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; Lifetime

1. Introduction

Within the nucleus of eukaryotic cells, nuclear deoxyribonucleic acid (DNA) is complexed with basic proteins (histones) and a variety of nonhistone proteins, to form chromatin, which is further orga- nized 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, especially in DNA-basic proteins (histones) complexes [2,3]. Despite great progress in the determination of chromatin substructures (the nucleosoms-complexes of DNA with five histones

* Corresponding author.

[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 emis- sion 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 life- times and precision calorimetry for the binding of the ligand ethidium bromide to normal and tumoral chro- matin, and fluorescence energy transfer measurements between a pair of fluorescent ligands, dansyl chloride and acridine orange, coupled to chromatin.

0022-2860/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO22-2860(96)0973 I- 1

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 concen- tration 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 uncova- lently coupled with acridine orange. For dansyl chlor- ide 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]:

E=($.($) where IA, Zg are the relative fluorescence intensities of the acceptor, in the absence and the presence, respec- tively, 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 while for Walker tumor chromatin these ratios are

r %R$ 1.75 and 1.45, respectively. 1

where R. = (Jk2.Qo~n”) i; x 9.79 x 103, J is the over- lap integral, k* is the orientation factor for dipole- dipole 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 spectrophot- ometer, an Aminco Bowman spectrofluorimeter, a time-resolved fluorimeter FL 900 CD and a LKB 8700 precision calorimeter were used.

3. Results and discussion

The liver chromatin spectrum (Fig. 1) presents the characteristics:

E260nm -=1.7and -= E

E2mm 14

280nm E ’ 240nm

220 240 260 280 300

Wavelength (nm)

Fig. 1. The spectra of chromatim from liver (1) and Walker tumor (2).

0 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.

Fig. 2 shows the relative fluorescence intensities for complexes of normal chromatin and of tumoral chro- matin with ethidium bromide. The values obtained indicate a greater availability to ligand binding in tumoral chromatin.

The fluorescence decay curves have three compo- nents, 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)

NC 7 (/ns) 2.24 2 0.06 8.63 + 0.05 24.11 + 0.08 f US) 12.10 42.73 45.17

TC r(/ns) 2.11 t 0.07 8.14 + 0.02 24.72 + 0.02 f U%) 11.82 38.15 50.03

L. Radu et al./Joumal of Molecular Structure 4OW409 (1997) 191-194 193

Table 2 The parameters deduced from calorimetric determinations

Sample CDNA x lo4 CM) n x 1O-5 (M) Q (Cal) AH (kcal mol-‘)

DNA 25.00 6.20 0.39945 - 6.44 DNA 10.25 2.53 0.17477 - 6.90 DNA 4.75 1.18 0.07486 - 6.30 NC 10.11 2.60 0.08242 - 3.17 TC 4.40 2.57 0.09465 - 3.68

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 com- plexation (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 trans- fer from dansyl chloride to acridine orange is taking place. For example, at 2.5 x 10” M liver chromatin

1

490 540

Wavelength (nm)

Fig. 3. The emission spectrum of dansyl chloride (1) and the excita- tion spectrum of acridine orange (2).

DNA concentration, the relative fluorescence inten- sity 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

0' 465 510

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)

denotes a less rigid structure of tumoral chromatin and intense genie activity.

Sample E r (/A, References

NC 0.274 i- 0.004 49.98 ? 0.012 TC 0.187 ? 0.006 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 euchro- matic 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

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