Conformational stability, reversibility and heat-induced aggregation of α-1-acid glycoprotein.

To investigate the relationship between conformational stability, reversibility of denaturation and aggregation of protein, we determined the conformation, melting temperature (Tm), and reversibility of heat-induced denaturation of α-1-acid glycoprotein (AGP) in aqueous solutions at various pH values using circular dichroism (CD) and differential scanning microcalorimetry. To quantitate and characterize heat-induced AGP aggregation under the same pH conditions, solutions of AGP were incubated at 50°C and then analysed by size exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CD and SEC in the presence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid. The conformational stability of AGP was reduced at lower pH, whereas the reversibility of protein denaturation was reduced at higher pH. AGP formed some large non-covalent aggregates during incubation at lower pH, whereas incubation at higher pH tended to cause the formation of dimer species without the formation of large aggregates. These results indicated that lower conformational stability was related to the formation of non-covalent large aggregates, whereas reduced reversibility was related to dimer formation. Thus, evaluating both conformational stability and reversibility is necessary for developing optimal formulations and to predict the kinds of aggregates that will be induced during protein storage.