Size-exclusion chromatography combined with solution X-ray scattering measurement of the heat-induced aggregates of water-soluble proteins at low ionic strength in a neutral solution.

The heat-induced (80 ± 1 ℃, 1 h) aggregates of bovine serum albumin and ovalbumin at neutral pH and low ionic strength (10 mM sodium phosphate buffer, pH 6.9) were characterized using size-exclusion chromatography combined with small-angle X-ray scattering measurement. The values calculated for the radius of gyration and molecular weight of the eluted aggregates of the bovine serum albumin were 9-11 nm and 540,000-820,000, respectively. Those of ovalbumin were 11-16 nm and 500,000-1,820,000, respectively. The overall linear conformation of the bovine serum albumin aggregates slightly differed from that of the ovalbumin aggregates since the mass fractal dimensions were found from calculation to be 1.63-1.7 for the bovine serum albumin and 1.36-1.51 for the ovalbumin. The surface property of the aggregates of both proteins was suggested to be similar to that of their native monomer since all the surface fractal dimensions were almost equivalent. The dimensionless Kratky plots of the eluted aggregates indicated that the non-globular conformation of the bovine serum albumin aggregates differs from that of the ovalbumin aggregates. These analyses using size-exclusion chromatography combined with the solution X-ray scattering measurement will be helpful for characterization of the components of the denatured protein aggregation in solution.