A robust and sensitive method for detecting subtle structural differences in bovine serum albumin.

Abstract

Bovine serum albumin (BSA) exhibits lot-to-lot variability, partly due to differences in fatty acid content. In this study, the structural properties of two BSA lots-fatty acid-bound and -free-were compared using electrophoretic, chromatographic, and spectroscopic techniques. No apparent differences in overall structure were detected by conventional methods, including UV absorbance spectroscopy, reducing and non-reducing SDS-PAGE, gel filtration chromatography, or agarose native gel electrophoresis. However, circular dichroism (CD) analysis of native BSA revealed small but significant differences in folded structure between the two lots, particularly in the microenvironment surrounding one of two tryptophan residues, a known fatty acid-binding region. These differences were further supported by the intrinsic fluorescence measurements. Under heat stress (73-76 °C), the two lots exhibited distinct behaviors. Native gel electrophoresis and CD spectroscopy conformational states with different patterns, including variations in aggregation propensity. These results demonstrate that, for the first time, combining CD and fluorescence spectroscopy with native electrophoresis under heat-stress conditions provides a sensitive and practical approach for detecting subtle conformational differences among BSA lots, offering a valuable tool for assessing protein quality and consistency.