectroscopy of Biological Molecules, Kluwer Academic Publishers, 1995, p. 327. B. Lewin, Cell 79 (1994) 397. E. Gajewski, G. Rao, Z. Nackerdien, M. Dizdaroglu, Biochemistry 29 (1990) 7876.
of
MOLECULAR STRUCTURE Journal of Molecular Structure 4081409 ( 1997) I9 I- 194
Fluorescence lifetime, precision calorimetry, and fluorescence energy transfer measurements in the study of normal and tumoral chromatin structure Liliana Raduav*, Vasile Preoteasaa, Irina Radulescu”, Serban Radub “Department of Molecular Genetics, Victor Babes Institute, Spl. Independentei 99-101, Bucharest 76201, Romania bDepartment of Electrical Engineering, Polytechnic University. Spl. lndependentei 313, Bucharest 77703, Romania
Received 30 August 1996; revised 19 November
1996; accepted 22 November
1996
Abstract Chromatin is a complex of deoxyribonucleic acid (DNA) with proteins, that exists in the nuclei of eukaryotic cells. Three methods have been used to study protein-DNA interactions in chromatin and to compare the chromatin from normal tissue with that from tumoral tissue: determination of the fluorescence lifetimes and measurement of the heats of reaction of complexation of the hgand ethidium bromide with chromatin, and evaluation of the fluorescence energy transfer between two ligands dansyl chloride and acridine orange when coupled with chromatin. 0 1997 Elsevier Science B.V.
Keywords: Calorimetry;
Chromatin;
Energy transfer; Fluorescence;
1. Introduction Within
the
nucleus
of eukaryotic
cells,
nuclear
(DNA) is complexed with basic proteins (histones) and a variety of nonhistone proteins, to form chromatin, which is further organized into higher-order structures and tightly packed inside the nucleus [l] In last twenty years, important progress has been made in the elucidation of the structure of chromatin, in DNA-basic proteins (histones) especially complexes [2,3]. Despite great progress in the determination of chromatin substructures (the nucleosoms-complexes of DNA with five histones deoxyribonucleic
* Corresponding 0022-2860/97/$17.00
acid
author. 0 1997 Elsevier
PIISOO22-2860(96)0973
I- 1
Science
Lifetime
[4]), some aspects of the structure of chromatin remain unknown. Our previous studies were oriented toward analysis of the structure of chromatin by absorption and emission spectroscopy of complexes of chromatin with specific DNA ligands [5,6] or by isotope uptake and ‘H NMR spectroscopic methods [7]. In this paper we analyze the structure of chromatin from normal tissue-the liver of Wistar rats-and that from tumoral tissue-Walker carcinosarcoma maintained on Wistar rats. The methods used for this purpose were determination of fluorescence lifetimes and precision calorimetry for the binding of the ligand ethidium bromide to normal and tumoral chromatin, and fluorescence energy transfer measurements between a pair of fluorescent ligands, dansyl chloride and acridine orange, coupled to chromatin.
B.V. All rights reserved
192
L. Radu et al./Journal of Molecular Siructure 408/409 (1997) 191 -I 94
2. Experimental
2 ,
The chromatin was extracted from the livers of Wistar rats and from the Walker carcinosarcoma, maintained on Wistar rats, by Lewin’s method[8]; purity was verified by an absorption test [9]. Ethidium bromide (c = lo-’ M) was complexed with chromatin samples (chromatin DNA concentration 2.5 x lOA M). In the experiments of double fluorescent labeling, the chromatin proteins were covalently bonded with dansyl chloride and the chromatin DNA was uncovalently coupled with acridine orange. For dansyl chloride hexcitation= 323 nm and Xemission= 505 nm and for acridine orange Xex,-itation= 505 nm and Xemission= 530 nm. The transfer efficiency between the two ligands is [6]:
220
240
260
280
300
Wavelength (nm) Fig. 1. The spectra of chromatim from liver (1) and Walker tumor (2).
E=($.($) where IA, Zg are the relative fluorescence intensities of the acceptor, in the absence and the presence, respectively, of the donor, and eA and eo are the molar extinction coefficients of the acceptor and donor, respectively, at the wavelength of excitation. The transfer efficiency is related to the distance, r, between the donor and the acceptor by the relationship: E=
re6 r %R$ 1
where R. = (Jk2.Qo~n”) i; x 9.79 x 103, J is the overlap integral, k* is the orientation factor for dipoledipole transfer, n the refractive index of the medium, and Q0 the quantum yield of the donor in the absence of transfer. A Pharmacia LKB Ultrospec III spectrophotometer, an Aminco Bowman spectrofluorimeter, a time-resolved fluorimeter FL 900 CD and a LKB 8700 precision calorimeter were used.
