Quantum yield of acridines interacting with DNA of defined base sequence

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-70°C were related to the duration of storage, not to the procedures of freezing and thawing [7]. At -SC, lipolysis persisted in frozen rat adipose tissue [3]. These enzymatic changes, however, may not be selective in nature, but selection is probable in tumour and heteroploid cell populations where chromosomal and antigenic changes are known [L 6, 8, 91. References 1. Ashwood-Smith, M J, Cry obiol 1 (1965) 242. 2. Bohm, F, Folia biol 18 (1972) 42. 3. Geyer, K G & Goodman, H M, Proc sot exptl biol med 133 (1970) 404. 4. Hol&kova, E, BaudySovl, M & Cinnerova, 0, Exptl cell res 40 (1965) 396. 5. McKee, M E, Harris, S E & Kihara, H, Proc sot exptl biol med 123 (1966) 499. 6. Morgan, J F, Gutrin, L F & Morton, H J, Cancer res 16 (1956) 907. 7. Peterson, W D Jr & Stulberg, C S, Cryobiol 1 (1964-5) 86. 8. Srb, V, Klen, R & Ht.&k, T, d fysiol 17 (1968) 621. 9. Wodinsky, I, Meaney, K F & Kensler, C J, Cryobiol 2 (1965-6) 44. Received November 16, 1971 Revised version received February 14, 1972

mosomes. It had already been noted, however, that quinacrine alone already exhibited the same banding pattern [I]. Also, related acridines such as proflavine gave similar results [2], indicating that the acridine ring as such must be related to the banding pattern. Initiated by the early observation of Tubbs et al. [4] that the quantum yield of acriflavine is dependent on the G-C content of DNA, we investigated the fluorescence properties of proflavine (PF) interacting with synthetic DNAs of different base sequence [5]. The quantum yield of PF interacting with poly[d(G-C)] decreased considerably, whereas an increase was observed when PF was bound to poly[d(A-T)]. These findings suggested that the banding pattern of acridine-labelled chromosomes, at least in part, could reflect a certain distribution of base pairs influencing the fluorescence properties of the label. These results are presented in greater detail in this communication and have been extended also for the case of quinacrine. Materials and Methods

Quantum yield of a&dines interacting with DNA of defined base sequence. A basis for the explanation of acridine bands in chromosomes U. PACHMANN and R. RIGLER, Institute for Cell Research and Genetics, Medical Nobel Institute, Karolinska Institutet, 104 01 Stockholm 60, Sweden

Fluorescence labelling of chromosomes with quinacrine derivatives has been introduced by Caspersson et al. [l-3] to identify individual chromosomes by their characteristic banding patterns. The reason for using quinacrine mustards was the idea of directing the fluorescence label by its functional groups specifically to certain regions in DNA which might be distributed differently in the individual chroExptI Cell Res 72

Proflavine (PF) was used as the hemisulfate (mol. wt 552.6; British Drug Houses, Poole, Dorset, UK). Quinacrine (QAC) (mol. wt 508.9) was a gift from the Winthrop Product Company, Surrey, UK. Calf thymus DNA was a preparation from Worthington Biochemical Corporation (lot 7 LB) characterized as follows; ePZBO= 6 400 M-l cm-l, protein content 0.6 %, intrinsic viscosity 60 dI/g. Poly[d(AT)] was synthesized according to [6]. Poly[d(G-C)], poly[d(I-C)] and poly[d(TG-AC)] were gifts from Dr T. Jovin, Max-Planck-Institut fur biophysikalische Chemie, Giittingen. Their approximate molecular weights as estimated from sedimentation velocity runs in a Spinco model E ultracentrifuge at 20°C were 17 x lo6 (poly[d(A-T)]), 6 x lo6 (poly[d(I-C)]) and 4 x IO* (poly[d(G-C)]). To determine their concentrations the extinction coefficients poly[d(A-T)]: 6 800 M-l cm-’ (260 nm), poly[d(G-C)]: 8 400 M-l cm-l (254 nm), poly[d(l -C)] 6 900 M-l cm-l (250 nm), poly[d(TG-AC)] 6 500 M-i cm-l (260 nm) according to [7] were used. Absorption and emission spectra, fluorescence titrations. All DNAs were dialysed against 0.03 M K phosphate; 0.1 M KCI, 0.5 mM EDTA, pH 7.0. The

