Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional Studies

• Published in: PloS one 2015-06, Vol.10 (6), p.e0130036-e0130036
• Main Authors: Wyatt, Amy R, Kumita, Janet R, Farrawell, Natalie E, Dobson, Christopher M, Wilson, Mark R
• Format: Article
• Language: English
• Subjects: alpha-Macroglobulins - chemistry; alpha-Macroglobulins - physiology; Amino acids; Binding; Blood plasma; Chlorine compounds; Cytokines; Denaturation; Dimers; Dissociation; Electrophoresis; Fluorescence; Freeze Drying; Freezing; Gel electrophoresis; Glucose - chemistry; Glycine; Growth factors; Humans; Hydrophobicity; Immune system; Ligands; Lipoprotein receptors; Low density lipoprotein receptors; Medical research; Molecular chains; Nucleophiles; Plasma; Protease inhibitors; Proteases; Protein Conformation; Protein denaturation; Proteinase inhibitors; Proteins; Receptors; Sodium; Sodium chloride; Sodium Chloride - chemistry; Storage; Structure-function relationships; Sucrose; Sucrose - chemistry; Sugar; Transformation; Trypsin; α2-Macroglobulin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0130036

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.