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Comparison between the in vivo and in vitro interaction of aminoacridines with nucleic acids and other compounds
COMPARISON
BETWEEN
INTERACTION
THE
V1VO
OF AMINOACRIDINES ACIDS
AND
OTHER
P. P. H. DE BRUYN Department
IN
of Anatomy,
The Received
AND WITH
IN
V1TRO
NUCLEIC
COMPOUNDS’ and
University November
N. H. SMITH
of Chicago,
Chicago,
Ill.,
U.S.A.
18, 1958
PHEVIOI’Swork
[ 1, 2, 31 has shovvn that (1) acridine dyes with amino groups in preferred positions are able to stain the cell nucleus in z~ir~o;(2) this staining is accompanied by spectral changes similar to those observed in the interactions of diaminoacridines with nucleic acids [4]; (3) the interactions of these dyes with nucleic acids, as measured by in uitro spectral changes, are more pronounced than with structurally similar dyes which are not capable of vital staining; (4) this interaction is not a simple acitl-base reaction; and (5) cnzymatic degradation of the nucleic acid diminishes the extent of interaction. When the nucleic acid used in formation of the dye complex is in an cnzymatically degraded form, much of its ability to shift the dye spectrum to longer wavelengths is lost. This behavior suggests that in addition to the primary forces which cause complex formation between dye and nucleic acid, an additional stability is obtained when the nucleic acid is intact. This stability is probably a result of the peculiar spatial orientation of the undegradcd nucleic acid, and it is lost through depolymerization. The purpose of the present study is to extend the observations in vitro to the interaction of aminoacridines with nucleic acids in living cells. In this paper are presented the results of spectrophotometric studies of acriflavine in suspensions of Ehrlich ascites tumor cells. In addition, it was hoped that the relation between staining in viuo and spectral effects could be elucidated by spectrophotometric examination of model system.
MATERIALS Acriflavine, sen’s phosphate
3,6-diamino-lo-methylacridinium buffer, pH 7.3 to give a solution
1 Aided by grants from the American on Growth of the National Research Cancer Fund. Erperimentul
AND
Cell Iiesecrrch
17
Cancer Council
METHODS chloride was dissolved in Siirenwhich was 2.0 i: 1O-4 M with respect
Society upon recommendation and from the Alexander and
of the Committee Margaret Stewart
In vivo and in vitro interaction
of aminoacridines
483
to the dye. This solution was used in the preparation of ascites cell suspensions for optical measurements. A cell suspension was obtained from the abdominal cavities of CF I female mice \vhich had been hosts to the Ehrlich ascites tumor for about four days. At this stage in the tumor’s development, it exists as a homogeneous and slightly bloody fluid of about 1.5 ml in volume. The fluid was repeatedly washed with Siirensen’s phosphate buffer solution and separated by mild centrifugation. After each centrifugation, the blood-free portion of the cell mass was selected for further washing. About five washings rendered the cells almost completely blood-free. These cells were then resuspended in buffer solution. Mixtures of the stock preparations of dye solution, cell suspension and neutral buffer, in known proportions, were prepared. Spectral measurements were made of the cell suspension alone, dye alone and the mixture of dye and cells. In addition, a portion of the dye-cell mixture was centrifuged, and the clear centrifugate was decanted for optical measurement. To estimate the viability of tumor cells at a given step in the procedure, cell counts were made using 0.025 per cent eosin in neutral buffer as the diluent. The criterion for cell death was stainability of the cells by eosin. The preparation of solutions containing a diaminoacridine and various other components was carried out by the usual procedures. Where non-aqueous solvents were employed the dye was used in a neutral, nonionic form. For aqueous solutions the hydrochloride salt was used. A Beckman model DU spectrophotometer was used for measurements of optical density of the preparations. Quartz cells of 1 cm path length were used. For the measurements of solutions of acriflavine at high concentrations, calibrated quartz spacers were employed to shorten the path length. For measuring optical densities of cell suspensions the cover of the cuvette compartment was replaced by a cover equipped with a mechanical mixing device to prevent the settling of cells during the measurements. RESULTS