Quantum yield of acridines interacting with DNA dQ(h) dh

603

a

1.0

0.6

b 1.0

08

0.K CT.-DNA p*y d&-T) po,y do-C) pdy dU4

-o-o-a-n-----.-.-

1.0

ae

0.6

06

a4

a4

a2

a2

0

0 A300

400

500

same buffer was used for all measurements. Absorption spectra were recorded in a Gary-14 absorptionspectrophotometer, the emission spectra in a modified Farrand spectrofluorometer equipped with two grating monochromators. The emission spectra were corrected for wavelength dependent monocbromator transmission and photomultiplier sensitivity by a calibrated standard light source. All emission spectra were excited at 455 nm. Fluorescence titrations were performed with an equipment as outlined in [S]. The fluorescence of PF and PF-DNA complexes was excited at 455 nm that of QAC and QAC-DNA complexes at 436 nm. The emission was recorded after passing a GG495 cut-off filter (Schott and Gen.). The pathlength of the cuvettes was 4.4 mm. The polarization of fluorescence was determined as the ratio between the difference and the sum of the light components vibrating parallel and perpendicular to the plane of polarization of the exciting light beam. To correct for depolarization of the optical system the polarizer was turned at right angles to both components of

Fig. 1. Ordinate: relative OD units. Absorption and quantumspectra of (a) PF; (b) QAC. PF, 2.25 x 10-O M (absorption); 1.0 x 1O-8 M (emission); QAC, 0.7 x 10-s M (absorption), 1.5 x lo-& M (emission); Calf thymus DNA 2.5 x lo-* M, poly[d(A-T)] 4.7 x lo-* M, poly[d(I-C)] 2.2 x lo-* M, poly[d(G-C)] 4.1 x lo+ M. Maximal optical density of unbound PF and QAC and maximal dQ/dL of poly [d(I-C)]-PF and poly[d(I-C)]-QAC adjusted to be unity. Amplification factors in the quantum spectra as indicated.

600 nm

the emitted light and their intensities were equalized by electronic adjustments. Lamp fluctuations were compensated by a beam-splitting arrangement.

Results Interactions between PF or QAC and DNAs of different base composition cause a red shift in the absorption spectrum and a corresponding blue shift in the emission spectrum (fig. 1). No significant differences can be detected in the distribution of absorption and emission bands in the different acridineDNA complexes. While the absorption of PF and QAC when bound to the different DNAs (except small differences) is decreased to about the same level, the inExptl Cell Res 72

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U. Pachmann & R. Rigler

TlTRRTIilN

PF-ONR

TIIRRTION

TITRRTION

PF-DGC

PF-DIG

Fig. 2. Abscissa: concentration of DNA added; ordinate: (left) fluorescence intensity; (right) polarization of fluorescence. A, Relative fluorescence intensity; O, degree of polarization. Fluorescence titrations of PF (1 x lo-@ M) with (a) calf thymus DNA; (b) poly[d (A-T)]; (c) poly[d(G-C)]; (d) poly[d(I-Q]. Computer printout. The PFconcentrations were kept constant during the whole experiment.

of emission is highly dependent on the type of DNA used. The quantum yields of fluorescence (q) as determined from integration of the quantum spectra in fig. 1 drop down to less than 0.05 in the PF-poly[d(G-C)]

tensity

Exptl Cell Res 72

complex and are enhanced in the PF-poly[d(A-T)] and PF-poly[d(I-C)] complexes. The quantum yields of the respective QACDNA complexes behave similarly (table 1). Preliminary experiments with poly[d(TG-

Quantum yield of acridines interacting

Table

1. Quantum yield (q~) and polarization

PF PF-calf thymus DNA PF-poIy[d(A-T)] PF-poly[d(G-C)] PF-poly[d(I-C)]

Pa

Pb

0.7 0.25 0.74 0.05 0.83

0.05 0.33 0.30 829

(p) of fluorescence

QAC

QAC-calf

QAC-wMd(A-VI

605

of acridine DNA complexes

thymus DNA

QAC-poly[d(G-C)] QAC-poly[d(I-C)]

with DNA

Pa

Pb

0.17 0.12 0.74 0.04 0.74

0.04 0.20 0.23 0.20 0.19

a q of PF was taken as 0.7 [9]. ’ Last values of titrations.

AC)] (not shown) indicate that p of the PF-poly[d(TG-AC)] complex is almost as low as that of the PF-poly[d(G-C)] complex. The extent of binding of PF and QAC can be determined from the change in intensity and polarization of fluorescence when both compounds are titrated with the different DNAs (figs 2, 3). While the intensity of fluorescence increases or decreases according to the type of DNA until a saturation level is reached, the polarization of fluorescence (p) is always increased, thus indicating immobilisation of PF and QAC when complexed with DNA. Discussion

