INTERACTION OF METHYLENE-BLUE WITH TRANSFER-RNA - A SPECTROSCOPIC STUDY

The binding of methylene blue (MB) with tRNA was studied using absorpt ion, fluorescence and circular dichroic spectroscopy. In the spectral titration of MB with tRNA, hypochromism was observed in the absorption maximum of the dye in the visible region till P/D = 4 and thereafter the intensity increased with a red shift at P/D > 9, indicating electr ostatic and intercalative binding at low and high P/D ratios, respecti vely. Analysis of absorption data, following Schwarz's procedure, show ed that the electrostatic binding is cooperative in nature (cooperativ ely parameter q = 50) with a binding constant K = 7.77 x 10(3) M(-1). A non-linear Scatchard plot was observed for the intercalative binding (at P/D > 4), probably due to a difference in the spectral characteri stics of the dye intercalated between the base pairs and that between the bases in the single stranded domains. Quenching of fluorescence wa s observed for both the binding processes. In the circular dichroism s pectra of tRNA-MB complexes at high P/D (similar to 30), nonconservati ve positive ICD bands were seen at 620 and 680 nm while at low P/D (si milar to 2), two conservative negative CD bands at 300 and 660 nm and two bisignate bands with cross overs at 565 and 605 nm were observed. The short wavelength component of the bisignate band at 565 nm is nega tive while that of the 605-nm band is positive, indicating that the fo rmer arises from left handed and the latter from the right handed heli cal disposition of dye molecules along the tRNA backbone. The changes in the CD spectrum of tRNA on dye binding could be due to a conformati onal change of the nucleic acid or a negative CD being induced at that region.