STRUCTURAL INVESTIGATION OF THE INTERACTION BETWEEN BOVINE SERUM ALBUMIN AND ACID GELATIN IN WATER - Biochemistry and Biotechnology: Research and Development - page 75

The ratio of the intensities of the two minima of the molecular ellipticity at 222 nm and
209 nm (> 1) obtained for BSA solution at 25 o C (Figure 1) indicates that BSA belongs to the
/ class of proteins, i.e., consists of mixed segments of alpha-helical and -structure [40].
Literature data indicate that the secondary structure of BSA contains about 68-50% alpha-
helices and 16-21% beta-sheets [41,42].
The comparison of the thermodynamic parameters presented in Table 2. for the thermal
unfolding of BSA with those reported in the literature is difficult not only because of different
experimental conditions but on account of different deconvolution procedures. The thermal
denaturation of BSA in aqueous solution was characterised by a transition with T m =62 o C,
H=335 kJ mol -1 [43,44] thus by a similar temperature but lower enthalpy changes than
reported here. The onset temperature of conformational changes, as found by DSC; is 58.1 o C
[45] . Other available literature results, obtained under more dissimilar conditions gave:
T m =68 o C, H = 535-600 kJ mol -1 (in 0.9% NaCI, pH 6.8 [46]; T m =61.5 o C, H = 568 kJ mol -

1 (in 0.067 M phosphate buffer, pH 6.0) [47,48], ; T m =63.9 o C,

H =785 kJ mol -1 (in 0.1 M

NaCI, pH 5.6) [49].
There are reports on the behavior of two different BSA samples in the literature. One is
practically pure BSA globulin containing some traces of fatty acids [50] and with the second
sample the fatty acids were also absent [39,51]. The former transition temperature was 69 o C-
70 o C, that for the latter sample was about 62 o C. The melting of the native structure of the
latter sample is the complex process which is described by two or three simple transitions
overlapping in temperature. From our data (Figure 10) we can therefore conclude that we are
working with the BSA sample containing some traces of fatty acids.

4.2. Behaviour of BSA Molecules in the Presence of Gelatin

The experimental findings demonstrate that the ratio of macromolecular components ( q)
is the key factor determining the interaction of gelatin with BSA at pH 5.4 (natural pH values
of pure BSA solutions in pure demineralized water) and the temperature above the
conformation transition of gelatin.
Taking into account that the maximal binding of BSA and gelatin in complex takes place
at q 2, knowing the weight-average molecular weights of BSA and gelatin we can roughly
evaluate the BSA/gelatin molar ratio in the complex in the selected conditions. Simple
calculation showed that at least 6 molecules of BSA join to 1 gelatin molecule, forming
BSA/gelatin complex. The question is appears: what is the structure of the complexes
formed? Usually, the sizes of interpolymeric complexes are larger then those of the
interacting polymers [19]. However in the given case, the sizes of the complex particles are at
least 10 times smaller then the size of gelatin molecules and approx. 50% larger then the sizes
of the BSA molecules. It is reasonable to suggest that at given conditions complex formation
results in the collapse of gelatin molecules due to sharp decrease of their total charge.The
collapse of the gelatin macromolecules in solution at isoelectric conditions due to electrostatic
attraction forces of oppositely charged groups has been shown early in study of the gelatin
self association [52]. Figure 11. presents schematically particles of the BSA/gelatin
complexes. The collapsed gelatin molecule wrap six BSA molecules forming the complex
particle containing BSA molecules in more folded state then before interaction with gelatin.