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 degrees -90 degrees 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 degrees -60 degrees 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 degrees -45 degrees 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.
Kueltzo
Lisa A
LA
Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047, USA.
Middaugh
C Russell
CR
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002626:Chemistry, Pharmaceutical; D002942:Circular Dichroism; D004355:Drug Stability; D005453:Fluorescence; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015394:Molecular Structure; D009994:Osmolar Concentration; D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D019999:Pharmaceutical Solutions; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor