Effect of Aggregation on the Hydrodynamic Properties of Bovine Serum Albumin
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To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity.
Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress. Bulk viscosity was measured by an oscillating piston viscometer.
As BSA was heated at a higher concentration or for a longer time, the relative contribution, molecular weight and intrinsic viscosity of aggregate species increased. Molecular shape was evaluated from intrinsic viscosity values, and aggregates were estimated to be more asymmetric than monomer species. The presence of aggregates resulted in an increase in bulk viscosity when relative contribution of very high molecular weight species exceeded 10%.
For model system and conditions studied, generation of higher order aggregate species was concluded to be associated with an increase in molecular asymmetry. Elevated viscosity in the presence of aggregated species points to molecular asymmetry being a critical parameter affecting solution viscosity of BSA.
KEY WORDSaggregation effective molecular shape intrinsic viscosity
Bovine serum albumin
High molecular weight
Low angle light scattering
Right angle light scattering
Size exclusion chromatography
Triple detector array
Very high molecular weight
ACKNOWLEDGMENTS AND DISCLOSURES
Authors would like to thank Lauren Fontana and Rui Fang for help with some experimental aspects, and Elizabeth Zecca for critical reading of the manuscript. Generous material and financial support from Genentech, Inc., as well as Outstanding Scholar and summer fellowships from graduate school at University of Connecticut are also acknowledged.
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