DNA measurement by mithramycin fluorescence in chromatin solubilized by heparin

ANALYTICAL

BIOCHEMISTRY

106,262-268

DNA Measurement

(1980)

by Mithramycin Fluorescence Solubilized by Heparin ANDRE GROYERAND

in Chromatin

PAULROBEL

ER 125 CNRS, Unit6 de Recherches SW le Mktabolisme Mol.kulaire et la Physio-Pathologie des Sttroides de l’lnstitut National de la SantP et de la Recherche Mkdicale, H6pital de Bicitre, 78 rue du GhnCral Leclerc, 94270 Bicstre, France Received March 19, 1980 The mithramycin fluorescence procedure described by B. T. Hill and S. Whatley (1975, FEBS Lett., 56, 20-23) for DNA measurement tends to underestimate DNA concentrations in biological samples as compared to the results obtained by the diphenylamine reaction. This discrepancy disappears when DNA is first solubilized, by buffer containing heparin, from either cell homogenates or nuclear preparations. The optimal conditions for maximal fluorescence are 8 mM Mg 2+, 10 &ml mithramycin, and heparin to DNA ratios 30.15 (w/w). Background fluorescence is reduced 90% by dextran-coated charcoal adsorption of unbound mithramycin. The limit of sensitivity of the assay is 0.3 &ml and fluorescence is linear up to 30 pg DNA/ml.

Deoxyribonucleic acid measurement by the widely used diphenylamine reaction (2) requires 16-20 h for color development with a lower limit of sensitivity of about 5 pg. More sensitive assays exploit the enhanced fluorescence intensity obtained when molecules such as ethidium bromide (EB)’ (3), diaminobenzoic acid (DABA) (4), or mithramycin (1,5) interact with DNA. Since EB also intercalates into RNA, DNA fluorescence can only be determined after digestion of RNA. Hydrolysis of DNA deoxyribose is necessary for DABA reaction. Mithramycin and related antibiotics (chromomycin As, olivomycin) interact directly with DNA. The interaction requires double-stranded DNA, Mg2+, occurs at guanine residues (6), and is not covalent (7), and there is no interference from proteins or RNA (1). 1 Abbreviations used: EB, ethidium bromide; DABA, diaminobenzoic acid; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; TE buffer, 10 mM Tris-HCl, pH 7.4 at 20°C 1 mM EDTA; TE-HlOO(200); TE buffer containing lOO(200) pg of heparin/ml; DCC, dextran-coated charcoal. 0003-2697/80/l 10262-07$02.00/O Copyright 0 19SO by Academic F’ress, Inc. All rights of reproduction in any form reserved.

262

In this work, we describe a modification of the mithramycin assay where nucleoprotein complexes are dissociated by heparin instead of by ultrasonic disruption of chromatin. In addition, the background fluorescence has been substantially reduced by dextran-coated charcoal adsorption of unbound mithramycin. MATERIALS

AND METHODS

Chemicals The commercially available preparation of mithramycin (Mithracin, Pfizer Laboratories) was used. Stock solutions (200 &ml) were prepared in either distilled water or 160 mM magnesium chloride and kept in the dark at 4°C. Unless otherwise stated, the final mithramycin concentration in the assay mixture was 10 pg/ml. Heparin (sodium salt) and sodium dodecyl sulfate (SDS) were obtained from Sigma Chemical Company. Calf thymus DNA was purchased from Worthington Biochemical Company.

