Towards the understanding of molecular mechanisms in the early stages of heat-induced aggregation of beta-lactoglobulin AB.
Heat-induced aggregation of bovine beta-lactoglobulin AB (10 mg/ml) was studied at 68.5 degrees C at two different pH values (6.7, 4.9) using gel electrophoresis techniques and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing and reducing conditions showed that in the early stages of the aggregation of beta-lactoglobulin disulfide linked aggregates were formed on heating at pH 6.7, but not at pH 4.9. We related this result to the pH-dependent activity of the free thiol group at C121. Mass spectrometric analyses were conducted in two steps. The first involved the analysis of intact non-native monomers and dimers following their ultrasonic passive elution into a suitable solvent mixture in order to confirm the identity of the different gel bands. The second step comprises the analysis of in-gel digests for the determination of disulfide patterns in non-native monomers, covalent dimers and trimers. The results of in-gel digestions analyzed by mass spectrometry suggest that non-native dimers could result from the formation of inter-molecular disulfide bonds C121-C66, C160-C160, or C121-C160. Moreover, two inter-molecular bonds C121-C66 and C160-C160 between two and the same monomer units have been detected, which may play an important role in limiting the process of covalent beta-lactoglobulin network formation. The combination of SDS-PAGE and MALDI-TOF MS enables us to understand the mechanism of beta-lactoglobulin aggregation at the macromolecular level.
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000737:chemistry*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D004220:Disulfides; D007782:Lactoglobulins