Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin

Bovine serum albumin (BSA) is a soft globular protein that undergoes conformational changes through several identified transition steps in the pH range 2–13.5. The ability to change conformation makes BSA capable of complexing different ligands from fatty acids to cations or drugs and carries them in the bloodstream. Microcalorimetric titration of BSA with NaOH solution was performed to measure the enthalpy of conformational changes. Two exothermic enthalpy changes were found in the course of the titration between pH 3 and 9.5, which can be identified with the E–F, and the F–N transitions. The enthalpy change at pH 3.5 (transition from the E to the F form of BSA, folding of intra-domain helices in domain I) is independent of the protein concentration. The second transition (F–N, folding of domain III) was observed at pH 4.8 for the 0.1% BSA solution, but it shifted to higher pH values as the protein concentration increased to 0.2% and 0.3%. The tightening of the protein structure with increasing pH was verified measuring intrinsic fluorescence of tryptophan residues. At even higher pH value, pH 10.5, fluorescence measurements revealed protein expansion. The BSA conformational changes were also measured by dynamic light scattering. The hydrodynamic diameter was smaller at the i.e.p. of BSA (5–7 nm at pH ~5) and larger at the two ends of the pH range (17.5 nm at pH 2 and 8.3 nm at pH 10).