Applications of fluorescence correlation spectroscopy: measurement of size-mass relationship of native and denatured schizophyllan

Abstract

Diffusion dynamics of a polysaccharide, schizophyllan has been studied by fluorescence correlation spectroscopy (FCS). Several different sizes of nondenatured and denatured schizophyllan have been labeled with rhodamine 6G in borate buffer. The length of the nondenatured schizophyllan was calculated from FCS data by using the Broersma's relationship for rod-like macromolecules. The obtained length was close to that obtained by atomic force microscopy (AFM) measurements. Denatured schizophyllan possesses a random coil conformation. Its hydrodynamic radius R(h) was measured by FCS. The relationship between R(h) and the molecular mass M has been studied and the scaling relationship R(h)--M(0.59) has been obtained, which is in agreement with the random coil model with excluded volume effect. The persistence length q(denat) of the denatured schizophyllan was determined by Hearst's relationship, to be equal to 5.16 +/- 0.75 (nm). The work demonstrates the utility of FCS method for dynamics investigations of biopolymers especially in diluted regime (concentration lower than 10(-8)M could be measured) where other techniques could not be used.