Denatured Bovine Serum Albumin Complex Process Of Partially Folded Intermediates

The unfolding and refolding of bovine serum albumins and the existence of intermediate stages were investigated by fluorescence phase diagram and high performance hydrophobic interaction chromatography, BSA aggregates and how they formed during the refolding procedure were studied by lauryl sodium sulfate-polyacrylamide gel electrophoreses (SDS-PAGE) and high-performance size-exclusion chromatography. This study will be helpful to understand how proteins folded rapidly and successfully to their unique native conformation in vitro and develop models of protein folding.The unfolding and refolding of bovine serum albumins and the existence of intermediates were studied by fluorescence phase diagram. In the presence of 2-mercaptoethanol, the BSA unfolding procedure induced by urea were directly unfolded from native state to unfolded state and no partially folded intermediates were detected in their unfolding procedure, in the absence of 2-mercaptoethanol, however, a partially folded intermediate was detected. Two intermediate stages were detected in the unfolding of BSA induced by GuHCL in the absence and presence of reduce agent, but GuHCL concentration at which intermediates formed were different. In the refolding of urea non-reduced denatured BSA, there were one partially folded intermediate, and two partially folded intermediates formed in the refolding procedure of GuHCL non-reduced denatured BSA. But the refolding procedure of urea and GuHCL reduced-denatured BSA are all four-step process and proceed via three intermediate stages.Unfolded bovine serum albumins induced by urea and its refolded intermediates were investigated by chromatography. There was one refolded intermediate during the refolding procedure of urea non-reduced denatured BSA in HPHIC. The retention time of this intermediate was longer than the native BSA;thereby its hydrophobicity was stronger than the native BSA. The unfolded BSA did not form aggregates during their refolding procedure and can be refolded successfully by SEC. Because of theexistence of macromolecular aggregates, the reduced-denatured bovine serum albumins can not be refolded successfully by HPHIC, and the most of urea reduced-denatured BSA aggregated during their refolding in SEC.The reason of aggregation during the dilute refolding of denatured BSA in the presence of different urea concentration was analyzed by SDS-PAGE. It was found that the unfolded BSA induced by urea in the absence of reduced agent formed soluble aggregates through intermolecular disulfide bonds. To the reduced-denatured BSA, aggregates were formed in the high urea concentration refolding system. In the low urea concentration, however, an amount of precipitates were formed in the refolding system. The reason of aggregation and precipitation of reduced-unfolded BSA was intermolecular hydrophobic interactions.Based on the results of experiments, we can draw the following conclusions: ( I ) the unfolding procedure of BSA denatured by urea in the presence of reduce agent obeys typical two-state model;In the absence of reduce agent, the unfolding procedure of BSA follows three-state model. (II) One intermediate formed in the refolding procedure of urea denatured BSA in the absence of reduce agent. The refolding procedure of BSA denatured by GuHCL in the absence of reduce agent obey four-state model. The refolding procedure of urea and GuHCL reduced-denatured BSA are all four-step process and proceed via three intermediate stages.(III) The hydrophobicity of the intermediate detected in the refolding of urea non-reduced denatured BSA was stronger than the native BSA, and it can form soluble aggregates through intermolecular disulfide bonds during the dilute refolding.(IV) The hydrophobicity of intermediates detected in the refolding of urea reduced-denatured BSA was so stronge that their intermolecular hydrophobic interactions can lead to the aggregation and precipitation of reduced-unfolded BSA during the refolding procedure.