TY - JOUR
T1 - Fundamental analysis of recombinant human epidermal growth factor in solution with biophysical methods
AU - Kim, Nam Ah
AU - Lim, Dae Gon
AU - Lim, Jun Yeul
AU - Kim, Ki Hyun
AU - Jeong, Seong Hoon
N1 - Publisher Copyright:
© 2014 Informa Healthcare USA, Inc.
PY - 2015/2/1
Y1 - 2015/2/1
N2 - Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.
AB - Correlation of thermodynamic and secondary structural stability of proteins at various buffer pHs was investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS) and attenuated total reflection Fourier-transform infrared spectroscopy (ATR FT-IR). Recombinant human epithelial growth factor (rhEGF) was selected as a model protein at various pHs and in different buffers, including phosphate, histidine, citrate, HEPES and Tris. Particle size and zeta potential of rhEGF at each selected pH of buffer were observed by DLS. Four factors were used to characterize the biophysical stability of rhEGF in solution: temperature at maximum heat flux (Tm), intermolecular β-sheet contents, zeta size and zeta potential. It was possible to predict the apparent isoelectric point (pI) of rhEGF as 4.43 by plotting pH against zeta potential. When the pH of the rhEGF solution increased or decreased from pI, the absolute zeta potential increased indicating a reduced possibility of protein aggregation, since Tm increased and β-sheet contents decreased. The contents of induced intermolecular β-sheet in Tris and HEPES buffers were the lowest. Thermodynamic stability of rhEGF markedly increased when pH is higher than 6.2 in histidine buffer where Tm of first transition was all above 70 °C. Moreover, rhEGF in Tris buffer was more thermodynamically stable than in HEPES with higher zeta potential. Tris buffer at pH 7.2 was concluded to be the most favorable.
KW - Biophysical analysis
KW - FT-IR
KW - pH
KW - Recombinant human epidermal growth factor
KW - Thermodynamic stability
UR - http://www.scopus.com/inward/record.url?scp=84921928391&partnerID=8YFLogxK
U2 - 10.3109/03639045.2013.859152
DO - 10.3109/03639045.2013.859152
M3 - Article
C2 - 24502269
AN - SCOPUS:84921928391
SN - 0363-9045
VL - 41
SP - 300
EP - 306
JO - Drug Development and Industrial Pharmacy
JF - Drug Development and Industrial Pharmacy
IS - 2
ER -