Pluronic F68 enhanced the conformational stability of salmon calcitonin in both aqueous solution and lyophilized solid form

Abstract

The effects of different surfactants on the conformational stability and structural similarity of salmon calcitonin (sCT) in aqueous solution and lyophilized forms were investigated by using microscopic Fourier transform infrared (FTIR) spectroscopy with second-derivative spectral analysis. Six surfactants, HCO-60, sodium dodecyl sulfate (SDS), Tween 80, PEG 400, Pluronic 68, and F127 were selected. The sCT aqueous solution with or without different surfactants was, respectively, incubated at 40°C for up to 35 h. sCT films were casted on the CaF(2) plates and IR spectra were collected as a function of incubation time. Second derivative analysis showed that the native sCT having a major α-helical structure was gradually changed to the combination of α-helix, random coil, and β-sheet conformations in aqueous solution at 40°C. Similar conformational changes with delayed β-sheet formation were obtained for sCT after co-incubation with all the surfactants except Pluronic F68. When the native sCT was freeze-dried alone, a marked conformational alteration was found as illustrated by a poor spectral correlation coefficient (r) value of 0.823 as compared to that of the unlyophilized native sCT. This r value was significantly deviated from 1, strongly indicating the influence of lyophilization stress on the surfactant-free sCT. The r value for sCT after lyophilizing with HCO-60, Pluronic F127, PEG 400, or Pluronic F68 was >0.9, suggesting the possible stabilization of these surfactants in the lyophilization process. The sCT sample after lyophilizing with Pluronic F68 showed a highest r value (>0.968), indicating the most optimal stabilization effect of Pluronic F68 for sCT sample via lyophilization. Pluronic F68 was found to be the preferential surfactant for preventing the secondary structure changes in aqueous solution at 40°C as well as in lyophilized powder.