E260nm
-=1.7and E 280nm
E2mm
spectrum
-= E 240nm
1
2 cor,*x
lo4
M
Fig. 2. The relative fluorescence intensities of the complexes of ethidium bromide with liver (1) and Walker tumor (2) chromatin, versus chromatin DNA concentration.
while for Walker tumor chromatin these ratios are 1.75 and 1.45, respectively. Fig. 2 shows the relative fluorescence intensities for complexes of normal chromatin and of tumoral chromatin with ethidium bromide. The values obtained indicate a greater availability to ligand binding in tumoral chromatin. The fluorescence decay curves have three components, with half-lives (7) of 2 ns for unbound ethidium bromide, 8 ns for ethidium bromide loosely bound to chromatin DNA, and 24 ns for intercalated ethidium bromide in chromatin DNA (Table 1). Table 1 The lifetimes (7) and the percentages of lifetimes v) of the chromatin-ethidium bromide complexes, for normal chromatin (NC) and for tumoral chromatin (TC)
3. Results and discussion The liver chromatin characteristics:
0
14
’
(Fig. 1) presents the
NC TC
7 (/ns) f US) r(/ns) f U%)
2.24 2 0.06 12.10 2.11 t 0.07 11.82
8.63 + 0.05 42.73 8.14 + 0.02 38.15
24.11 + 0.08 45.17 24.72 + 0.02 50.03
193
L. Radu et al./Joumal of Molecular Structure 4OW409 (1997) 191-194
Table 2 The parameters
deduced from calorimetric
Sample
CDNA
DNA DNA DNA NC TC
25.00 10.25 4.75 10.11 4.40
x
lo4
determinations n x
CM)
6.20 2.53 1.18 2.60 2.57
These three similar components were observed both in liver chromatin and in the Walker tumor chromatin, but with different relative contributions (f). In tumoral chromatin, a 9% increase of the 24 ns component was observed, indicative of a less rigid chromatin structure. The heat of interaction (Q) of the ethidium bromide with chromatin is dependent on the concentration of DNA (Table 2, in which n is the number of moles of ligand. The mean value obtained for the enthalpy (AH) of complexation of ethidium bromide with DNA is -6.54 kcal mall’. Table 2 also includes values for Q and AH for normal chromatin (NC) from liver and for tumoral chromatin (TC) from Walker carcinosarcoma. The values obtained for the enthalpy of complexation (AH) indicate also the greater possibility of coupling in tumoral chromatin. The emission spectrum of dansyl chloride can be perfectly superimposed on the excitation spectrum of acridine orange (Fig. 3), indicating that energy transfer from dansyl chloride to acridine orange is taking place. For example, at 2.5 x 10” M liver chromatin
AH (kcal mol-‘)
Q (Cal)
1O-5 (M)
0.39945 0.17477 0.07486 0.08242 0.09465
-
6.44 6.90 6.30 3.17 3.68
DNA concentration, the relative fluorescence intensity of the donor was lo = 1.93, of the acceptor IA = 4.12, of the donor in the presence of the acceptor Zk = 1.4 1, and of the acceptor in the presence of the donor Zi = 5.95 (Fig. 4). The values obtained for the transfer efficiency between the two fluorescent ligands and for the mean donor-acceptor distance for normal chromatin (NC) and for tumoral chromatin (TC) are listed in Table 3. The lower efficiency and the greater donor-acceptor distance in the tumoral chromatin, compared with normal chromatin, is an indication of a less rigid structure in tumor chromatin. 4. Conclusions The fluorescence lifetime and precision calorimetry determinations for DNA stain-chromatin complexes
-. 1.8
1
0' 465
490
540
Wavelength (nm) Fig. 3. The emission spectrum of dansyl chloride (1) and the excitation spectrum of acridine orange (2).
510
Wavelength (nm) Fig. 4. The relative fluorescence intensities of the donor and of the acceptor, either alone or in the presence of the other ligand: I, I,; 2, I$ 3, IA; 4, I:.
194
L. Radu et al./Journal of Molecular Structure 408/409 (1997) 191-194
Table 3 The transfer efficiency (E) between dansyl chloride and acridine orange and the mean donor-acceptor distance (r) for normal chromatin (NC) and for tumoral chromatin (TC) Sample
E
r (/A,
NC TC
0.274 i- 0.004 0.187 ? 0.006
49.98 ? 0.012 57.14 ? 0.015
and also the energy transfer measurements are useful in the study of the protein-nucleic acid interactions in chromatin. The results indicate a higher proportion of euchromatic regions in tumor cells, a conclusion important for the understanding of tumoral development mechanisms. The greater distance between the two ligands in tumoral chromatin compared with normal chromatin
denotes a less rigid structure of tumoral chromatin intense genie activity.
and
References [I] [2] [3] [4] [5] [6] [7]
[8] [9]
S. Weibrod, Nature 297 (1982) 289. R.D. Komberg, Ann. Rev. B&hem. 46 (1977) 931. G. Felsenfeld, J.D. McGhee, Cell 44 (1986) 375. G.W. Braddock, J.P. Baldwin, E.M. Bradbury, Biopolymers 20 (1981) 327. L. Radu, V. Preoteasa, Z. Lenghel, J. Mol. Struct. 348 (1995) 29. L. Radu, I, Mihailescu, B. Constantinescu, J. Int. Sot. Opt. Eng. 4 ( 1994) 405. L. Radu, 0. Horer, B. Constantinescu, V. Preoteasa, in: Spectroscopy of Biological Molecules, Kluwer Academic Publishers, 1995, p. 327. B. Lewin, Cell 79 (1994) 397. E. Gajewski, G. Rao, Z. Nackerdien, M. Dizdaroglu, Biochemistry 29 (1990) 7876.