title: Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies.
abstract: Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a protease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transformed (i.e. adopts a more compact conformation after interactions with proteases or small nucleophiles), and are also influenced by dissociation of the native alpha-2-macroglobulin tetramer into stable dimers. Alpha-2-macroglobulin is predominately present as the native tetramer in vivo; once purified from human blood plasma, however, alpha-2-macroglobulin can undergo a number of conformational changes during storage, including transformation, aggregation or dissociation. We demonstrate that, particularly in the presence of sodium chloride or amine containing compounds, freezing and/or lyophilization of alpha-2-macroglobulin induces conformational changes with functional consequences. These conformational changes in alpha-2-macroglobulin are not always detected by standard native polyacrylamide gel electrophoresis, but can be measured using bisANS fluorescence assays. Increased surface hydrophobicity of alpha-2-macroglobulin, as assessed by bisANS fluorescence measurements, is accompanied by (i) reduced trypsin binding activity, (ii) increased chaperone activity, and (iii) increased binding to the surfaces of SH-SY5Y neurons, in part, via lipoprotein receptors. We show that sucrose (but not glycine) effectively protects native alpha-2-macroglobulin from denaturation during freezing and/or lyophilization, thereby providing a reproducible method for the handling and long-term storage of this protein.
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Amy R
authors.initials: AR
authors.lastname: Wyatt
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Natalie E
authors.initials: NE
authors.lastname: Farrawell
authors.affiliation: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.affiliation: Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
authors.forename: Mark R
authors.initials: MR
authors.lastname: Wilson
mesh_terms: D005612:Freeze Drying; D005615:Freezing; D005947:Glucose / Q000737:chemistry; D006801:Humans; D011487:Protein Conformation; D012965:Sodium Chloride / Q000737:chemistry; D013395:Sucrose / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000737:chemistry / Q000502:physiology*
chemical_list: D000511:alpha-Macroglobulins; D012965:Sodium Chloride; D013395:Sucrose; D005947:Glucose