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Alpha-2-Macroglobulin Is Acutely Sensitive to Freezing and Lyophilization: Implications for Structural and Functional
Amy R Wyatt1, Janet R Kumita2, Natalie E Farrawell1
1Illawarra Health and Medical Research Institute and School of Biological Sciences, University of Wollongong, Wollongong, NSW 2522, Australia.
Plos One
|June 24, 2015
Summary
Alpha-2-macroglobulin (A2M) undergoes conformational changes during storage, impacting its functions. Sucrose effectively protects native A2M from denaturation during freezing and lyophilization, ensuring its stability and functional integrity.
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Alpha-2-macroglobulin (A2M) is a key secreted protein with protease inhibitor and molecular chaperone functions.
- A2M's activity depends on its native or transformed conformation and tetramer/dimer state.
- Purified A2M can undergo conformational changes (transformation, aggregation, dissociation) during storage.
Purpose of the Study:
- To investigate the effects of freezing and lyophilization on A2M conformation and function.
- To identify methods for preserving native A2M structure and activity during storage.
- To characterize conformational changes using bisANS fluorescence assays.
Main Methods:
- Purification of A2M from human blood plasma.
- Induction of conformational changes using freezing/lyophilization in the presence of NaCl or amine compounds.
- Assessment of conformational changes using bisANS fluorescence assays and native polyacrylamide gel electrophoresis.
- Measurement of trypsin binding and chaperone activity.
- Evaluation of A2M binding to SH-SY5Y neurons.
Main Results:
- Freezing and/or lyophilization induced conformational changes in A2M, particularly with NaCl or amine compounds.
- These changes, indicated by increased surface hydrophobicity (measured by bisANS fluorescence), correlated with reduced trypsin binding and increased chaperone activity.
- A2M also showed increased binding to SH-SY5Y neurons via lipoprotein receptors.
- Sucrose, but not glycine, effectively protected native A2M from denaturation during these processes.
Conclusions:
- Conformational changes in A2M during storage can be induced by freezing/lyophilization and are not always detected by standard electrophoresis.
- BisANS fluorescence is a sensitive method for detecting these hydrophobic changes.
- Sucrose provides effective cryoprotection for A2M, enabling reproducible handling and long-term storage.
