J. Phoiochem. Phofobioi. A: Chem, 78 (1994) 31-37 31 Energy transfer and formation of exciplex between thiacyanine and acridine orange facilitated by anionic biopolymers and synthetic polymers M.K. Pal+ and J.K. Ghosh Deparimen~ zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Biochemistry and Biophysia, Kalyani University,Kalyani 741 23.5 (hzdia) (Received May 19, 1993; accepted September 9, 1993) Ab&aet The fluorescence of thiacyanine (TWA) dye is greatly enhanced on binding to polyanions due to the reduction in internal conversion of the excitation energy of the dye. Glycosaminoglycans (chondroitin sulphates ((35) A, B and C; heparin) and the synthetic polyanion poly(vinylsulphate) (PVS), . m addition to enhancing the fluorescence’ of THIA, facilitate the formation .of an exciplex between excited THIA and cool acridine orange (AO), both bound to the potymers. Of the three chondroitin sulphates, chondroitin sulphate C (CSC) is the most efficient in enhancing the fluorescence of THIA and facilitating the formation of an exciplex. The efficiency of hepar& in this respect is comparable with that of CSC, and poly(vinyl sulphate) is even more efficient. In 20% (v/v) ethanol the association constant of THIA-CSC is reduced to one-tenth of its value in water. Aqueous ethanul also partially inhibits exciplex formation between the two dyes bound to chondroitin sulphates, although, the effect is less prominent in the case of CSC. 1. Introduction 2. Experimental details During the preparation of a fluorometric probe for the competitive binding of the cationic dyes thiacyanine (THIA) and acridine orange (AO) to polyanions, we observed that the fluorescence of THIA was greatly enhanced when bound to gly- cosaminoglycans, DNA etc. This fluorescence en- hancement is due to the reduction in internal conversion of THIA on binding to the polymer matrix, since it has also been observed in aqueous solution on increasing the viscosity of the solvent by adding sucrose or glycerol and on freezing a methanol solution of the dye [l]. The enhanced fluorescence of THIA caused by polyanions is reduced on addition of A0 to the system, with a congruent enhancement of A0 fluorescence, indicating a transfer of energy from THIA to A0 both bound to polyanions. It has also been observed that the fluorescence peak of A0 in the chondroitin sulphate-THIA-A0 system, excited at the A,, value of THIA, is red shifted from 522 to 560 nm, indicating the formation of an exciplex between the two dyes, both bound to polyanions. Heparin (Hep) and chondroitin sulphates of types A, B and C (CSA, CSB, CSC), purchased from Sigma Chemical Company, and poly(vinylsulphate) (K salt) (PVS), purchased from Serva Feinbiochemica, Germany, were used as received. 3,3’-Diethylthiacyanine iodide (THIA) (Koch-Light, UK) was used without further puri- fication. Acridine orange (AO, as Z&l, double salt, E. Merck, Germany) was made zinc free and purified by the method of Armstrong et al. [2]. Stock solutions of dyes and polymers were prepared in double distilled water; the dye solutions were stored in the dark and polymer solutions in a freezer. All the experimental solutions were buff- ered at pH 7.4 using 2.0 mM phosphate buffer. The molarities of the polymers are expressed in terms of the respective equivalent weights, defined as the respective average weights containing one anionic site (CSA, CSB, CSC: 252; Hep: 242; PVS (K salt: 162). ‘Author to whom correspondence should be addressed. The absorption and fluorescence spectra were recorded with a Shimadzu spectraphotometer (model UV-150-02) and a Shimadzu spectrofluo- rometer (model RF-540) respectively using quartz cuvettes (path length, 1.0 cm). lOlO-6030/94/$07.00 @ 1994 Elsevier Sequoia. All rights reserved SSDI 1010-6030(93)03705-L ,,