Examples of conformational dependent α₂M activities.

<p>* α₂M dimers generated by hypochlorite treatment.</p><p>† α₂M dimers generated by thiocyanate treatment.</p><p>‡ α₂M dimers generated by SDS treatment.</p><p>Many of the activities of α₂M are dependent on whether or not the protein is in its native conformation or in its transformed state. Additionally, dissociation of the native α₂M tetramer into dimers (that can be induced using several different chemical methods) has also been demonstrated to influence the activities of α₂M.</p

The effect of lyophilization from Tris buffer on purified α₂M.

<p>Images of native PAGE (3–8% Tris-acetate) gels showing the migration of (A) reconstituted α_<b>2</b>M that had been lyophilized from 20 mM Tris, 130 mM glycine, 80 mM trehalose, pH 8.0 and (B) α_<b>2</b>M 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 α_<b>2</b>M are also shown in (A). (C) Matched α_<b>2</b>M 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.</p

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

<p>(A) Images of native PAGE (3–8% Tris-acetate) analyses of α_<b>2</b>M 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 α_<b>2</b>M 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 α_<b>2</b>M 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.</p

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

<p>(A) Image of a native PAGE (3–8% Tris-acetate) gel showing the migration of α_<b>2</b>M 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 α_<b>2</b>M as described in (A). (C) BisANS fluorescence measurements (excitation 360 nm, emission 490 nm) for α_<b>2</b>M as described in (A). * Denotes significant increases or decreases in soluble α_<b>2</b>M, bisANS fluorescence or trypsin binding compared to a matched α_<b>2</b>M sample stored at 4°C (Student’s t-test p < 0.01).</p

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

<p>(A) Image of a native PAGE (3–8% Tris-acetate) gel showing α_<b>2</b>M 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 α_<b>2</b>M 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 α_<b>2</b>M 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 α_<b>2</b>M stored at -20°C compared to a batch matched sample stored at 4°C. v Denotes significant decreases in cell surface binding of α_<b>2</b>M as a result of pre-incubation of the cells with RAP.</p

The effect of NaCl on frozen α₂M preparations.

<p>(A) Images of native PAGE (3–8% Tris-acetate) gels showing α_<b>2</b>M 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 α_<b>2</b>M* (generated by treatment with 400 mM NH_<b>4</b>Cl in PBS overnight). (B) Corresponding bisANS fluorescence measurements for α_<b>2</b>M 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-α_<b>2</b>M complexes generated using α_<b>2</b>M as described in (A). The results shown are the mean BAPNA conversion rates (n = 3±SD). * Denotes significant increases in bisANS fluorescence of α_<b>2</b>M 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-α_<b>2</b>M complexes generated using α_<b>2</b>M stored at -20°C compared to a matched sample stored at 4°C (both Student’s t-test p < 0.01).</p

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

<p>Images of native PAGE (3–8% Tris-acetate) gels showing α_<b>2</b>M (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 α_<b>2</b>M 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 α_<b>2</b>M (α_<b>2</b>M*; generated by treatment with 400 mM NH_<b>4</b>Cl in PBS overnight) is shown. (D) bisANS fluorescence measurements for α_<b>2</b>M 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-α_<b>2</b>M complexes. For this assay α_<b>2</b>M was stored as described in (D). * Denotes significant increases in bisANS fluorescence as a result of storing native α_<b>2</b>M at -20°C, or FD compared to a matched α_<b>2</b>M sample stored at 4°C (Student’s t-test p < 0.01).</p

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

<p>(A) Image of a native PAGE (3–8% Tris-acetate) gel showing α_<b>2</b>M 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. α_<b>2</b>M was lyophilized from buffer only, with sucrose present at the indicated concentrations, or with glycine at the indicated mass ratios (α_<b>2</b>M-to-glycine). As references, the positions of native α_<b>2</b>M, transformed α_<b>2</b>M and dimeric α_<b>2</b>M (generated by incubation with 8 M urea) are shown. (B) Recovery of soluble α_<b>2</b>M after lyophilization from the conditions described in (A) as assessed by the BCA assay. (C) Corresponding bisANS fluorescence measurements for α_<b>2</b>M after lyophilization from the conditions described in (A). The results shown are the values of the mean bisANS fluorescence (n = 3±SD) in AFU.</p

Effects of biotinylation on lysozyme.

<p>(A) MALDI mass spectrometry of lysozyme incubated in the absence (upper) and the presence (lower) of 100-fold molar concentration of BioNSE. (B) Primary sequence of human lysozyme showing the sites where trypsin digestion has occurred in BioHuL (open grey arrows) and WTHuL (filled black arrows). (C) Secondary structure of BioHuL (dashed grey line) and WTHuL (solid black line) by far-UV CD. (D) Thermal denaturation curves of BioHuL (open grey circles) and WTHuL (filled black squares) monitored by near-UV CD. Mid-point T_m values are defined as the temperatures at which 50% of the population of protein molecules is unfolded.</p

<i>d</i>STORM images of BioHuL fibrils.

<p>(A)–(C) Super-resolution <i>d</i>STORM images of different BioHuL fibrils (formed <i>in vitro</i>). (D) An overlay of a straight BioHuL fibril with its fluorescence sum image. (E) The cross-sections of the individual fluorescence sum and the super-resolved <i>d</i>STORM image of the BioHuL fibril displayed in panel (D). The full-width half-maximum (FWHM) of the fluorescence intensity distribution of the unresolved sum image depicts a fibril diameter of 783 nm whereas the super-resolved image depicts a fibril diameter of 133±20 nm; the latter showing 6 times better resolution. (F) (left panel) DIC image of BioHuL present within SH-SY5Y mammalian cells after probing with streptavidin-Alexa647. (middle panel) Fluorescence sum image of a region within the SH-SY5Y cells (right panels) Super-resolution <i>d</i>STORM images of BioHuL fibrils in the same region of the SH-SY5Y mammalian cells as the fluorescence sum image. All scale bars all represent 1 μm.</p