In Fig. 1, curve A, shows the absorption spectrum of at suspension of Ehrlich ascites cells (9.8 x 106 cells/ml) in a neutral buffered solution containing 3.38 x IO-5 M 3,6-diamino-lo-methylacridinium chloride. Curves B and C are the spectra of the dye and cells separately, in the same concentration as
in curve A. Curve D is the spectrum of the centrifugate of the mixture the spectrum of which is represented by curve A. The dead cell population in the stock cell suspension just prior to use was 15 per cent, based on nuclear stainability by 0.025 per cent eosin. After measurement of their spectra, solution il and C contained 29 and 20 per cent of dead cells, respectively. In Fig. 2 are absorption curves derived from the data shown in Fig. 1. Curve A of Fig. 1 may be considered the sum of the absorption and scatter due to the cell suspension, the absorption by uncomplexed or “free” dye in Exprimentul
Cell
Research
17
484
P. P. H. De Bruyn
and N. H. Smith
solution, and absorption by complexed dye. Curve E of Fig. 2 is curve A minus the sum of curves C and D of Fig. 1. This curve represents the absorption spectrum of only the dye contained within the cells. Curve D of Fig. 1 shows a shift in absorption to longer wavelengths as compared to curve B, the solution of dye alone. This shift is probably caused by complexing of dye with nucleic acid, present in the centrifugate probably
Fig. Fig. 1.-A, cell suspension pension; and D, centrifugate Fig. 2.-E, (A-C-D), complex in cells only.
1.
Fig.
+ 3.38 x 1O-6 A1 acriflavine; of A.
complex
in cells;
F, (D-0.408@,
B, 3.38 x 10-b complex
2.
,II acriflavine;
in centrifugate;
C, cell susG, (E-1.31
F),
as a result of some cell destruction. However, in the region of 400 m,u to 430 m,u, the ratio of absorption of curves B and D is constant and equal to 0.408, indicating that the same substance is absorbing in both solutions. Since curve B is of free acriflavine, curve D is in that region, also of free acriflavine. Curve F of Fig. 2 is an estimate of the complexed dye in the centrifugate, based on the assumption that in curve D, only the free dye and not the long wavelength complex absorbs in the region of 400-430 m,u in the centrifugate. Curve F is obtained by subtracting 0.408 times the optical density of Curve B from Curve D. Comparison of curves E and F yields the fact that the ratio of absorptions is constant above 465 m,u, curve E being 31 per cent higher than curve F in this region. If we assume that the two peaks of curve E are due to the sum of absorptions by a complex found only within the cells and Experimentd
Cell Research
17
In vivo and in vitro interaction
485
of aminoacridines
a complex found both in the cells and in solution, the curve E minus 1.31 times F will represent the cellular complex which has no counterpart in solution. This resulting curve (G in Fig. 2) has an absorption peak at 435 mp. In Fig. 3 are the long wavelength spectra of acriflavine in distilled water over a thousand-fold concentration change. These data demonstrate that Beer’s law deviations, which are usually associated with metachromasia,
4.03.63.2
-
2.8 P0
2.4-
j.
201.6 1.20.80.4-
01 400
, 10
, 20
, 30
, 40
, 50
, 60
I 10
, 80
I 90
0 360
,
,
(
,
,
,
,
80
400
20
40
60
80
500
v
Y Fig. Fig.
3.PAcriflavine.
Fig. 4.-A absorption
and A’, maximum;
Fig.
3.
(1) 5 x 1O-6 M;
(2) 5 x 1O-5 M;
DNA 0.5 per cent, C, A’ minus B.
acriflavine
4.
