the mechanism of complex formation for the native and thermally aggregated BSA is different.
We can formulate important question: what is the nature of complex formation BSA TA
with acid gelatin? The interaction energy in the presence of salt has been considered by de Kruif et al. [37].
It was expected that the protonated amino groups of the protein associate with deprotonated carboxyl group of the polyelectrolyte. Ball et al. [38] argued that this requires energy to be put into the system, because the average distance between the macro ions is larger due to steric hidrances.
The low ionic strength will suppress dissociation of the carboxylic groups (the effective pKa increases) and may be suppresses protonation of the protein, an energy which is released on mixing.
So it seems that at low ionic strength the lowering of the free energy is due to enthalpic effects while at higher ionic strength entropic effects largely contribute [37].
Increase of the ionic strength of the binary water–BSATA and water–gelatin systems before their subsequent mixing up
to 0.25/NaCl/do not leads to formation of the complex particles. (Fig. 4a). The intensity size distribution functions for
such ternary systems is very similar to those obtained for the ternary water–gelatin–BSATA system if to suppose that there is no interaction between these macromolecules (see Figs. 2 and 4).
Many scientists suppose that nonelectrostatic forces, hydrophobic and (or) hydrogenbonds, play a determinant role in this
process. To answer to this question, absorption values of (BSATA+NaCl) + gelatin + NaCl), and (BSATA+ urea) +
(gelatin + urea) mixtures at 400 nm were determined as a functions of the concentrations of NaCl and urea (Fig. 4b). Sodium chloride suppress only electrostatic interactions between biopolymers, while 6 M urea
suppress both hydrophobic and hydrogen interactions in biopolymer systems. This allow us to analyze a contributions of different intermacromolecular bonds in the process of complex formation. Introduction of NaCl in water results in full insensitivity of the BSATA solutions to the presence of gelatin. On the other hand, an addition of 6 M urea in the binary BSATA+ urea, and gelatin + urea solutions does not prevent complex formation. This indicates that
the complexes are formed via electrostatic interaction, rather than through hydrogen bonds formation or hydrophobic interaction. The role of salt is to “soften” the interactions, which is equivalent to making the electrostatic binding constant smaller. On the other hand, an addition of 0.04–027 M NaCl in the ternary water–gelatin–BSATAsystem results in sharp decrease in the average size of the main peak characterizing complex particles up to 200–220 nm (Fig. 5a). However the size of the particles in the presence of NaCl remains at least twice larger that the size of BSATA.
Absorbtions of the BSATA + gelatin system at q = 1 as a function concentration of NaCl and urea are presented in Fig. 5b. One can see that absorbtion values of the complex forming mixture decreases with the increase of both concentration of urea and NaCl. It means that the presence of salt in the ternary water–gelatin–BSATAsystem do
not lead to complete dissociation of the complex particles. In other words, the secondary forces (hydrophobic interaction and hydrogen bonds) play an appreciable role in stabilization of the complex BSATA–gelatin-particles.
3.2. Circular dichroism measurements
CD is a sensitive technique to monitor conformational changes in protein upon interaction with ligands. The raw CD spectra of BSATA in the absence and presence of gelatin after subtraction the CD spectra of gelatin are shown in Fig. 6. The CD spectra of BSATA exhibited two negative bands in the far-UV region at 208 and
222 nm, characteristic of -helical structure of protein. The reasonable explanation is that the negative peaks at 208 and 222 nm are both contributed to n/* transition for the peptide bond of -helix