Our data indicate that the quantum yields of PF and particularly of QAC-both known to give characteristic banding patterns in chromosomes-are enhanced in DNA of alternating AT sequence whereas they are reduced in DNA of alternating GC sequence. The higher value of ~1for the PF-poly[d(AT)] complex given in [S] was measured as the intensity of the emission peak related to the absorption in the exciting wavelength. These observations and those of other authors [9-121 lead to the conclusion that the quantum yield of these and related acridines -such as acriflavine-is mainly determined by the type of nucleotide base pairs involved in binding. The quantum yields given for the different acridine-DNA complexes have to be

regarded as lower limits for the poly[d(A-T)] and poly[d(I-C)] complexes and as upper limits for the poly[d(G-C)] complexes. Dealing with an equilbrium situation even at great excess of DNA a certain amount of unbound acridine is present. The data on the quantum yield of PFpoly[d(TG-CA)] complexes indicate, in accordance with an assumption of [12], that a GC pair adjacent to an AT pair is able to quench the PF-fluorescence almost as effectively as two adjacent GC pairs, Apparently at least two adjacent AT pairs are needed to enhance the quantum yield of PF. If one calculates from the data of table 1 and the known distribution of two adjacent AT, GC and GC-AT pairs of calf thymus DNA [13], the quantum yield of the complex, one arrives at a value close to the observed one. In this calculation possible differences in the quantum yield between A-T/T-A and A-T/A-T pairs as well as the respective GC and CC-AT combinations are neglected. This would mean that the fluorescence of AT and GC containing DNA-to adopt a rather simplified picture-is mainly determined by PF which is bound to AT regions possibly by intercalation and is not adjacent to a GC pair. To obtain congruence between observed and calculated p of QAC-calf thymus DNA, one must assume that at least 4 adjacent AT pairs are needed to enhance the 40 of QAC. Comparison between poly[d(G-C)] and Exptl Cell Res 72

606

U. Pachmann & R. Rigler

IllRRTION

QRC-DNA

TIlRATION

OAC-DGC

3. Fluorescence titrations of QAC (1.5 x 1O-B M) with (a) calf thymus DNA; [d(G-Cl; (d) poly[d(I-C)]. Same symbols as in fig. 2.

Fig.

poly[d(I-C)] complexes indicate that the guanine apparently is responsible for the quenching of the PF and QAC fluorescence. Based on theoretical arguments [14], formation of charge transfer complexes have been discussed in relation to the quenching of Exptl

Cell Res 72

(b) poly[d(A-T)];

(c) poly-

fluorescence [4, Ill. However, a rigorous experimental proof including the analysis of the excited-state kinetics of different acridine-DNA complexes is still lacking. One has also to keep in mind that poly[d(I-C)] might have a rather unusual DNA-helix 1151,

Quantum yield of acridines interacting with DNA

which could be the reason, in addition to the NH,-group lacking in 2-position, why the quantum yield of PF(QAC)-poly[d(I-C)] complexes differs from that of the corresponding poly[d(G-C)] complexes. In evaluating binding constants and number of binding sites from fluorescence data, one has to exclude that the quantum yield of acridines in the bound state-even if binding occurs to homogeneous polymers-is not represented by a distribution of molecules bound to sites with different quantum yields. Studies of the luminescence decay characteristics of these acridine-DNA complexes are in progress to answer this question. From saturation functions calculated from the observed degree of polarization, which is less dependent on the quantum yield of the bound-state [16], preliminary values for the number of binding sites n (given as number of acridines bound per phosphate residue) and for the stability constant, can be derived. They do not show major differences when PF (n =0.06-0.1, K =0.9-2 X lo6 M-l) or QAC (n=O.O8-0.16, K=0.6-1 x lo6 M-l) is bound to the different DNAs, indicating that these compounds must be rather evenly distributed along an AT and GC containing DNA. The increase in p of PF and QAC in the titration experiments shows that the changes in the quantum yields are actually caused by the binding of both compounds to the various DNAs. Amongst the acridine-DNA complexes the poly[d(G-C)] exhibits a significantly lower p. Apart from the fact that in this case the lower value of p partly is due to unbound acridine with much higher v than in the bound state, it might also be caused by an increased rotational freedom of either poly[d(G-C)] and/or the bound acridine. Studies of the fluorescence-depolarization kinetics should provide an answer [17].

607

Any extrapolation from the data obtained from the interaction between acridines and synthetic DNAs to the situation in the chromosome has to consider that the structure of these DNAs in solution might be different from DNA of comparable base sequence in the highly organized state of a chromosome. In addition, the structure of chromosomal DNA is to a great extent determined by the interaction of different kinds of proteins known to interfere with the binding of acridines to nucleic acids [18, 191. However, instrumentation and methods [l, 20, 211 are available to decide if afluorescent chromosome band is due to increased binding or increased quantum yield of the bound fluorescence label. In those instances where the distribution of PF along the chromosome including the fluorescent bands (PF-bands in Vicia faba) was measured by light absorption [22] a homogeneous distribution was found indicating that PF in the banding regions exhibits a significantly higher quantum yield. It is tempting to conclude that the fluorescent bands in these cases might correspond to AT rich regions possibly enlarged by gene amplification. However, more detailed biophysical studies on the actual chromosome bands using techniques as mentioned above are required to support this suggestion. Regarding the rather detailed information fluorescence analysis can give, as well as the extensive knowledge about the interactions between acridines and nucleic acids [18, 23, 241, it is evident that these compounds are superior to nonfluorescent dyes for the analysis of the nature of chromosome bands. We thank Professor T. Caspersson for very valuable discussions, Dr V. Pigiet for sedimentation velocity determinations as well as Mrs B. Larsson and Mrs R. Jakabffy for expert technical assistance. This work was supported by grants from the Swedish Cancer Society (proj. no. 56%B71-OlP and no. 150-B71-06P). Exptl Cell Res 72