(3) 5 x 1O-4 -14; and (4) 5 x 1W3 M. 5.06 x 1O-5 ‘Tc B, reflection
of curve
A about
occur as well as long wavelengths shifts. In curve 1 of Fig. 3, it is apparent that there is a hump in the short wavelength side of the peak, although not as pronounced as in the spectra of the complexed dye. That this hump is associated with polymerization of the dye and consequently with metachromasia (accepting the theory generally proposed for metachromasia) is borne out by the shift of the absorption maxima to the region of the hump at higher dye concentration. Another condition under which short wavelength peaks occur under conditions favorable to dye aggregation is illustrated in Fig. 4. This figure shows the spectrum, A and A’, of the DNA-acriflavine complex. The symmetrical curve A-B and curve C represent hypothetical components of curve A-A’. Table I lists the wavelengths of maximum absorption in the visible region Experimental
Cell Research
17
486
P. P. H. De Bruyn
and N. H. Smith
of the spectrum for the diaminoacridines proflavine and acriflavine in a variety of solvents, including solutions of nucleic acid degradation products. This experiment was undertaken in order to determine, if possible, which components of nucleic acid are active in the complexing action which results
Solvent
Solvent Proflavine max (mp)
Water 0.05 % DNA in water pH 7.0 95 % methanol Glacial acetic acid Phenol pH 4.0 Phenol pH 8.0 6.5 M pyridine in water 6.5 ,II pyridine in HCl pH 6.0 Triethylphosphate 0.053 dl triethylphosphate in water Benzaldehyde
Water pH 7.0 and pH 3.2 0.05 % DNA in water pH 7.0 Adenine in H,O pH 3.2, 8.6 g/l Adenine in H,O pH 3.2, 4.3 g/l Adenine in H,O pH 3.2, 2.1 g/l Guanine sulfate in H,O pH 2.0,
444 462 456 454 455 454 457 462 462 444 459
Acriflavine max (w) 452 46X 461 458 456 10 g/l 459
Arriflavine Max w) (cont.) Cytosine in H,O pH 4.5, 10 g/l Cytosine in HZ0 pH 4.5, 5 g/l Thymine in H,O pH 4.5, 7.0 g/l Adenosine in H,O pH 3.2, 9.3 g/l Adenosine in H,O pH 3.2, 4.6 g/l Cytidine in H,O pH 4.5, 36.5 g/l Cytidine in H,O pH 4.5, 18.2 g/l Cytidine in H,O pH 4.5, 9.1 g/l Guanosine in H,O pH 3.2, 5.2 g/l Yeast adenylic acid in H,O, 9.9 g/l Cytidilic acid in H,O pH 4.5, X.6 g/l Sodium guanylate in H,O, 10.7 g/l Pyridine 1.58 ,?I pyridine in H,O fructose 1,6-diphosphate in H,O, 45 g/l Ribose in H,O, 34 g/l Triethyl phosphate
458 454 456 458 455 458 456 455 457 458 454 460 472 459 452 452 470
in in z&o staining. The criterion of complexing was the longwave length shift of the spectra of acriflavine or proflavine in solutions of components similar in structure to component parts of the DNA molecule. As a control group dissimilar compounds were also tested. From Table I, it is evident that there is no correlation between the ability to shift the spectrum to longer wavelengths and the structural similarities between DNA and the compounds tested. DISCUSSION
The spectral shifts which accompany the complex formation between diaminoacridines and nucleic acids in solution are opposite those associated with metachromasia. The metachromatic effect is generally attributed to Experimental
Cell
Research
17
In vivo and in vitro interaction
of aminoacridines
487
polymerization of cationic dye molecules which are bound to an anionic polymer. Such polymerization results in shifts of maximum absorption to shorter wavelengths. These shifts are presumably related to those acccompanying Beer’s law deviations at high dye concentrations. Although acriflavine, a typical diaminoacridine, does not obey Beer’s law, in aqueous solutions containing nucleic acid a shift to longer bvavelengths occurs. There is nevertheless, evidence of metachromasia in the acriflavine-DNA complex. In Fig. 4, the spectrum of the dye-nucleic acid complex, curve A-A’, may be considered the composite of the symmetrical curve A-B and curve C. The position of the peak of curve C coincides with that of acriflavine at high concentrations as is shown in Fig. 3. An analysis of the spectral curves for acriflavine in suspensions of living cells leads to similar results. In Fig. 1, curve A, that of the living cell suspension plus acriflavine, shows a pronounced hump in the region of 440 m,u. \Vhen this spectrum is corrected for the absorption and scatter of the cells alone (curve C) and absorption by dye in solution (curve LJ), the resulting curve (E, Fig. 2) shows a peak at 440P413 111,~as xvell as one at 470 mp. another metachromatic effect is seen in curve G of Fig. 2, lvhich represents the cellular complex which is not found in the free solution. The shift of the absorption peak to 43.5 rnp is a