Disulfide bond formation is not crucial for the heat-induced interaction between β-lactoglobulin and milk fat globule membrane proteins
2020
Hansen, Steffen F. | Nielsen, Søren D. | Rasmusen, Jan T. | Larsen, Lotte B. | Wiking, Lars
During heat treatment of milk, β-lactoglobulin (β-LG) associates with the milk fat globule membrane (MFGM). The objective of this study was to examine different binding types that could be involved in this process. First, we tested the thiol-disulfide bond interchange between β-LG and MFGM by heating raw milk (87°C, 8 min) in the presence of different reagents capable of preventing this interaction, and then evaluated the presence of β-LG in resulting MFGM preparations by sodium dodecyl sulfate-PAGE. Contrary to commonly accepted theory, β-LG still associated with MFGM when milk was heated in the presence of 10 mM N-ethylmaleimide, dithiobis-nitrobenzoic acid, or dithioerythritol. This finding indicated that noncovalent binding could be involved in the interaction, and therefore these were studied next. Preventing noncovalent interactions by heating milk in the presence of 8 M urea (to inhibit formation of hydrogen bonds) or 2 M NaCl (to inhibit electrostatic and hydrophobic interactions) reduced the association of β-LG and MFGM. Inhibiting both hydrogen and disulfide bond formation by addition of 8 M urea and 10 mM dithioerythritol or inhibiting hydrophobic interactions with 0.2% sodium dodecyl sulfate completely prevented the association. In contrast to the simple thiol-disulfide interaction model, the results suggest a more complex understanding of the interactions between β-LG and MFGM during heating of milk. This indicates that disulfide formation between β-LG and proteins in the MFGM is not required for the association, but that hydrophobic interactions and hydrogen bonding may be crucial. This novel insight into β-LG and MFGM association is in contrast to the current literature and requires further study.
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