Structural Characterization of Bovine Granulocyte Colony Stimulating Factor: Effect of Temperature and pH

The protein bovine granulocyte colony stimulating factor (bGCSF) was studied in solution as a function of pH (2–7) and temperature (10°–90°C) using fluorescence, circular dichroism, and Fourier transform infrared spectroscopies, as well as differential scanning calorimetry and optical density as a measurement of aggregation. bGCSF possesses significant conformational lability under the solution conditions examined. Under all pH conditions examined, a major conformational change is observed as a function of temperature at 50°–60°C, although the magnitude and precise temperature at which this occurs varies with pH. Three major conformations are adopted with changing pH. One is observed at pH 2 and 3, a second at pH 4, and a third at pH 5–7. At low pH (2–3), bGCSF adopts a molten globule-like conformation at moderate temperatures (25°–45°C), whereas at pH 4 the protein appears to form a non-molten globule extended conformation. The use of this type of study as complementary data for protein phase diagram development as well as the relationship between the conformational lability demonstrated by bGCSF and that observed for recombinant human granulocyte colony stimulating factor and other similar cytokines is discussed. © 2003 Wiley-Liss, Inc. and the American Pharmacists Association.