DNA

Buffers

MEASUREMENT

BY MITHRAMYCIN

FLUORESCENCE

263

diluted to 950 Z.L~with TE-HlOO buffer, then 50 ~1 of mithramycin solution (200 Z&ml The buffers used were: Dulbecco’s cal- in 160 mM MgC&) was added. The mixture cium and magnesium-free phosphate-buffered was vigorously stirred with a Vortex mixer saline (PBS), 0.136 M NaCl, 2.6 mM KCl, and left at room temperature for 5 min. This 6.4 mM N%HPO,, 1.4 mrvr KH,PO,; TE time length was sufficient to obtain steady buffer, 10 mM Tris-HCl, pH 7.4 at 20°C state of antibiotic binding to DNA. 1 mM EDTA. TE-HlOO and TE-H200 buffers Dextran-coated charcoal adsorption. One consisted of TE buffer containing 100 and milliliter of ice-cold dextran-coated charcoal 200 pg of heparin/ml, respectively. suspension (0.25% Norit A, 0.025% dextran T70, w/v, in TE buffer) was centrifuged at Tissues 3600g for 10 min. The supernatant was discarded and the pellet was resuspended Purified rooster liver nuclei were preby vigorous stirring in 1 ml of assay mixture pared according to the method of Blobel after completion of mithramycin binding. and Potter (8) with minor modifications. The suspension was left at 0°C for 5 min Human embryo fibroblasts were harvested + 15 s and charcoal particles were removed from confluent monolayers by trypsinizaby centrifugation at 3600g for 20 min. The tion (trypsin 1:250, Difco) and collected by centrifugation at SOOg for 10 min. The resulting supernatant was carefully decanted and left at room temperature. pellet was resuspended in 1 ml of TE buffer Fluorescence measurements. Fluorescence and the cells were disrupted by freezeintensity (IF) was measured with an Amincothawing (four times). DNA was measured Bowman spectrofluorometer. Excitation in the homogenate or in the crude nuclear and emission wavelengths were 440 and 540 pellet prepared by centrifugation at SOOg nm, respectively. Depending on the amount for 10 min. of DNA present in the reaction mixture, the meter multiplier was set at higher (0.01) Sonication or lower (0.03) sensitivity. DNA specific fluorescence (IF,,,) was calculated as the The samples were sonicated at 0°C with difference between total fluorescence (IF,) a Branson B 12 sonifier by two 5-s bursts, and mithramycin intrinsic fluorescence (i.e., at a 100-W setting. fluorescence of identical sample in absence of DNA). Other DNA Measurement Techniques When dextran-coated charcoal treatment The diphenylamine reaction (2) and the was applied, the intensity of fluorescence of the final supernatant decreased gradually measurement of DNA phosphorus (9) were with time. Steady state was reached after used as reference techniques. 20 min at room temperature, and then IF remained constant during the following 40 Assay Procedure min. Measurements of IF were routinely Solubilization of chromatin. Purified performed after a 30-min interval, and will rooster liver nuclei, total homogenates, be referred to asZF,c, in the Results section. or crude nuclei from cultured human fibroCalfthymus DNA. Calf thymus DNA (10 blasts were prepared in TE buffer and mixed and 400 Z.&ml of TE buffer) was used as with an equal volume of TE-H200 buffer. standard. The samples were left at 0°C for 30 min. Calculations. Linear regressions and Mithramycin binding to DNA. Triplicate their 95% confidence limits were calculated aliquots (lo-80 ~1) were removed and with a Mitra 15 computer.

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RESULTS

Effects of Mg2+, Mithramycin, EDTA, Heparin, and SDS Concentrations on DNA Specific Fluorescence Preliminary experiments were carried out with calf thymus DNA (20 pg/ml) in PBS, in order to define optimal experimental conditions for the mithramycin assay. Mithramycin (10 CLg/ml) intrinsic fluorescence decreased 4.6-fold when Mg2+ concentrations were increased from 0.04 to 25 mM. However, since mithramycin does not bind to DNA in the absence of Mg2+ (6), maximal specific fluorescence was reached with Mg2+ concentrations 3 4 mM (Fig. 1A). The removal of unbound mithramycin by dextran-coated charcoal was nearly complete (96 2 0.9%) within 5 min at 0°C at all Mg2+ concentrations tested (Fig. 1B). ZFDcc increased when magnesium concentrations were raised from 0.2 to 1 mM, then re-

FIG. 1. Effect of Mg*+ concentration on DNA specific fluorescence. Incubations were performed in PBS (A) or in TE buffer (B) containing mithramycin (10 &ml) and MgZ+ concentrations ranging from 0.04 to 25 mM. In experiment B, all samples were treated with dextran-coated charcoal at 0°C for 5 min, as reported under Materials and Methods. (1) 0, without DNA; (2) A, with 20 pg/ml calf thymus DNA (IF,); (3) 0, specific fluorescence (IFDNA) calculated from the difference between incubations (2) and (1). Each point represents the mean of two determinations.