result of the highly aggregated form of the acriflavine, strongly bound within the cell, where the highest concentration of nucleic acids occurs. These data suggest that more than one interaction resulting in spectral shifts occurs. In solutions of nucleic acids a shift to longer wavelengths in the acriflavine spectrum is accompanied by a hump in the absorption curve at a shorter wavelength than the peak for free dye (Fig. 4). In the case of absorption by dye-cell suspensions, due to the nature of the absorbant, this short wavelength hump (G in Fig. 2) can be separated graphically from the rest of the absorption curve (E in Fig. 2). It is apparent that the shift to shorter wavelengths is more pronounced in the intracellular complex, as is evident from a comparison of curves E and G with F in Fig. 2 or with the dye-DNA spectra. If this shift is due to a polymerization of absorbed dye, then the loss of complexing ability with nucleic acids through degradation of the nucleic acid may be explained. In intact nucleic acids, the regularity in structure allo\\-s the formation of long chains of the polymerized dye. If this regularity is disrupted, the weak forces holding the dye molecules together are not sufficient to maintain the polymerized dye structure. It can be shown that the long lvavelength shift in absorption by diamino3” - 593706
Experimental
Cell
Research
17
P. P. H. De Bruyn
and N. H. Smith
acridines is not specific for nucleic acids. In Table I, the wavelength of maximum absorption is given for proflavine and acriflavine in aqueous and nonaqueous solutions containing a variety of acidic, basic, and neutral substances. All of the substances tested, excepting solutions of ribose and fructose 1,6-diphosphate caused a shift in the spectrum of proflavine or acriflavinr to longer wavelengths. It must be noted, however, that except for solutions of nucleic acids, relatively large concentrations of the interacting material are necessary to obtain a spectral shift. It is, of course, possible that the spectral changes to longer wavelengths which occur in the interaction of dyes with small amounts of nucleic acids are basically unrelated to similar spectral effects observed using high concentration of other substances. In staining, there is an aggregation of dye molecules on the nucleic acid molecule. The interaction between dye molecules in the aggregate results in increased absorption at a shorter wavelength than the absorption maximum of free dye. The interaction between the dye and nucleic acid molecules results in increased absorption at longer wavelengths. It is suggested that these two observed spectral effects are secondary to the staining reaction between diaminoacridines and cellular constituents. If the long wavelengths shifts observed in solutions of proflavine or acriflavine with other materials, including nucleic acid degradation products are a direct consequence of the same complex formation which causes similar spectral shifts of acridines in dilute nucleic acid solutions, one is left with the conclusion that the complexing interaction is of a non-specific type. Compounds as widely dissimilar as acetic acid, phenol, pyridine, triethyl phosphate, benzaldehyde and nucleic acid cause similar spectral shifts to longer wavelengths. Also there would be little reason to associate complex formation with a dyenucleic acid phosphate interaction. Apparently purines, pyrimidines, nucleosides and nucleotides are equally effective in causing a change in the absorption spectrum of acriflavine. This shift in spectra to longer wavelengths may then be regarded as a solvent effect which can occur in the absence of complexing or in the case of solutions containing nucleic acids, in addition to complexing.
SUMMARY
The spectrum of acriflavine in living Ehrlich ascites tumor cells has been determined. The changes in spectra which accompany staining are similar to those obtained in acriflavine-nucleic acid solutions. Two characteristics of the interaction of diaminoacridine dyes with nucleic acid are noted. One is Experimental
Cell Research
17
In vivo and in vitro interaction the appearance a metachromatic wavelength peak specific spectral
of aminoacridines
489
of a shorter wavelength peak which may be associated with type of staining interaction. The second is a shift of the long to longer wavelengths. This shift is compared with the nonchanges which accompany changes in the solvent. REFERENCES
1.
DE
BRUYN,
P. P. H.,
FARR,
R.
S., BANKS,
H.
and
MORTHLAKD,
F. W.,
Ezpll.
Cell
Research
4, 174 (1953). 2. DE BRUYN, 3. 4.
MORTHLAND, LOESER, CH.
