On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex
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ORIGINAL ARTICLE
On the utility of fluorescence‑detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk Jennifer M. Crowther1,2 · Marita Broadhurst3 · Thomas M. Laue4 · Geoffrey B. Jameson1,2,5 · Alison J. Hodgkinson3,7 · Renwick C. J. Dobson1,2,6 Received: 15 June 2020 / Revised: 25 September 2020 / Accepted: 29 September 2020 © European Biophysical Societies’ Association 2020
Abstract β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of β-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled β-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these β-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein. Keywords β-Lactoglobulin · Immunoglobulin · Fluorescence · Analytical ultracentrifugation · Protein interactions Abbreviations FITC Fluorescein isothiocyanate IgG Immunoglobulin G IgA Immunoglobulin A IgM Immunoglobulin M AUC Analytical ultracentrifugation
Introduction β-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milk, including cow and goat milk, yet is absent in human milk. β-Lactoglobulin is known to affect the processing of ruminant milk, for instance due to heatinduced aggregation during heat treatment (Manderson et al. 1998). It is also one of the main immunogenic proteins that contribute to milk allergies (Hufnagl et al. 2018). Considerable effort has been made to elucidate the biological and biophysical properties of β-lactoglobulin and as such it is
Special Issue: Analytical Ultracentrifugation 2019. * Jennifer M. Crowther [email protected]
3
Food and Bio‑Based Products, AgResearch Limited, Ruakura Research Centre, Hamilton, New Zealand
* Alison J. Hodgkinson [email protected]
4
Center To Advance Molecular Interaction Science, University of New Hampshire, Durham, NH, USA
* Renwick C. J. Dobson [email protected]
5
School of Fundamental Sciences, Massey University, Palmerston North, New Zealand
6
Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia
7
On-Farm R&D, Farm Source, Fonterra Co-Operative Group, Hamilton 3200, New Zealand
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