Fig 1.
DSC profiles for BSA (1 mg/ml) preheated at 60°C (0.1 M Na-phosphate buffer, pH 7.0).
The dependences of the excess heat capacity (Δ) on temperature for BSA preincubated at 60°C for different time intervals (tinc): (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 Tmax for preheated BSA preparations.
Fig 2.
Kinetics of thermal denaturation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The dependences of the portions of the native protein (γnat) on time. The γnat value was calculated as a Q/Q0 ratio (Q0 and Q are the denaturation heats determined from the area under the DSC profiles). The solid curves were calculated from the Eq 4 containing parameters B, k1,den and k2,den.
Table 1.
Parameters of thermal denaturation and aggregation of bovine serum albumin ([BSA] = 1 mg/ml, 0.1 M Na-phosphate buffer, pH 7.0).
Fig 3.
Fractograms of BSA (1 mg/ml) preheated at 65°C.
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.
Fig 4.
Kinetics of thermal aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The dependences of portions of the non-aggregated protein (γnon-agg) on time. The γnon-agg values were calculated from the AF4 data. The dotted horizontal lines correspond to the γnon-agg,lim values. The dash-dotted line in panel B corresponds to the value of γUlr = 1 –γUhr.
Fig 5.
Relationship between portions of the aggregated protein (γagg) and the denatured protein (γden).
The γagg vs γden plots are constructed at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C. The values of γden = 1 –γnat were calculated from Eq 4. For each temperature parameters B, k1,den and k2,den indicated in corresponding panels of Fig 2 were used. The γagg vs γden plot at 65°C (panel B) was used for determination of the portion of the highly reactive BSA form (γUhr) at this temperature.
Fig 6.
Mechanism of thermal aggregation of BSA.
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, Uhr) is characterized by a high rate of aggregation; aggregation leads to formation of primary aggregates with the hydrodynamic radius (Rh,1). The second form (low reactive unfolded form, Ulr) participates in the aggregation process by its attachment to the primary aggregates produced by the Uhr form and possesses ability for self-aggregation with formation of stable small-sized aggregates (Ast). The Ast form corresponds to non-aggregated unfolded species of BSA in AF4 experiments. At full exhaustion of the Ulr form, secondary aggregates with the hydrodynamic radius (Rh,2) are formed. Further aggregation of the protein is a result of sticking of the secondary aggregates.
Fig 7.
Dependences of the hydrodynamic radius (Rh) on time for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The dotted horizontal lines on panels A, B, and C correspond to Rh,1 or Rh,2 values calculated from the dependences of Rh on the portions of denatured and aggregated BSA, respectively. The solid curve on panel D was calculated from Eq 3 at df = 1.76.
Fig 8.
Dependences of the hydrodynamic radius (Rh) on the portion of the denatured protein (γden) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The values of γden were calculated from Eq 4. For each temperature parameters B, k1,den and k2,den indicated in corresponding panels of Fig 2 were used. The Rh vs γden plots at 65°C and 70°C (panels B and C) were used for determination of the hydrodynamic radius of the primary aggregates (Rh,1).
Fig 9.
Dependences of the hydrodynamic radius (Rh) on the portion of the aggregated protein (γagg) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The vertical dotted lines correspond to γagg = γUhr + γUlr,agg. The Rh vs γden plots at 60°C, 65°C and 70°C (panels A–C) were used for determination of the hydrodynamic radius of the secondary aggregates (Rh,2).
Fig 10.
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.
The dotted horizontal lines on the panels A, B and C correspond to I2 values calculated from the dependences of the light scattering intensity on the portion of aggregated BSA.
Fig 11.
Dependences of the light scattering intensity (I) on the portion of the aggregated protein (γagg) for aggregation of BSA at (A) 60°C, (B) 65°C, (C) 70°C and (D) 80°C.
The vertical dotted lines correspond to γagg = (γUhr + γUlr,agg). The I vs γagg plots at 60°C, 65°C and 70°C (panels A–C) were used for determination of parameter I2 corresponding to the value of the light scattering intensity after completion of the secondary BSA aggregates formation. The solid lines were calculated from Eq 5.
Fig 12.
Sedimentation behavior of native BSA at 20°C.
The differential sedimentation coefficient distributions c(s) for BSA (1 mg/ml). The inset shows c(M) distribution. Rotor speed was 48000 rpm.
Fig 13.
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.
The c(s) and ls-g*(s) distributions were obtained at 24°C. Rotor speed was 52000 rpm.
Fig 14.
TEM images of BSA preparations pre-heated at 65°C for 110 min (A) and 330 min (B).
BSA concentration was 1 mg/ml. Preparation preheated for 110 min was washed in deionized water. Image in panel B contains particles of tobacco mosaic virus (TMV).
Fig 15.
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.
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.