P. P. H., ROBERTSON, R., C. and FARR, F. W., DE BRUYN, P. P. H. and SMITH, N., Federation Proc. 14, 245 (1955).
R. S., And. Rec. 108, 279 (1950). N. H., Expfl. Cell Research 1, 201(1954).
Experimental
Cell Research
17
BETWEEN
INTERACTION
THE
V1VO
OF AMINOACRIDINES ACIDS
AND
OTHER
P. P. H. DE BRUYN Department
IN
of Anatomy,
The Received
AND WITH
IN
V1TRO
NUCLEIC
COMPOUNDS’ and
University November
N. H. SMITH
of Chicago,
Chicago,
Ill.,
U.S.A.
18, 1958
PHEVIOI’Swork
[ 1, 2, 31 has shovvn that (1) acridine dyes with amino groups in preferred positions are able to stain the cell nucleus in z~ir~o;(2) this staining is accompanied by spectral changes similar to those observed in the interactions of diaminoacridines with nucleic acids [4]; (3) the interactions of these dyes with nucleic acids, as measured by in uitro spectral changes, are more pronounced than with structurally similar dyes which are not capable of vital staining; (4) this interaction is not a simple acitl-base reaction; and (5) cnzymatic degradation of the nucleic acid diminishes the extent of interaction. When the nucleic acid used in formation of the dye complex is in an cnzymatically degraded form, much of its ability to shift the dye spectrum to longer wavelengths is lost. This behavior suggests that in addition to the primary forces which cause complex formation between dye and nucleic acid, an additional stability is obtained when the nucleic acid is intact. This stability is probably a result of the peculiar spatial orientation of the undegradcd nucleic acid, and it is lost through depolymerization. The purpose of the present study is to extend the observations in vitro to the interaction of aminoacridines with nucleic acids in living cells. In this paper are presented the results of spectrophotometric studies of acriflavine in suspensions of Ehrlich ascites tumor cells. In addition, it was hoped that the relation between staining in viuo and spectral effects could be elucidated by spectrophotometric examination of model system.
MATERIALS Acriflavine, sen’s phosphate
3,6-diamino-lo-methylacridinium buffer, pH 7.3 to give a solution
1 Aided by grants from the American on Growth of the National Research Cancer Fund. Erperimentul
AND
Cell Iiesecrrch
17
Cancer Council
METHODS chloride was dissolved in Siirenwhich was 2.0 i: 1O-4 M with respect
Society upon recommendation and from the Alexander and
of the Committee Margaret Stewart
In vivo and in vitro interaction
of aminoacridines
483
to the dye. This solution was used in the preparation of ascites cell suspensions for optical measurements. A cell suspension was obtained from the abdominal cavities of CF I female mice \vhich had been hosts to the Ehrlich ascites tumor for about four days. At this stage in the tumor’s development, it exists as a homogeneous and slightly bloody fluid of about 1.5 ml in volume. The fluid was repeatedly washed with Siirensen’s phosphate buffer solution and separated by mild centrifugation. After each centrifugation, the blood-free portion of the cell mass was selected for further washing. About five washings rendered the cells almost completely blood-free. These cells were then resuspended in buffer solution. Mixtures of the stock preparations of dye solution, cell suspension and neutral buffer, in known proportions, were prepared. Spectral measurements were made of the cell suspension alone, dye alone and the mixture of dye and cells. In addition, a portion of the dye-cell mixture was centrifuged, and the clear centrifugate was decanted for optical measurement. To estimate the viability of tumor cells at a given step in the procedure, cell counts were made using 0.025 per cent eosin in neutral buffer as the diluent. The criterion for cell death was stainability of the cells by eosin. The preparation of solutions containing a diaminoacridine and various other components was carried out by the usual procedures. Where non-aqueous solvents were employed the dye was used in a neutral, nonionic form. For aqueous solutions the hydrochloride salt was used. A Beckman model DU spectrophotometer was used for measurements of optical density of the preparations. Quartz cells of 1 cm path length were used. For the measurements of solutions of acriflavine at high concentrations, calibrated quartz spacers were employed to shorten the path length. For measuring optical densities of cell suspensions the cover of the cuvette compartment was replaced by a cover equipped with a mechanical mixing device to prevent the settling of cells during the measurements. RESULTS
