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

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. Lot-dependent conformational differences between fatty acid–bound and –free bovine serum albumin (BSA) were evaluated using a robust and sensitive method that has been streamlined in analytical workflow by combining agarose native gel electrophoresis with circular dichroism (CD) and intrinsic fluorescence spectroscopy. CD and fluorescence spectroscopy detected subtle structural differences under native conditions, while mild heating (73–76 °C) induced intermediate conformational states exhibiting distinct lot-dependent behaviors. These differences, including altered aggregation propensity, were clearly visualized by agarose native gel electrophoresis, demonstrating the utility of this simple, robust, and sensitive approach for assessing lot-to-lot structural variability, the effects of post-translational modifications, or conformational changes due to molecular interactions in proteins. Fatty acid–bound and –free BSA lots show subtle structural differences.CD and fluorescence spectroscopy detected conformational variation in aromatic tryptophan signal near the fatty acid–binding site.Heating revealed distinct and different intermediate and aggregation patterns between the BSA lots by CD and agarose native gel electrophoresis.CD and fluorescence spectroscopy combined with native electrophoresis can assess lot-dependent protein quality with high sensitivity and precision. Fatty acid–bound and –free BSA lots show subtle structural differences. CD and fluorescence spectroscopy detected conformational variation in aromatic tryptophan signal near the fatty acid–binding site. Heating revealed distinct and different intermediate and aggregation patterns between the BSA lots by CD and agarose native gel electrophoresis. CD and fluorescence spectroscopy combined with native electrophoresis can assess lot-dependent protein quality with high sensitivity and precision.