Urea-Induced Dissociation of Nerve Growth Factor from Venom of Chinese Cobra by SEC

Abstract

The urea-induced dissociation of nerve growth factor from venom of Chinese cobra (cNGF) was studied by intrinsic fluorescence emission spectra, SEC, urea-gradient polyacrylamide gel electrophoresis, assays of biological activity and thermodynamic parameters. The results showed that when urea concentration was lower than or equal to 4.0 mol L−1 or higher than or equal to 8.0 mol L−1, cNGF existed only in native homodimer form or monomer form, respectively; whereas when urea concentration was higher than 4.0 mol L−1 and lower than 8.0 mol L−1, they existed simultaneously in the native homodimer and monomer forms and the former decreased, while the latter increased with the increase in urea concentration. Based on the association–dissociation equilibrium between cNGF and urea molecules, an equation, which includes two characteristic dissociation parameters K and ∆m, was presented to describe the urea-induced dissociation process of cNGF. As the reaction temperature increased from 15 to 35 °C, positive enthalpy and entropy changes were observed, and the parameter K increased from 2.72 × 10−13 to 5.18 × 10−12 (L mol−1), while the parameters ∆m and ∆G, respectively, decreased from 10.18 to 8.42 and from −10.27 to −18.67 (kJ mol−1), which means that the urea-induced dissociation of cNGF was spontaneous and entropy-driven and the higher temperature was favorable for the dissociation process. Using the procedures and equations mentioned in the paper, the urea-induced dissociation of cNGF is first comprehensively described. Furthermore, this work presents a useful method for people to study the dissociation of dimer or multimer proteins induced by denaturants, inducers, pH, etc.