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.

Fig 1. The effects of storage temperature on the conformation of purified α₂M in PBS/Az.

Images of native PAGE (3–8% Tris-acetate) gels showing α₂M (A) freshly purified in PBS/Az (lane 1), after storage in PBS/Az (4°C, 4 months) (lane 2) and after storage in PBS/Az (4°C, 8 months) (lane 3). In all samples, there is a small amount of higher molecular weight (HMW) species present; (B) in PBS/Az (4°C or -20°C, 10 days). Also shown is α₂M in PBS/Az after rapid freezing in liquid nitrogen (LN) and subsequent storage (-20°C, 10 days), and (C) in PBS/Az (4°C, 2 months) or after freeze-drying (FD) and storage (-20°C, 10 days). In images (A-C) the position of transformed α₂M (α₂M*; generated by treatment with 400 mM NH₄Cl in PBS overnight) is shown. (D) bisANS fluorescence measurements for α₂M in PBS/Az (4°C, -20°C, or freeze dried and stored at -20°C, all for 10 days). The results shown are the mean bisANS fluorescence (n = 3±SD) in arbitrary fluorescence units (AFU). (E) Trypsin activity assay showing the conversion of BAPNA to p-nitroaniline by trypsin-α₂M complexes. For this assay α₂M was stored as described in (D). * Denotes significant increases in bisANS fluorescence as a result of storing native α₂M at -20°C, or FD compared to a matched α₂M sample stored at 4°C (Student’s t-test p < 0.01).

Fig 2. The effect of NaCl on frozen α₂M preparations.

(A) Images of native PAGE (3–8% Tris-acetate) gels showing α₂M stored in 20 mM sodium phosphate buffer, pH 7.4, in the presence or absence of 150 mM NaCl (4°C or -20°C, 20 days). LN indicates that the sample was rapidly frozen in liquid nitrogen prior to storage at -20°C. Also shown is the position of α₂M* (generated by treatment with 400 mM NH₄Cl in PBS overnight). (B) Corresponding bisANS fluorescence measurements for α₂M as described in (A). The results shown are the mean bisANS fluorescence (n = 3±SD) in AFU. (C) Trypsin activity assay showing the rate of BAPNA conversion to p-nitroaniline by trypsin-α₂M complexes generated using α₂M as described in (A). The results shown are the mean BAPNA conversion rates (n = 3±SD). * Denotes significant increases in bisANS fluorescence of α₂M stored at -20°C compared to a matched sample stored at 4°C. ^v Denotes significant decreases in the rate of BAPNA conversion to p-nitroaniline by trypsin-α₂M complexes generated using α₂M stored at -20°C compared to a matched sample stored at 4°C (both Student’s t-test p < 0.01).

Fig 3. The effect of freezing and thawing on the structure and surface hydrophobicity of α₂M.

(A) Image of a native PAGE (3–8% Tris-acetate) gel showing the migration of α₂M in 20mM sodium phosphate buffer, pH 7.4 after 0–4 cycles of rapid freezing in liquid nitrogen followed by thawing at 37°C (F/T cycles). (B) CD spectra of α₂M as described in (A). (C) BisANS fluorescence measurements (excitation 360 nm, emission 490 nm) for α₂M as described in (A). * Denotes significant increases or decreases in soluble α₂M, bisANS fluorescence or trypsin binding compared to a matched α₂M sample stored at 4°C (Student’s t-test p < 0.01).

Fig 4. The effect of storage at -20°C on the ability of α₂M to bind to SH-SY5Y cells.

(A) Image of a native PAGE (3–8% Tris-acetate) gel showing α₂M stored in 20 mM sodium phosphate buffer, pH 7.4 containing 100 mM NaCl or in PBS/Az (4°C or -20°C, 10 days). LN indicates that the sample was rapidly frozen in liquid nitrogen prior to storage at -20°C. (B) Corresponding bisANS fluorescence measurements for α₂M stored as described in (A). The results shown are the mean values of bisANS fluorescence (n = 3±SD) in AFU. (C) Flow cytometry analysis showing the binding of α₂M preparations stored as described in (A) to SH-SY5Y cells. The results shown are the composite geometric mean values of FITC fluorescence for 5000 viable cells (n = 3 ± SD) in AFU and are adjusted for background fluorescence. * Denotes significant increases in cell surface binding of α₂M stored at -20°C compared to a batch matched sample stored at 4°C. v Denotes significant decreases in cell surface binding of α₂M as a result of pre-incubation of the cells with RAP.

Fig 5. The effect of storage at -20°C on α₂M chaperone activity.

(A) Images of native PAGE (3–8% Tris-acetate) analyses of α₂M stored in 20 mM phosphate buffer, pH 7.4 (4°C or -20°C, 1 month). The latter sample was rapidly frozen in LN prior to storage at -20°C. (B) Corresponding bisANS fluorescence measurements for the α₂M samples described in (A). The results shown are the values of the mean bisANS fluorescence (n = 3±SD) in AFU. * Denotes significantly increased bisANS fluorescence as a result of storage at -20°C (Student’s t-test p < 0.01) (C) Turbidity measurements of CPK aggregation in the presence or absence of α₂M which had been stored at 4°C or -20°C as described in (A). The data are from individual measurements and are representative of several different experiments.

Fig 6. The effect of lyophilization from Tris buffer on purified α₂M.

Images of native PAGE (3–8% Tris-acetate) gels showing the migration of (A) reconstituted α₂M that had been lyophilized from 20 mM Tris, 130 mM glycine, 80 mM trehalose, pH 8.0 and (B) α₂M stored in solution at 4°C in 20 mM Tris, pH 8.0 for 2 months or following reconstitution after it had been lyophilized from 20 mM Tris, pH 8.0 and stored at -20°C for 7 days. As references, the positions of native and transformed α₂M are also shown in (A). (C) Matched α₂M samples in 20 mM phosphate, pH 7.4 or 20 mM Tris, pH 8.0 stored at 4°C for 2 months. Both samples contained 0.02% (w/v) sodium azide.

Fig 7. The effect of sucrose or glycine on the preservation of native α₂M characteristics after lyophilization.

(A) Image of a native PAGE (3–8% Tris-acetate) gel showing α₂M stored in 20 mM phosphate buffer, pH 7.4 (4°C, 2 months) or after lyophilization and storage at -20°C for 7 days prior to reconstitution. α₂M was lyophilized from buffer only, with sucrose present at the indicated concentrations, or with glycine at the indicated mass ratios (α₂M-to-glycine). As references, the positions of native α₂M, transformed α₂M and dimeric α₂M (generated by incubation with 8 M urea) are shown. (B) Recovery of soluble α₂M after lyophilization from the conditions described in (A) as assessed by the BCA assay. (C) Corresponding bisANS fluorescence measurements for α₂M after lyophilization from the conditions described in (A). The results shown are the values of the mean bisANS fluorescence (n = 3±SD) in AFU.