Abstract
The unfolding process of human serum albumin (HSA) was studied by thermal effect on the native fluorescence of the protein, thermal inactivation of the hydrolase activity of albumin and differential scanning calorimetry using the high sensitive calorimeter developed by Privalov. The denaturation process can be described by an approximation of the model of Eyring and Lumry: native ⇌ unfolded reversible ⇀ unfolded irreversible. It was found that the rate of irreversible step was very slow (at temperatures below 74°C), allowing the resolution of the denaturation process as a reversible one on the basis of two states approximation. However, the presence of intramolecular cooperation in the thermal denaturation process at temperatures above 74°C cannot be discarded, which might be favoring the aggregation of albumin molecules. The midpoint temperature of unfolding obtained by differential scanning calorimetry was of 63.1°C ± 0.4 at pH 7.4. This value was independent of the rate of scanning and it is in agreement with those obtained by techniques such as thermal effect on the protein fluorescence and on the hydrolase activity of albumin. The enthalpy of unfolding at pH 7.4 was 88.9 ± 4 Kcal/mol. This value was low compared with those obtained for other proteins, suggesting the presence of a molten globule in the unfolding pathway of albumin. The neutral-basic conformational change (pH 7.4) of albumin did not modify the thermal stability and the enthalpy of denaturation of the protein. A pH below 4.3 (transition acid-neutral) the presence of a second peak in the thermogram of albumin with a T M of 46.2°C ± 0.9 would be suggesting a lost of cooperativity between the various domains of albumin in the unfolding.
Published Version
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