FIG. 2. Effect of mithramycin concentration on DNA specific fluorescence. Incubations were performed in TE buffer containing 8 mM Mgl+ and increasing concentrations of mithramycin (0.1-22 &ml). IF was measured (A) without or(B) with dextran-coated charcoal adsorption. (1) 0, without DNA; (2) A, with calf thymus DNA (20 pglml) (IF,); (3) 0, specific fluorescence (ZFDNA) calculated from the difference between incubations (2) and (1). Each point represents the mean of two determinations.

mained constant up to 10 mM Mg2+. For Mgz+ concentrations > 10 mM, IF,,, decreased, likewise because high magnesium concentrations accelerate the dissociation of mithramycin-DNA complexes. Intrinsic and DNA specific fluorescence were also measured at increasing mithramycin concentrations (0.1-22 pg/ml) in the presence of 8 mM Mg2+ (Fig. 2A). Maximal specific fluorescence required antibiotic concentrations 3 10 pg/ml. After treatment with dextran-coated charcoal, IF,,, reached a plateau for initial mithramycin concentrations of lo-12 &ml, then increased at higher concentrations. This increase was related to incomplete adsorption of unbound mithramycin (Fig. 2B). Consequently, the mithramycin-DNA binding equilibrium achieved after 30 min at room temperature was modified, and ZFocc was exponentially correlated with ZFDNA: log ZFocc

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fered with intrinsic or DNA specific mithramycin fluorescence. On the contrary, increasing SDS concentrations (O. l-2%) decreased intrinsic and completely inhibited DNA specific fluorescence. Also, heparin (0.03-1.2 mg/ml) did not modify mithramycin adsorption by charcoal particles either in the presence or absence of DNA in the assay mixture (data not shown).

20

60

Effect of Heparin on Nuclear DNA Specific Mithramycin Fluorescence

rvctt?a

FIG. 3. Dissociation kinetics of mithramycin-DNA complexes in the presence of dextran-coated charcoal. (A) The values of IFDNA obtained in Fig. 2A are plotted versus the log of IFocc obtained in Fig. 2B for the same concentration of mithramycin. (B) Serial samples of TE-HlOO buffer contained 8 mM Mg9+, 20 &ml of DNA, and either 10 &ml (0) or 22 &ml (0) mithramycin. After completion of mithramycin binding to DNA, the incubation mixtures were mixed with dextran-coated charcoal particles (0.25-0.025%, final concentrations). At the indicated time intervals, duplicate l-ml samples of each series were centrifuged at 3600g for 20 min. The supematant was carefully decanted and left at room temperature for 30 min, then was measured at 540 nm. When appropriate, IF,c background fluorescence was substracted from total fluorescence. For each time point, ZFDNA was then calculated from the relationship described in (A).

= 0.022 IFDNA + 1.11; r = 0.987 (Fig. 3A). When reaction mixtures containing 20 pg of DNA was incubated with either 10 or 22 pg of mithramycin per milliliter, then exposed to charcoal at 0°C for various time lengths, ZFDcc decreased with a firstorder law. ZFDNA was calculated from ZFDcc at each time interval, and the actual dissociation kinetics of mithramycin-DNA complexes were plotted (Fig. 3B), confirming that their initial concentrations were identical at both mithramycin concentrations. In all following experiments 8 mM Mg*+ and IO-pg/ml mithramycin concentrations were used, whereas the duration of the charcoal step was 5 f 0.25 min. Neither EDTA (0- 1.5 mM) nor Tris-HCl (10 mM) nor heparin (O-2 mg/ml) inter-

Purified rooster liver nuclei were resuspended in TE buffer, and chromatin was solubilized by a 10-s sonication. Longer sonication times did not result in increased antibiotic DNA interaction (i.e., specific fluorescence remained unchanged). Then nucleoprotein complexes were dissociated by mixing samples of the sonicated nuclear suspension with equal volumes of TE buffer containing increasing heparin concentrations (0.03-1.2 mg/ml final concentrations). Nuclear DNA specific fluorescence increased (Fig. 4). Maximal fluorescence was about twice that of sonicated nuclei (2.07 ? 0.08,

0.5 tk&n/DNA

rata

FIG. 4. DNA specific fluorescence of heparintreated nuclei. Purified rooster liver nuclei were resuspended in TE buffer. Two samples were diluted with an equal volume of TE buffer and sonicated. They were taken as controls without heparin. The remaining samples were diluted in equal volumes of TE buffer containing increasing concentrations of heparin, such that the range of heparin/DNA ratios (w/w) was O-3. ZFDNA was measured in duplicate in all samples.

