Influence of Protein Hydrolysis on the Growth Kinetics of β-lg Fibrils

Abstract

Recently it was found that protein hydrolysis is an important step in the formation of β-lactoglobulin fibrils at pH 2 and elevated temperatures. The objective of the present study was to further investigate the influence of hydrolysis on the kinetics of fibril formation. Both the hydrolysis of β-lactoglobulin and the growth of the fibrils were followed as a function of time and temperature, using SDS polyacrylamide gel electrophoresis and a Thioflavin T fluorescence assay. As an essential extension to existing models, the quantification of the effect of the hydrolysis on the fibrillar growth was established by a simple polymerization model including a hydrolysis step.

β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils

“…This value is lower than that reported by Kroes-Nijboer et al 18 for β-Lg hydrolysis at 80°C during self-assembly (3.9 × 10 −3 min −1 ) (R 2 = 0.88). The latter study involved heating with continuous shearing, whereas we heated without shearing in order to minimize secondary nucleation, which would have interfered with a close analysis of events during the lag phase.…”

Formation of heat-induced cottonseed congossypin(7S) fibrils at pH 2.0

“…This increase indicates the formation of aggregates or fibril that use these peptides as building blocks. It has been reported that β ‐lactoglobulin fibrils formed at pH 2.0 are composed of peptides, and that the formation of peptides precedes their incorporation into fibrils . Furthermore, the particle size homogeneity of the formed aggregates gradually increased as the protein concentration increased.…”

On the role of peptide hydrolysis for fibrillation kinetics and amyloid fibril morphology

“…aer full hydrolysis). The mechanistic breakpoint for when hydrolysis becomes rate-limiting is at l ¼ 1, which occurs between 5 and 10 g l À1 for pure b-lactoglobulin at 90 C. 18 With a 50-60% b-lactoglobulin content in WPI, this would correspond to 8-20 g l À1 WPI. Hence, it is not likely that the morphological switch occurring between 40 and 60 g l À1 is related to the switch in the rate-limiting mechanism of the brillation reaction.…”

“…16 Fibrillation of WPI at low pH and high temperature has been suggested to proceed through hydrolysis of the proteins into smaller peptide fragments that spontaneously assemble into PNFs. [17][18][19] Although other whey proteins, e.g. a-lactalbumin, can also form amyloid-like brils, 20 PNFs formed under the applied conditions have been shown to be built exclusively from b-lactoglobulin-derived peptides.…”

“…a-lactalbumin, can also form amyloid-like brils, 20 PNFs formed under the applied conditions have been shown to be built exclusively from b-lactoglobulin-derived peptides. 21 The bril assembly has been correlated with the polypeptide hydrolysis reaction 18,22 and mass spectrometry (MS) has been used to identify the peptide building blocks of the brils. 17,19,23 The consensus of these investigations is that the two polypeptide segments, one in the most N-terminal part of b-lactoglobulin (residues 1-53) and one in the C-terminus (residues 138-162), constitute the key building blocks.…”