Ferguson plot analysis of multiple intermediate species of thermally unfolded bovine serum albumin

Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) upon thermal unfolding. Differential scanning calorimetry showed an irreversible melting of BSA in Tris-HCl and phosphate buffers with a mid-transition temperature, Tm, of similar to 68 degrees C. Thermally unfolded BSA was analyzed by agarose native gel electrophoresis stained by Coomassie blue and SYPRO Orange staining as a function of pH or protein concentration. SYPRO Orange was used to stain unfolded proteins. BSA heated at 70 and 80 degrees C, i.e., above the Tm, formed multiple intermediate species, which depended on the pH between 7.0 and 8.0, protein concentration and which buffer was used. These intermediate species were analyzed by Ferguson plot, which showed that BSA heated at 60 degrees C had a similar size to the native BSA, indicating that they are either native or native-like state consistent with no SYPRO Orange staining. The intermediate species observed at higher temperatures with the mobility less than that of the native BSA showed a steeper Ferguson plot and were stained by SYPRO Orange, indicating that these species had a larger hydrodynamic size than the native BSA and were unfolded.

Automated summary

The Ferguson plot was used to characterize the multiple intermediate species of bovine serum albumin (BSA) during thermal unfolding. Different factors such as pH, protein concentration, and buffer type influenced the formation of these intermediate species. By analyzing the size and staining properties of the intermediate species, it was determined that some species were native or native-like in structure, while others had unfolded structures and larger hydrodynamic size compared to native BSA.