Download Absorption and Fluorescence Properties of Some Basic

Absorption and Fluorescence Properties of Some Basic Dyes Complexing
with Nucleosides or Nucleic Acids
G
iit i
T o m ita
Institute of Biophysics, Faculty of Agriculture, K yushu University, Fukuoka (Japan)
(Z . Naturforschg. 23 b, 922— 925 [1968] ; eingegangen am 21. August 1967)
The absorption and fluorescence spectra of some basic dyes were m easured in the pre­
sence of large excess of adenosine or nucleic acids. The visible absorption band of dye shifts to
longer wavelengths than that in free dye solution. T h e fluorescence spectral shape and peak posi­
tion remain unchanged on the addition of nucleoside or nucleic acid, while the fluorescence inten­
sity is enhanced or quenched to some extent depending on the nature of dyes. These results give
us some information about the binding nature of basic dyes to nucleosides or nucleic acids.
Basic
dyes
exhibit
characteristic
bindings
Materials and Experimental
to
various polynucleotides or nucleic a c id s 1_4. T w o
types o f bindings are known, depending on the con­
centration ratio of dye to nucleotide. A t low ratio
o f nucleotide to dye, the visible absorption peak of
dye
appears
at
much
shorter
wavelength
than
that o f free dye, and this type of complex is called
Com plex I. A t high ratio of nucleotide to dye, on
the other hand, the visible absorption band shifts
to somewhat longer wavelengths than that o f free
dye. This type o f complex is named Com plex II.
Com plex I is non-fluorescent and dye molecules are
thought to be
metachromatically bound to phos­
phates in polynucleotide. Com plex II is fluorescent
and is thought to be produced by the intercalation
o f dye molecule between base pairs 5 or bases 6.
W e found that the fluorescence emission of C om ­
plex I I is enhanced or quenched to some extent, de­
pending
on the nature
of basic
dyes,
while
the
fluorescence shape and peak position are unchanged
by the complex formation. V arious nucleosides can
also form m olecular complexes with basic dyes 7’ 8.
These
complexes
exhibit
the
characteristic
b e­
haviours of absorption and fluorescence sim ilar to
Acridine orange, proflavine, thionine and methylene
blue purchased from Merck were used for the absorp­
tion and fluorescence measurements without further
purification. Adenosine was obtained from Sigma Che­
mical Co. The herring sperm DNA and baker’s yeast
RN A were also obtained from the same Co.
The visible absorption spectrum was determined
with a Beckman model spectrophotometer. The fluores­
cence spectrum was determined with a grating fluores­
cence spectrophotometer. The exciting monochromatic
light absorbed by dye was isolated from a mercury
lamp with a grating monochromator and the fluores­
cence emitted at the rectangular direction to the ex­
citing light was analysed with a grating monochroma­
tor. The emission spectrum was normalized for the
equal absorbed light quanta and corrected for the re­
absorption by the dye itself. The fluorescence intensity
was determined by the height of the fluorescence peak,
since the fluorescence spectral shape and the wave­
length at the maximum emission did not change over
the experimental error by the state of dye, free or
bound. The dimension of the fused silica cell con­
taining the solution was 1 x 1 x 5 cm3. A ll the measure­
ments were carried out in the phosphate buffered solu­
tions (pH 7.0) at 25 °C. Sodium chloride was added,
for the solution containing nucleic acid, to obtain the
final ionic strength of 0.5.
those o f Com plex II.
T he m ain purpose of this article is to find the
Results and Discussion
characteristic properties of absorption and fluores­
cence in m olecular complexes of some basic dyes
and nucleosides or nucleic acids, and to obtain some
inform ation about the binding nature in these com­
plexes.
1 D. B r a d l e y and M . W o l f , Proc. nat. A cad. Sei. U S A 45,
944 [1959],
2
R. S t e i n e r and R. B e e r s , Science [W ashington ] 124, 355
[1958].
3 R. S t e i n e r and R. B e e r s , Arch. Biochem. Biophysics 81, 75
[1959].