In Fig. 1, curve A, shows the absorption spectrum of at suspension of Ehrlich ascites cells (9.8 x 106 cells/ml) in a neutral buffered solution containing 3.38 x IO-5 M 3,6-diamino-lo-methylacridinium chloride. Curves B and C are the spectra of the dye and cells separately, in the same concentration as
in curve A. Curve D is the spectrum of the centrifugate of the mixture the spectrum of which is represented by curve A. The dead cell population in the stock cell suspension just prior to use was 15 per cent, based on nuclear stainability by 0.025 per cent eosin. After measurement of their spectra, solution il and C contained 29 and 20 per cent of dead cells, respectively. In Fig. 2 are absorption curves derived from the data shown in Fig. 1. Curve A of Fig. 1 may be considered the sum of the absorption and scatter due to the cell suspension, the absorption by uncomplexed or “free” dye in Exprimentul
Cell
Research
17
484
P. P. H. De Bruyn
and N. H. Smith
solution, and absorption by complexed dye. Curve E of Fig. 2 is curve A minus the sum of curves C and D of Fig. 1. This curve represents the absorption spectrum of only the dye contained within the cells. Curve D of Fig. 1 shows a shift in absorption to longer wavelengths as compared to curve B, the solution of dye alone. This shift is probably caused by complexing of dye with nucleic acid, present in the centrifugate probably
Fig. Fig. 1.-A, cell suspension pension; and D, centrifugate Fig. 2.-E, (A-C-D), complex in cells only.
1.
Fig.
+ 3.38 x 1O-6 A1 acriflavine; of A.
complex
in cells;
F, (D-0.408@,
B, 3.38 x 10-b complex
2.
,II acriflavine;
in centrifugate;
C, cell susG, (E-1.31
F),
as a result of some cell destruction. However, in the region of 400 m,u to 430 m,u, the ratio of absorption of curves B and D is constant and equal to 0.408, indicating that the same substance is absorbing in both solutions. Since curve B is of free acriflavine, curve D is in that region, also of free acriflavine. Curve F of Fig. 2 is an estimate of the complexed dye in the centrifugate, based on the assumption that in curve D, only the free dye and not the long wavelength complex absorbs in the region of 400-430 m,u in the centrifugate. Curve F is obtained by subtracting 0.408 times the optical density of Curve B from Curve D. Comparison of curves E and F yields the fact that the ratio of absorptions is constant above 465 m,u, curve E being 31 per cent higher than curve F in this region. If we assume that the two peaks of curve E are due to the sum of absorptions by a complex found only within the cells and Experimentd
Cell Research
17
In vivo and in vitro interaction
485
of aminoacridines
a complex found both in the cells and in solution, the curve E minus 1.31 times F will represent the cellular complex which has no counterpart in solution. This resulting curve (G in Fig. 2) has an absorption peak at 435 mp. In Fig. 3 are the long wavelength spectra of acriflavine in distilled water over a thousand-fold concentration change. These data demonstrate that Beer’s law deviations, which are usually associated with metachromasia,
4.03.63.2
-
2.8 P0
2.4-
j.
201.6 1.20.80.4-
01 400
, 10
, 20
, 30
, 40
, 50
, 60
I 10
, 80
I 90
0 360
,
,
(
,
,
,
,
80
400
20
40
60
80
500
v
Y Fig. Fig.
3.PAcriflavine.
Fig. 4.-A absorption
and A’, maximum;
Fig.
3.
(1) 5 x 1O-6 M;
(2) 5 x 1O-5 M;
DNA 0.5 per cent, C, A’ minus B.
acriflavine
4.
