Quantification of anti-aggregation activity of chaperones: a test-system based on dithiothreitol-induced aggregation of bovine serum albumin.

The methodology for quantification of the anti-aggregation activity of protein and chemical chaperones has been elaborated. The applicability of this methodology was demonstrated using a test-system based on dithiothreitol-induced aggregation of bovine serum albumin at 45°C as an example. Methods for calculating the initial rate of bovine serum albumin aggregation (v agg) have been discussed. The comparison of the dependences of v agg on concentrations of intact and cross-linked α-crystallin allowed us to make a conclusion that a non-linear character of the dependence of v agg on concentration of intact α-crystallin was due to the dynamic mobility of the quaternary structure of α-crystallin and polydispersity of the α-crystallin-target protein complexes. To characterize the anti-aggregation activity of the chemical chaperones (arginine, arginine ethyl ester, arginine amide and proline), the semi-saturation concentration [L]0.5 was used. Among the chemical chaperones studied, arginine ethyl ester and arginine amide reveal the highest anti-aggregation activity ([L]0.5 = 53 and 58 mM, respectively)

Effect of ArgAd on DTT-induced aggregation of BSA ([BSA] = 1.0 mg/ml; 2 mM DTT).

<p>(A) The dependences of the light scattering intensity on time obtained at the following concentrations of ArgAd: (1) 0, (2) 75 and (3) 150 mM. Points are the experimental data. The solid curves were calculated from Eq. (5). At [ArgAd] = 75 mM the fitting procedure gave the following values of parameters: <i>k</i>_agg = 40.1 (counts/s) min⁻², <i>t</i>₀ = 12.1 min and <i>K</i> = 1.18·10⁻³ min⁻². The dotted line was calculated from Eq. (3) at <i>k</i>_agg = 40.1 (counts/s) min⁻² and <i>t</i>₀ = 12.1 min. (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on time obtained in the absence of ArgAd (1) and in the presence of 150 mM ArgAd (2). (C) The dependence of the <i>K</i>_agg/<i>K</i>_agg,0 ratio on the concentration of ArgAd. Points are the experimental data corresponding to the following concentrations of BSA: (1) 0.5, (2) 1 and (3) 2 mg/ml. The solid curve was calculated from Eq. (20). Inset shows the dependence of the duration of the lag period (<i>t</i>₀) on the concentration of ArgAd.</p

Analysis of combined action of α-crystallin and Arg.

<p>The dependences of (<i>k</i>_agg)^1/<i>n</i> on the [ α -crystallin]/[BSA] ratio in the absence (squares) and in the presence of 100 mM Arg (circles). Conditions: [BSA] = 1 mg/ml, [DTT] = 2 mM, 0.1 M Na-phosphate buffer pH 7.0, 45°C.</p

Fractograms of BSA (1 mg/ml) heated at 45°C in the presence of 2 mM DTT.

<p>The heating times were the following: 20 (A), 45 (B) and 90 (D) min. AF4 conditions were the same as described in legend to Fig. 11.</p

The values of refractive index (<i>n</i>), density (ρ) and dynamic viscosity (η) of solutions of arginine, arginine ethylester, arginine amide and proline at 45°C (0.1 M Na-phosphate buffer, pH 7.0).

<p>The values of refractive index (<i>n</i>), density (ρ) and dynamic viscosity (η) of solutions of arginine, arginine ethylester, arginine amide and proline at 45°C (0.1 M Na-phosphate buffer, pH 7.0).</p

Effect of Pro on DTT-induced aggregation of BSA ([BSA] = 1.0 mg/ml, 2 mM DTT).

<p>(A) The dependences of the light scattering intensity on time obtained at the following concentrations of Pro: (1) 0, (2) 500 and (3) 1000 mM. Points are the experimental data. The solid curves were calculated from Eq. (5). (B) The dependences of the hydrodynamic radius (<i>R</i>_h) of the protein aggregates on time obtained in the absence of Pro (1) and in the presence of 1000 mM Pro (2). (C) The dependence of the <i>K</i>_agg/<i>K</i>_agg,0 ratio on the concentration of Pro. Points are the experimental data. The solid curve was calculated from Eq. (20). Inset shows the dependence of the duration of the lag period (<i>t</i>₀) on the concentration of Pro.</p

Initial rate of DDT-induced aggregation of BSA as a function of α-crystallin concentration (45°C; 2 mM DTT).

<p>The dependence of (<i>K</i>_agg/<i>K</i>_agg,0)^1/<i>n</i> on α-crystallin concentration (lower abscissa axis) or the ratio of the molar concentrations of α-crystallin and BSA (upper abscissa axis <i>x</i> = [α-crystallin]/[BSA]; <i>n</i> = 1.6). Points are the experimental data. The solid line in the interval of <i>x</i> values from 0 to <i>x</i>₁ = 0.17 was calculated from Eq. (12) at <i>S</i>₀ = 0.40 subunits of α-crystallin per one BSA molecule. The solid line in the interval of <i>x</i> values from <i>x</i>₁ = 0.17 to <i>x</i>₂ = 2.6 was calculated from Eq. (13) at <i>Y</i>₀ = 0.94 and <i>x</i>_0.5 = 0.093. The inset shows the dependence of the adsorption capacity (AC) of α-crystallin with respect to the target protein on <i>x</i>.</p

Kinetic parameters for DDT-induced aggregation of BSA (45°C; 2 mM DTT).

<p>(A) The dependence of parameter <i>k</i>_agg on BSA concentration. The solid curve was calculated from Eq. (9) at <i>n</i> = 1.6. Inset shows the dependence of <i>K</i>_agg on BSA concentration in the logarithmic coordinates. (B) The dependence of duration of the lag period (<i>t</i>₀) on BSA concentration.</p

SEC elution profiles of cross-linked and intact α-crystallin on a TSK-gel HW-55f column.

<p>The fraction of cross-linked protein marked with gray color was isolated for further testing the chaperone-like activity. Triangles point out retention time of the protein standards: thyroglobulin (660 kDa), catalase (440 kDa), aldolase (158 kDa), BSA (67 kDa), α-crystallin (20 kDa).</p