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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 Unauthenticated Download Date | 6/15/17 6:57 AM 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. Unauthenticated Download Date | 6/15/17 6:57 AM 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 ]. Unauthenticated Download Date | 6/15/17 6:57 AM 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. Unauthenticated Download Date | 6/15/17 6:57 AM