Fig. 1 shows the visible absorption and fluores­
cence spectra of proflavine in the absence and pre­
sence of adenosine or D N A or RN A. The addition
of large excess of adenosine gives rise to the red
4 R. S t e i n e r and R. B e e r s , Polynucleotides, Elsevier, N ew
Y ork 1961, p. 301.
L. S. L e r m a n , J. m olecular B iol. 3 ,1 8 [1 9 6 1 ] .
6 N . J. P r i t c h a r d , A . B l a k e , and A . R. P e a c o c k e , Nature
[L o n d o n ] 212, 1360 [1 9 6 6 ].
7 G. T o m i t a , Experientia [B a s e l] 23, 614 [196 7].
8 G. T o m i t a , Biophysik [H e id e lb e rg ] 4, 23, 118 [196 7].
5
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shift of absorption band by about 4 nm and slight
hypochromicity. With increasing the temperature of
solution, the absorption band undergoes the blue
shift and finally coincides with that of free pro­
flavine at that temperature. This fact suggests that
proflavine can form a sort of molecular complex
brings about the red shift (about 16 nm) of absorp­
tion band and a certain degree of hypochromicity.
These phenomena are derived from the formation of
Complex II in which proflavine molecule is inter­
calated between base pairs in D N A or bases in
RNA.
The spectral shape and peak position of pro­
flavine fluorescence do not depend on whether dye
is free or bound to various complexing partners.
The fluorescence emission, however, is quenched
to some extent by the complex formation of dye
molecule with D N A or R N A. The very slight
enhancement of fluorescence emission occurs in the
case of proflavine-adenosine system.
Similar behaviours of absorption and fluores­
cence can be seen for the other dyes; acridine
orange (Fig. 2 ), thionine (Fig. 3) and methylene
nm — ►
Fig. 1. Absorption and fluorescence spectra of free and bound
proflavine. Curves 1, 2, 3 and 4, absorption spectra of p ro­
flavine, free and bound to adenosine, D N A and R N A , respec­
tively; curves 1', 2', 3' and 4', fluorescence spectra of pro­
flavine, free and bound to adenosine, D N A and R N A , respec­
tively; concentration of proflavine, 1 .6 2 -1 0 _ 5 M ; concentra­
tion of adenosine, 1 .5 -1 0 - 2 M ; concentration of D N A ,
2• 1 0 ~ 3 M in nucleotide; concentration of R N A , 2 • 10“ 3 M in
nucleotide; light for fluorescence excitation, 436 nm H g line.
with adenosine. The other nucleosides also have the
similar effect to adenosine on the absorption spec­
trum of dye. The presence o f high concentration of
D N A or R N A (nucleotide-to-dye ratio ^ 100)
nm
— ►
Fig. 2. Absorption and fluorescence spectra of free and bound
acridine orange. Curves 1, 2, 3 and 4, absorption spectra of
acridine orange, free and bound to adenosine, D N A and R N A ,
respectively; curves 1', 2', 3 ' and 4', fluorescence spectra of
acridine orange, free and bound to adenosine, D N A and R N A ,
respectively; concentration of acridine orange, 8 - 1 0 ~ 6 M .
Conditions are the same as for F ig. 1 unless otherwise stated.
blue, except for the strong fluorescence-enhancemend in acridine orange-adenosine and -nucleic acid
complexes. The absorption and fluorescence maxima
and the relative fluorescence intensity are given in
Table 1. W e can summarize the optical behaviours
observed as follows: (1 ) the absorption spectrum
shifts to longer wavelengths by complexing of basic
dyes with adenosine or nucleic acids, (2 ) the
fluorescence peak appears at the same wavelength
for the definite dye, independent of whether dye is
free or bound,
(3 )
the fluorescence intensity is
enhanced or quenched, depending on the nature of
dye.
