Based on Fourier transform infrared (FTIR) microspectroscopy, the conformation of rhEGF under the influence of pH, heat treatment, chaotropic salts, concentration of salt and protein structure perturbants was studied. The FTIR spectrum of rhEGF showed that major secondary structures from amide I bands composed of 40.6% β-sheets, 25.0% reverse turns, 16.5% random coils, 13.0% loops and 4.9% side-chain structures. At extreme pH conditions (pH < 4 and pH > 8), there were changes in intensity of the bands attributed to loop (1658 cm^-1) and random coil structures, and these bands shifted to lower wavenumbers, indicating changes in protein conformation. Thermal denaturation of rhEGF occurred at 40-76 °C and the formation of intermolecular β-aggregates was revealed by the FTIR spectra. Thermal-irreversible property of rhEGF after second-heating treatment suggested that rhEGF has a poor thermal stability. While investigating the stability of rhEGF in the presence of chaotropic salts, anions induced protein unfolding of rhEGF more significantly than cations. The optimal stabilizing effect was found at the 2 M NaCl added to rhEGF, and expressed the structure of rhEGF more stable on the many components. The bands of loop structure (1654 cm^-1), β-sheet (1638 cm^-1) and intermolecular antiparallel β-aggregation formation (1694, 1619 and 1612 cm^-1) seem to be ?marked? to be more sensitive in determining environmental changes of rhEGF for FTIR microspectroscopy.

Key words: Recombinant human epidermal growth factor; FTIR microspectroscopy; Protein aggregation; Thermodynamics; Preformulation.