Melting Temperature (Tm)
Circular Dichroism (CD)
10 mM sodium phosphate, 10 mM NaCl, 25 mM (NH4)2SO4
Thermal and chemical unfolding of hFGF1 mutants were performed on a Jasco-1500 spectrophotometer through which intrinsic fluorescence and far-UV CD measurements can be made simultaneously. Equilibrium unfolding experiments were performed using a protein concentration of 0.5 mg/mL in 10 mM sodium phosphate buffer containing 10 mM NaCl and 25 mM (NH4)2SO4 at pH 7.2.
Experiments including heparin were performed at a heparin to protein ratio of 10:1. Far UV CD data was acquired using a 1 mm quartz cuvette. Spectra were collected every 5 degrees from 20 to 80 °C. Molar ellipticity values were recorded at 228 nm and the fraction unfolded was plotted as a function of temperature. The denaturation temperature (Tm) was determined as the temperature at which 50% of the protein population was denatured.