Kinetics of Thermal Denaturation and Aggregation of Bovine Serum Albumin

<div><p>Thermal aggregation of bovine serum albumin (BSA) has been studied using dynamic light scattering, asymmetric flow field-flow fractionation and analytical ultracentrifugation. The studies were carried out at fixed temperatures (60°C, 65°C, 70°C and 80°C) in 0.1 M phosphate buffer, pH 7.0, at BSA concentration of 1 mg/ml. Thermal denaturation of the protein was studied by differential scanning calorimetry. Analysis of the experimental data shows that at 65°C the stage of protein unfolding and individual stages of protein aggregation are markedly separated in time. This circumstance allowed us to propose the following mechanism of thermal aggregation of BSA. Protein unfolding results in the formation of two forms of the non-native protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation. Aggregation of U_hr leads to the formation of primary aggregates with the hydrodynamic radius (<i>R</i>_h,1) of 10.3 nm. The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). At complete exhaustion of U_lr, secondary aggregates with the hydrodynamic radius (<i>R</i>_h,2) of 12.8 nm are formed. At 60°C the rates of unfolding and aggregation are commensurate, at 70°C the rates of formation of the primary and secondary aggregates are commensurate, at 80°C the registration of the initial stages of aggregation is complicated by formation of large-sized aggregates.</p></div

Mechanism of thermal aggregation of BSA.

<p>The first step of a general aggregation process is unfolding of the native form (N), which results in the formation of two forms of the unfolded protein with different propensity to aggregation. One of the forms (highly reactive unfolded form, U_hr) is characterized by a high rate of aggregation; aggregation leads to formation of primary aggregates with the hydrodynamic radius (<i>R</i>_h,1). The second form (low reactive unfolded form, U_lr) participates in the aggregation process by its attachment to the primary aggregates produced by the U_hr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (A_st). The A_st form corresponds to non-aggregated unfolded species of BSA in AF4 experiments. At full exhaustion of the U_lr form, secondary aggregates with the hydrodynamic radius (<i>R</i>_h,2) are formed. Further aggregation of the protein is a result of sticking of the secondary aggregates.</p

Fractograms of BSA (1 mg/ml) preheated at 65°C.

<p>The heating times were the following: (1) 0, (2) 5, (3) 15, (4) 90 and (5) 600 min. AF4 conditions: 23°C, axial (detector) flow 1 ml/min, focus flow 5 ml/min, cross flow 5 ml/min for 10 min and then linear decay to 0.1 ml/min within 20 min plus 8 min at 0 ml/min.</p

Dependences of the hydrodynamic radius (<i>R</i>_h) on the portion of the denatured protein (γ_den) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The values of γ_den were calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.e007" target="_blank">Eq 4</a>. For each temperature parameters <i>B</i>, <i>k</i>_1,den and <i>k</i>_2,den indicated in corresponding panels of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.g002" target="_blank">Fig 2</a> were used. The <i>R</i>_h <i>vs</i> γ_den plots at 65°C and 70°C (panels B and C) were used for determination of the hydrodynamic radius of the primary aggregates (<i>R</i>_h,1).</p

Dependences of the light scattering intensity on time for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The dotted horizontal lines on the panels A, B and C correspond to <i>I</i>₂ values calculated from the dependences of the light scattering intensity on the portion of aggregated BSA.</p

Dependences of the light scattering intensity (<i>I</i>) on the portion of the aggregated protein (γ_agg) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The vertical dotted lines correspond to γ_agg = (γ_Uhr + γ_Ulr,agg). The <i>I vs</i> γ_agg plots at 60°C, 65°C and 70°C (panels A–C) were used for determination of parameter <i>I</i>₂ corresponding to the value of the light scattering intensity after completion of the secondary BSA aggregates formation. The solid lines were calculated from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153495#pone.0153495.e013" target="_blank">Eq 5</a>.</p

Fluorescence emission spectra of ThT (20 μM; curve 1) and ThT solution incubated with heated BSA (0.4 mg/ml) for 30 min at 25°C.

<p>BSA (1 mg/ml) was preincubated for 12 h at 60°C, 65°C, 70°C and 80°C (curves 2–5, respectively). Excitation wavelength was 450 nm.</p

Sedimentation behavior of BSA (1 mg/ml) preincubated for 12 h at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.

<p>The <i>c</i>(<i>s</i>) and ls-g*(<i>s</i>) distributions were obtained at 24°C. Rotor speed was 52000 rpm.</p

DSC profiles for BSA (1 mg/ml) preheated at 60°C (0.1 M Na-phosphate buffer, pH 7.0).

<p>The dependences of the excess heat capacity (Δ) on temperature for BSA preincubated at 60°C for different time intervals (<i>t</i>_inc): (1) 0, (2) 5, (3) 15, (4) 30, (5) 60 and (6) 90 min. Each DSC profile is the average of three measurements. The heating rate was 1°C/min. The inserted table gives the value of <i>T</i>_max for preheated BSA preparations.</p