F ig. 3. Absorption and fluorescence spectra of free and bound
thionine. Curves 1, 2 and 3, absorption spectra of thionine,
free and bound to adenosine and D N A , respectively; curves 1',
2' and 3', fluorescence spectra of thionine, free and bound to
adenosine and D N A , respectively; concentration of thionine,
1 .5 -1 0 - 5 M ; light for fluorescence excitation, 546 nm H g
line. Conditions are the same as for Fig. 1 unless otherwise
stated.
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D ye
C o m p lex - A b s o rp tio n Fluorescence
R e la tiv e
in g
m a x im u m
m a x im u m
fluorescence
p a rtn e r
[n m ]
[n m ]
intensity
A c rid in e
o ran ge
None
A d en o sin e
DNA
RNA
492
496
504
506
530
530
530
530
1.0
2.7
2.2
3.7
P ro fla v in e
None
Ad enosin e
DNA
RNA
444
448
460
461
506
506
506
506
1.0
1.02
0.45
0.2 i
Th ion in e
None
A d en o sin e
DNA
598
605
613
615
615
615
l.ls
0.73
M eth y le n e N o n e
blu e
Ad enosin e
DNA
665
670
672
675
675
675
1.1»
0.34
1.0
1.0
T a b le 1. Absorption and fluorescence maxima, and relative
fluorescence intensities of some basic dyes in free and bound
states. L ight for fluorescence excitation, 436 nm H g line for
acridine orange and proflavine and 546 nm H g line for thionine and methylene blue. Concentrations of dyes and complexing partners are given in Fig. 1 — 3, except for methylene
blue (1.5 ‘ 10- 5 M ) .
Generally, the basic dye in aqueous solution has
a tendency to form dimer through V a n d e r
Waals-London
interactions. The transition
moments of monomers in dimer are parallel to each
other and perpendicular to the direction o f a line
connecting their centers of gravity. In this type of
dimer, the upper molecular exciton level is opti­
cally allowed, but the lower forbidden9-11. For this
reason, dimer is non-fluorescent and its absorption
band appears at the shorter wavelength than
the monomer absorption band. In the basic dye
we are dealing with, the dimer band overlaps
upon the monomer 0 — 1 band. The addition of
large excess of adenosine or nucleic acids gives rise
to the dissociation of dimer into monomers. Mono­
mer is bound to adenosine or nucleic acid. This is
confirmed by the coincidence of the fluorescenceexcitation spectrum with the absorption spectrum.
The visible absorption band o f bound dye is com­
posed of the 0 — 0 and 0 —> 1 absorption bands as
seen in the Figures. (The dimer and 0 — 1 absorp­
tion bands are not clear for proflavine at the con­
centration used in Fig. 1.) Such bound dyes are
fluorescent, and the fluorescence emission is en9 M . K a s h a , Discuss. F araday Soc. 9 ,1 4 [1 9 5 0 ].
10 S . E. S h e p p a rd and A . L. G e d d e s, J. Amer. diem. Soc. 66,
1995 [1 9 4 4 ].
hanced for acridine orange by the complex forma­
tion, while considerably quenched for the other
dyes. The equilibrium constant for monomer-dimer
in aqueous acridine orange solution was calculated
to be about 2*104Z/mol at 25 °C from the concen­
tration change of extinction coefficient8. Therefore,
at the concentration (8 10~6M ) of acridine orange
employed here, the concentrations of monomer and
dimer are estimated to be 6.4 -10-6 and 8 10~7M,
respectively. For this reason, the enhancement of
fluorescence observed can not be attributable to
merely the dissociation o f dimer to monomers. The
strong binding interaction between acridine orange
and adenosine or nucleic acid may result in the en­
hancement of fluorenscence emission and the red
shift of the monomer absorption band.
There are two possible arrangements for dye and
adenosine to form molecular complex: the side-by-side
arrangement in a plane and face-to-face arrangement.
In the former type of complex, the direct binding
through hydrogen bridge between dye and nucleo­
side may be essential. This complex seems to be
stabilized by the V a n d e r W a a l s - L o n d o n in­
plane interactions between hydrogen bonded pair.