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Note added in proof Data similar to those reported in this article have also been obtained by Weisblum & de Haseth [25] and have led them to comparable conclusions.

References 1. Caspersson, T, Zech, L, Modest, E J, Foley, G E, Wagh, U & Simonsson, E, Exptl cell res 58 (1969) 128. 2. Caspersson, T, Zech, L, Wagh, U, Modest, E J & Simonsson, E, Exptl cell res 58 (1969) 141. 3. Caspersson, T, Exptl cell res 58 (1969) 451. 4. Tubbs, R K, Ditmars, W E & Van Winkle, Q, J mol biol 9 (1964) 545. 5. Rigler, R, Exptl cell res 58 (1969) 460. 6. Schachman, H K, Adler. J. Raddina. C M, Lehman, J R & Kornberg, A, J biol &em 235 (1960) 3242. 7. Wells, R D, Larson, J E, Grant, R C, Shortle, B E & Cantor, C R, J mol biol 54 (1970) 465. 8. Ehrenberg, M, Cronvall, E & Rigler, R, FEBS letters 18 (1971) 199. 9. Weill, G, Biopolymers 3 (1965), 567. 10. Chan, L M & Van Winkle, Q, J mol biol 40 (1969) 491. 11. Thomes, J C, Weill, G & Daune, M, Biopolymers 8 (1969) 647.

The American Type Culture Collection Registry of Animal Cell Lines The second edition of the Registry of Animal Cell Lines was released April 1,1972 and contains descriptions of 134 animal cell lines derived from over 30 different species. Represented in the collection are normal diploid cells, malignant cells, cells grown in chemically-defined media, virus-transformed cells, cells with special chromosome configurations, biochemical markers, and virus susceptibilities and cells that produce specialized products such as hormones, immunoglobulins, mucopolysaccharides, and pigments. The Registry contains a full-page description ERRATUM Exptl cell res 68 (1971) 1, l-10. The Abscissa to Fig. 1 should commence “pmoles diamide/109 CHO cells . . .” In line 10 of the caption to Fig. 4, “stained” should read “starved”, and the penultimate line should read “The control (100%) NPSH content is 1 x lo-* ~moles/cell.”

Exptl Cell Res 72

12. Chan, L M & McCarter, J A, Biochim biophys acta 204 (1970) 252. 13. Jesse, J, Kaiser, A D & Kornberg, A, J biol them 236 (1961) 864. 14. Pullman, B & Pullman, A, Quantum biochemistry. Wiley, New York (1963). 15. Mitsui, Y, Langridge, R, Grant, R C, Kodama, M, Wells, R D, Shortle, B E & Cantor, R C, Nature 228 (1970) 1166. 16. Ellerton, N F & Isenberg, I, Biopolymers 8 (1969) 787. 17. Ehrenberg, M & Rigler, R, Chem phys letters 14 (1972). In press. Rigler, R, Acta physiol Stand 67 (1966) 1. ii: Gittelson, B L & Walker, I 0, Biochim biophys acta 138 (1967) 619. 20. Caspersson, T, Lomakka, G & Rigler, R, Acta histochem, suppl. 6 (1965) 123. 21. Caspersson, T & Lomakka, G, Instrumentation in biochemistry (ed T W Goodwin) p. 25. Academic Press, New York and London (1966). 22. Caspersson, T et al. To be published. 23. Blake, A & Peacocke, A R, Biopolymers 6 (1968) 1225. 24. Ltiber, G & Achtert, G, Biopolymers 8 (1969) 595. 25. Weisblum, B & de Haseth, P L, Proc natl acad sci US 69 (1972) 629. Received March 5, 1972

of each cell line, citing historical information, culture medium, references, and characterization tests performed on the cells. New features in this edition include: The description of 47 newly accessioned cell lines; additional characterization of certain cell lines; recommendations on the freezing of animal cell lines; a new index by tissue of origin; a special “use” list classifying cells by special uses or properties. $2.50 per copy for shipment by book mail; $4.00 per copy for shipment by air parcel post. Please send orders to: American Type Culture Collection, Cell Repository, 12301 Parklawn Drive, Rockville, Md 20852, USA.