PNNL

Abstract

The adsorption and unfolding of proteins on rigid surfaces is characterized by numerous chemical and physical interactions such as hydrogen bonds, disulfide bridges, hydrophobic effects, and London forces. The kinetics of unfolding is dependent on pH, temperature, surface chemistry, as well as protein deformability and structure. In practical applications, this fundamental process has broad implications in biomedical engineering (i.e. artificial implants, drug delivery, and surgical equipment), nanotechnology, maritime construction, and chromatography. However, little is known about the mechanisms behind unfolding because of the atomic lengths and rapid time scales associated with the surface-mediated pathway. Therefore, the unfolding kinetics of myoglobin, ß-glucosidase, and ovalbumin were investigated by adsorbing the proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography (HPLC). The elution profiles and retention times were obtained by UV/Vis spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to protein unfolding on the non-porous surfaces. This data, and those of previous studies, fit a linear trend between percent unfolding after a fixed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, ß-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and ß-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confirms that unfolding can be described by experimental techniques, thereby allowing for the better elucidation of the relationships between the structure and function of soluble proteins as well as other macromolecules.