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mean + SEM, n = 11) and was reached at heparin to DNA ratios Z= 0.15. Specific fluorescence was proportional to the amount of DNA present in total homogenates and crude or purified nuclei prepared in TE-HlOO buffer (Fig. 5). The lower limit of sensitivity was 0.3 pg DNA/ml.

DNA @g/m’ 1

Comparison with Spectrophotometric Techniques

5

The DNA content of purified rooster liver nuclei and human embryo fibroblast homogenates or crude nuclei was measured by the mithramycin and diphenylamine assays. Samples volumes were lo-80 ~1 for the former and 100-500 ~1 for the latter. DNA concentrations measured by both techniques were linearly correlated (Fig. 6) and the slope of the straight line was 1.07 f 0.07 (mean f SD). In one experiment with purified liver nuclei, DNA phosphorus was measured in addition to the mithramycin

,/,II

20 Sample

40 vdume

60

60

(pII

FIG. 5. Linear correlation of DNA specific fluorescence with sample volume. Human embryo fibroblast homogenate (3 1.3 fig of DNA/ml, 0) or crude nuclear pellet (24.5 pg of DNA/ml, A) and purified rooster liver nuclei (42.1 /.~g of DNA/ml, Cl) were prepared and chromatin was solubilized with TE buffer containing 100 pg of heparin/ml. Aliquots were then removed (lo-80 ~1) and diluted to 950 ~1 with TEHlOO buffer and IFDNA was measured.

15 Mtthranycm

20

25

FIG. 6. Comparison of mithramycin and diphenylamine assays. Human embryo fibroblast homogenates and crude nuclear pellets and purified rooster liver nuclei were prepared in TE-HlOO buffer. DNA was measured by either the mithramycin assay (lo- to 80-~1 aliquots) or the diphenylamine reaction (lOOto 5OQl aliquots) and the results were expressed in micrograms of DNA/milliliter of suspension. Each point represents the mean of nine determinations for the mithramycin assay and 10 determinations for the diphenylamine reaction.

and diphenylamine reactions. The values obtained by the three methods were 137 ? 2, 133 + 3, and 128 t 2 pg (mean f SEM), respectively. On the contrary, when the mithramycin assay was applied to sonicated nuclei, the amount of DNA detected by fluorescence was significantly lower than that obtained by the diphenylamine reaction (Table 1). Removal

I

10

of Unbound

Mithramycin

At high sensitivity setting (meter multiplier = O.Ol), the background fluorescence was approximately equal to the specific fluorescence of 5 pg DNA/ml. Such large background reduced precision for low DNA concentrations. Therefore unbound mithramycin was removed by exposure of the assay mixtures to 0.25% dextran-coated charcoal at 0°C for 5 min. was still linear with increasing DNA ~FDCC concentrations (inset, Fig. 7), and the slope of the straight line was 30% smaller than that obtained in the absence of charcoal treatment. Nevertheless, a good linear correlation was obtained when nuclear DNA was measured by the mithramycin assay

DNA

MEASUREMENT

TABLE COMPARISON

BY MITHRAMYCIN

1

OF THE MITHRAMYCIN

ASSAY,

PERFORMED

ON SONICATED OR HEPARIN-TREATED NUCLEI, WITH THE DIPHENYLAMINE REACTION” DNA

b.cdml)

Mithramycin fluorescence Experiment No. 1 2 3

Sonicated nuclei 54 -+ 5 47 It 3 53 rf: 3

Heparintreated nuclei 140 +- 5 105 + 2 130 2 2

Diphenylamine 127 2 2 103 2 1 127 + 1

a Purified liver nuclei were resuspended in heparinfree TE buffer and were sonicated for 10 s at 0°C. Duplicate lOO-, 200-, 300-, 400-, and XJO-~1 aliquots were removed and DNA was measured using the diphenylamine reaction. IEDNA was measured on triplicate 20-, 40-, and 60-~1 samples in the presence or absence of heparin (100 &ml) in the assay mixture. In each case, DNA content per milliliter of nuclear suspension was calculated from the slope of the linear plots (DNA contents vs sample volume).

