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.