In the complex with the face-to-face arrangement of
complexing pair, the V a n d e r W a a l s - L o n d o n
interactions are considered as being the essential
factor for stabilization of the two interacting mole­
cules which lie above one another in parallel plane.
However, even in the latter type of complex, if the
purine and ribose rings are mutually perpendicular
as found by A l v e r and F u r b e r g 12 in the case of
cytidylic acid b, basic dye may have the possibility
of linking with ribose, perhaps by the hydrogen
bond. A t present, however, it seems to be too early
to decide definitely which is the true arrangement.
As for the binding interaction between dye and
nucleic acid at high nucleotide-to-dye ratio, L e r m a n 5
postulated that dye molecule is intercalated between
the ordered array of base pairs in D N A and located
over acrossing a hydrogen bonded base pair. On the
basis of this intercalation model, the binding inter­
action may be expected to be reduced by the denatura­
tion o f D N A . However, many observations do not
support this expectation. For this reason, F r i t c h a r d
and others6 proposed a modified intercalation
11 W . T. Simpson and D. L. P e t e rs o n , J. chem. Physics 25, 588
[1 9 5 7 ].
12 E. A l v e r
and
S. F u r b e r g ,
Acta chem. scand. 13, 910
[1 9 5 9 ].
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model. According to them, the dye molecule does
not interact with a hydrogen bonded pair, but the
interaction was assumed to occur between dye and
two adjacent bases on the same polynucleotide chain
and a negatively charged oxygen atom on the phos­
phate group between the two bases interacts directly
with the positively charged ring nitrogen of the
amino acridine. The interaction between the oxygen
and nitrogen may occur through the hydrogen
bridge. The fact that R N A and various polynucleo­
tides 3,4 can also exhibit strong binding interactions
with basic dyes seems to support the latter modified
intercalation model.
It is known that when the fluorescent dye is
bound to the n electron system through hydrogen
bond, the fluorescence emission is quenched (dis­
sipation of excitation energy through hydrogen
bridge) or enhanced, depending on whether the
hydrogen bond can conjugate directly with the zi
electron system or n o t13. The partial fixation o f the
non-bonding electron by the intermolecular hydro­
gen bonding gives rise to the blue shift o f n — n *
absorption band and the red shift of j i — j i * absorp­
tion band 9. This effect leads to the enhancement of
fluorescence emission, when n — n * state appears at
higher energy than n — vi* state. However, when the
shift of n — n * state is not large enough and n — n *
state still lies under n — zi* state, the fluorescence
quenching may be expected owing to the smaller
energy separation between j i — j i * and n — n * states
than that before the formation of hydrogen bonding.
The above considerations seem to be helpful for the
understanding of the fluorescence enhancement and
quenching observed, depending on the kind of dyes.
The more definite explanation in each case remains
in the future investigation.
13 N . M a t a g a and S. T s u n o , B u ll. d i e m . Soc. Japan 3 0 , 7 1 1
[1 9 5 7 ].
Next as mentioned above, although the absorption
band shifts to longer wavelengths upon complex
formation, the wavelength of the maximum emission
remains constant within the experimental error.
Such a behaviour can be explained by the assump­
tion that, while the potential slope of the excited
state is appreciably changed by the complex forma­
tion, the ground state and the equilibrium position
of the excited state are less influenced. Namely, the
F r a n c k - C o n d o n state for the absorption is the
different position on the different potential curve o f
the excited state, depending on the complexing part­
ner, but the emission transition occurs from nearly
the same equilibrium position as in the case of free
dye. These circumstances are simply shown in
Fig. 4.
F ig. 4. Sim plified potential diagram for absorption and emis­
sion in free and bound dyes. W g , ground state for free or
bound d ye; W t*, excited state for free dye; W\)*, excited
state for dye bound to nucleoside or nucleic acid.
The results obtained in the present investigation
seem to be o f interest as offering some useful infor­
mation about the binding properties of basic dyes
with nucleosides or nucleic acids.
The author is grateful to the Ministry of Education
for a grant in aid for special project research on bio­
physics covering part of the expenses.
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