(3) 5 x 1O-4 -14; and (4) 5 x 1W3 M. 5.06 x 1O-5 ‘Tc B, reflection
of curve
A about
occur as well as long wavelengths shifts. In curve 1 of Fig. 3, it is apparent that there is a hump in the short wavelength side of the peak, although not as pronounced as in the spectra of the complexed dye. That this hump is associated with polymerization of the dye and consequently with metachromasia (accepting the theory generally proposed for metachromasia) is borne out by the shift of the absorption maxima to the region of the hump at higher dye concentration. Another condition under which short wavelength peaks occur under conditions favorable to dye aggregation is illustrated in Fig. 4. This figure shows the spectrum, A and A’, of the DNA-acriflavine complex. The symmetrical curve A-B and curve C represent hypothetical components of curve A-A’. Table I lists the wavelengths of maximum absorption in the visible region Experimental
Cell Research
17
486
P. P. H. De Bruyn
and N. H. Smith
of the spectrum for the diaminoacridines proflavine and acriflavine in a variety of solvents, including solutions of nucleic acid degradation products. This experiment was undertaken in order to determine, if possible, which components of nucleic acid are active in the complexing action which results
Solvent
Solvent Proflavine max (mp)
Water 0.05 % DNA in water pH 7.0 95 % methanol Glacial acetic acid Phenol pH 4.0 Phenol pH 8.0 6.5 M pyridine in water 6.5 ,II pyridine in HCl pH 6.0 Triethylphosphate 0.053 dl triethylphosphate in water Benzaldehyde
Water pH 7.0 and pH 3.2 0.05 % DNA in water pH 7.0 Adenine in H,O pH 3.2, 8.6 g/l Adenine in H,O pH 3.2, 4.3 g/l Adenine in H,O pH 3.2, 2.1 g/l Guanine sulfate in H,O pH 2.0,
444 462 456 454 455 454 457 462 462 444 459
Acriflavine max (w) 452 46X 461 458 456 10 g/l 459
Arriflavine Max w) (cont.) Cytosine in H,O pH 4.5, 10 g/l Cytosine in HZ0 pH 4.5, 5 g/l Thymine in H,O pH 4.5, 7.0 g/l Adenosine in H,O pH 3.2, 9.3 g/l Adenosine in H,O pH 3.2, 4.6 g/l Cytidine in H,O pH 4.5, 36.5 g/l Cytidine in H,O pH 4.5, 18.2 g/l Cytidine in H,O pH 4.5, 9.1 g/l Guanosine in H,O pH 3.2, 5.2 g/l Yeast adenylic acid in H,O, 9.9 g/l Cytidilic acid in H,O pH 4.5, X.6 g/l Sodium guanylate in H,O, 10.7 g/l Pyridine 1.58 ,?I pyridine in H,O fructose 1,6-diphosphate in H,O, 45 g/l Ribose in H,O, 34 g/l Triethyl phosphate
458 454 456 458 455 458 456 455 457 458 454 460 472 459 452 452 470
in in z&o staining. The criterion of complexing was the longwave length shift of the spectra of acriflavine or proflavine in solutions of components similar in structure to component parts of the DNA molecule. As a control group dissimilar compounds were also tested. From Table I, it is evident that there is no correlation between the ability to shift the spectrum to longer wavelengths and the structural similarities between DNA and the compounds tested. DISCUSSION
The spectral shifts which accompany the complex formation between diaminoacridines and nucleic acids in solution are opposite those associated with metachromasia. The metachromatic effect is generally attributed to Experimental
Cell
Research
17
In vivo and in vitro interaction
of aminoacridines
487
polymerization of cationic dye molecules which are bound to an anionic polymer. Such polymerization results in shifts of maximum absorption to shorter wavelengths. These shifts are presumably related to those acccompanying Beer’s law deviations at high dye concentrations. Although acriflavine, a typical diaminoacridine, does not obey Beer’s law, in aqueous solutions containing nucleic acid a shift to longer bvavelengths occurs. There is nevertheless, evidence of metachromasia in the acriflavine-DNA complex. In Fig. 4, the spectrum of the dye-nucleic acid complex, curve A-A’, may be considered the composite of the symmetrical curve A-B and curve C. The position of the peak of curve C coincides with that of acriflavine at high concentrations as is shown in Fig. 3. An analysis of the spectral curves for acriflavine in suspensions of living cells leads to similar results. In Fig. 1, curve A, that of the living cell suspension plus acriflavine, shows a pronounced hump in the region of 440 m,u. \Vhen this spectrum is corrected for the absorption and scatter of the cells alone (curve C) and absorption by dye in solution (curve LJ), the resulting curve (E, Fig. 2) shows a peak at 440P413 111,~as xvell as one at 470 mp. another metachromatic effect is seen in curve G of Fig. 2, lvhich represents the cellular complex which is not found in the free solution. The shift of the absorption peak to 43.5 rnp is a result of the highly aggregated form of the acriflavine, strongly bound within the cell, where the highest concentration of nucleic acids occurs. These data suggest