whether or not unbound antibiotic was removed (Fig. 7). Removal of unbound mithramycin did not alter the reproducibility of the assay, provided the exposure of the samples to dextran-coated charcoal was timed very carefully. When DNA content per milliliter was calculated for two nuclear suspensions from the values obtained with various sample volumes (20,40, and 60 pl), the standard deviation did not exceed 7% of the mean (129.7 + 9.7 and 168.6 ? 12.8 pg of DNA/ml). The assay was sensitive to as little as 0.25 pg DNA and specific fluorescence was proportional to the amount of calf thymus DNA added up to 30 pg. DISCUSSION

Approximately half the EB binding sites are available in native chromatin as compared to protein-free DNA. Heparin forms stable complexes with basic chromatin

267

FLUORESCENCE

proteins. This provokes the dissociation of DNA-protein complexes, resulting in complete solubilization of chromatin (lo), and renders practically all binding sites in chromatin available for the chromophore (11,12). Similar results were obtained after Pronase stripping or proteinase K digestion of chromatin for EB (13) and DABA (14) fluorescence, respectively. Identical conclusions can be drawn for mithramycin-DNA interaction since fluorescence measurements in sonicated chromatin preparations are about two times lower than those in heparin-solubilized preparations. In the latter case, the mithramycin assay gives results identical to those of the diphenylamine reaction or the measurement of DNA phosphorus. However, it should be mentioned that the original paper by Hill and Whatley (1) demonstrated a good correlation using two cell types (TA 3B, a mouse tumor line, and human embryo lung fibroblasts) between the mithramycin fluorescence assay after

L

I

FIG. 7. The effect

r.

2

4 WDNn”

8

of removal of unbound mithramycin on DNA measurement by the mithramycin assay. Purified rooster liver nuclei were resuspended in TE buffer and treated with heparin (100 &ml, final concentration). Triplicate aliquots (20, 40, and 60 ~1) were removed. ZFDNAand ZFDcc were measured before and after dextran-coated charcoal adsorption of unbound mithramycin, respectively. Inset: Calibration curves. Incubations were performed in TE buffer containing mithramycin (10 Z&ml), Mg2+ (8 mM), and increasing concentrations of calf thymus DNA (O.l8 wdml). 0, IFmA; 0, ZFDcc.

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sonication of the samples and the diphenylamine reaction. It is possible that various cell types behave differently in this respect. As shown here, the addition of heparin appears to overcome these differences. The main advantage of the modified mithramycin assay is increased sensitivity. Furthermore, the assay precision can be improved for low DNA concentrations by dextran-coated charcoal adsorption of unbound mithramycin. ACKNOWLEDGMENTS We thank J. C. Courvalin for stimulating discussions and encouragements, and F. Boussac, J. C. Lambert, and C. Barrier for the preparation of the manuscript.

REFERENCES 1. Hill, B. T., and Whatley, S. (1975) FEBS Lett. 5x&20-23.

2. Burton, K. (1956) Biochem. J. 62,315-323. 3. Le Pecq, J. B., and Paoletti, C. (1966) Anal. Biochem. 17, 100-107. 4. Kissane, J. M., and Robins, E. (1958) J. Biol. Chem. 233, 184-188. 5. Hill, B. T. (1976) Anal. Biochem 70,635-638. 6. Ward, D. C., Reich, E., and Goldberg, I. H. (1%5) Science 149, 1259- 1263. 7. Behr, W., Honikel, K., and Hartmann, G. (1969) Eur. J. Biochem. 9,82-92. 8. Blobel, G., and Potter, V. R. (1%6) Science 154,1664- 1666. 9. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466468. 10. Bomens, M. (1973) Nature (London) 244,28-30. 11. Saiga, H., and Kinoshita, S. (1976) Exp. CelL Res. 102, 143-152. 12. Karsten, U., and Wollenberger, A. (1977) Anal. Biochem. 77,464-470. 13. Karsten, U., and Wollenberger, A. (1972) Anal. Biochem. 46, 135- 147. 14. Barth, C. A., and Willershausen, B. S. (1978) Anal. Biochem. 90, 167-173.