that more than one interaction resulting in spectral shifts occurs. In solutions of nucleic acids a shift to longer wavelengths in the acriflavine spectrum is accompanied by a hump in the absorption curve at a shorter wavelength than the peak for free dye (Fig. 4). In the case of absorption by dye-cell suspensions, due to the nature of the absorbant, this short wavelength hump (G in Fig. 2) can be separated graphically from the rest of the absorption curve (E in Fig. 2). It is apparent that the shift to shorter wavelengths is more pronounced in the intracellular complex, as is evident from a comparison of curves E and G with F in Fig. 2 or with the dye-DNA spectra. If this shift is due to a polymerization of absorbed dye, then the loss of complexing ability with nucleic acids through degradation of the nucleic acid may be explained. In intact nucleic acids, the regularity in structure allo\\-s the formation of long chains of the polymerized dye. If this regularity is disrupted, the weak forces holding the dye molecules together are not sufficient to maintain the polymerized dye structure. It can be shown that the long lvavelength shift in absorption by diamino3” - 593706
Experimental
Cell
Research
17
P. P. H. De Bruyn
and N. H. Smith
acridines is not specific for nucleic acids. In Table I, the wavelength of maximum absorption is given for proflavine and acriflavine in aqueous and nonaqueous solutions containing a variety of acidic, basic, and neutral substances. All of the substances tested, excepting solutions of ribose and fructose 1,6-diphosphate caused a shift in the spectrum of proflavine or acriflavinr to longer wavelengths. It must be noted, however, that except for solutions of nucleic acids, relatively large concentrations of the interacting material are necessary to obtain a spectral shift. It is, of course, possible that the spectral changes to longer wavelengths which occur in the interaction of dyes with small amounts of nucleic acids are basically unrelated to similar spectral effects observed using high concentration of other substances. In staining, there is an aggregation of dye molecules on the nucleic acid molecule. The interaction between dye molecules in the aggregate results in increased absorption at a shorter wavelength than the absorption maximum of free dye. The interaction between the dye and nucleic acid molecules results in increased absorption at longer wavelengths. It is suggested that these two observed spectral effects are secondary to the staining reaction between diaminoacridines and cellular constituents. If the long wavelengths shifts observed in solutions of proflavine or acriflavine with other materials, including nucleic acid degradation products are a direct consequence of the same complex formation which causes similar spectral shifts of acridines in dilute nucleic acid solutions, one is left with the conclusion that the complexing interaction is of a non-specific type. Compounds as widely dissimilar as acetic acid, phenol, pyridine, triethyl phosphate, benzaldehyde and nucleic acid cause similar spectral shifts to longer wavelengths. Also there would be little reason to associate complex formation with a dyenucleic acid phosphate interaction. Apparently purines, pyrimidines, nucleosides and nucleotides are equally effective in causing a change in the absorption spectrum of acriflavine. This shift in spectra to longer wavelengths may then be regarded as a solvent effect which can occur in the absence of complexing or in the case of solutions containing nucleic acids, in addition to complexing.
SUMMARY
The spectrum of acriflavine in living Ehrlich ascites tumor cells has been determined. The changes in spectra which accompany staining are similar to those obtained in acriflavine-nucleic acid solutions. Two characteristics of the interaction of diaminoacridine dyes with nucleic acid are noted. One is Experimental
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In vivo and in vitro interaction the appearance a metachromatic wavelength peak specific spectral
of aminoacridines
489
of a shorter wavelength peak which may be associated with type of staining interaction. The second is a shift of the long to longer wavelengths. This shift is compared with the nonchanges which accompany changes in the solvent. REFERENCES
1.
DE
BRUYN,
P. P. H.,
FARR,
R.
S., BANKS,
H.
and
MORTHLAKD,
F. W.,
Ezpll.
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4, 174 (1953). 2. DE BRUYN, 3. 4.
MORTHLAND, LOESER, CH.
P. P. H., ROBERTSON, R., C. and FARR, F. W., DE BRUYN, P. P. H. and SMITH, N., Federation Proc. 14, 245 (1955).
R. S., And. Rec. 108, 279 (1950). N. H., Expfl. Cell Research 1, 201(1954).
Experimental
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