BSA structure

Based on the information that BSA is homologous to human serum albumin (HSA) (McLachlan and Walker, 1977), the location of helices in domains I, II and III were extracted from the amino acid sequence of BSA based on the crystallographic co-ordinates of HSA, and were found to be;

Effect of Guanidine HCl

Protein denaturation by pH is caused by the ionisation of the side chains whereas small ions or molecules other than protons, e.g., urea and guanidinium ion, affect both the side chain and backbone hydrogen bonds (Scheraga, 1961). In this study, the denaturation of BSA by guanidine hydrochloride (Gdn) can be approximated to the E form (Figure 5.10) but with more random coils depending on the concentration of Gdn., for two reasons:

This means that the disulphide bonds will be intact in both cases of unfolding i.e., due to pH effects below neutral pH, and unfolding due to Gdn. at pH 7. Thermal denaturation is different from the above mentioned mechanism because of thermal motion and the rupture of disulphide bonds. The gel structure formed by heating different unfolded states of BSA by Gdn. was more elastic than BSA heated without prior unfolding. The maximum elastic modulus occurred with a 1 molar [Gdn] and diminished with increasing [Gdn] (Figure 5.14). This shows that some secondary structural elements were required for gel formation. Wang and Damodaran (1991), observed that beta-sheets were essential for gel network formation, and a critical value of about 25% was required. As [Gdn] was increased, BSA became more unfolded (random form) and the elastic modulus decreased (Figure 5.14).

Effect of Heating

In this study it is proposed that native BSA when heated passed through two stages (Kuznetsow et al., 1975; Lin and Koenig, 1976; Oakes, 1976; Wetzel et al., 1980), an initial reversible stage where some of the alpha-helices unfolded and the molecules aggregated through inter-beta-sheet formation (Astbury .,1935; Lin and Koenig, 1976; Clark et al.,1981b). As the temperature was increased further, Cys-34 was exposed and disulphide bonds were formed between different molecules, which formed the irreversible stage (Wetzel et al., 1980).

Unfolding by Gdn. exposed hydrophobic groups. Therefore, the effect of addition of 1 molar Gdn. to BSA caused an increase in hydrophobic interactions in both the first and second stages of gelation. Although the charge distribution on the tertiary structure seems fairly uniform (Figure 5.8), the primary structure is not uniformly charged. The calculated net charges on domain I, II and III were -10, -8 and 0 (Peters, 1985). It is proposed that small quantities of SWP, which are negatively charged (chapter 2), were preferentially bound to domain III through electrostatic interactions resulting in a new aggregate.

The forces involved in the aggregation were hydrogen bonding between the beta-sheets of BSA molecules, disulphide bonds between cys-34, hydrophobic interaction between exposed nonpolar residues and electrostatic interactions between the proteins in SWP and domain III of BSA. It is proposed that this extra enthalpic energy was responsible for the effect of small quantities of SWP on the elastic modulus of BSA (Figure 5.17). It is likely that a critical value of SWP was needed to saturate domain III in BSA. As the concentration of SWP increased, the elastic modulus decreased because domains I and II, being negatively charged, repelled SWP. SWP was found to aggregate on hydration which was proposed to be due to hydrophobic interaction (chapter 3). Heating SWP, caused a reduction in hydrophobic interactions (chapter 3). It is proposed that the exposed hydrophobic groups on SWP would preferably bind helix (10) of domain III of BSA, because helix (10) is made up of hydrophobic groups and two lysine residues.

Urea behaves similarly to Gdn. except that, because it is not charged like Gdn, permeates the interior of protein molecules, occupying small cavities and somewhat perturbing the close-packed interior (Creighton, 1993). Treatment of BSA with urea caused an increase in the elastic modulus of BSA compared to the control (Table 5.3). This was due to the extra hydrophobic interactions made possible by urea denaturation. When urea was added in the presence of iodoacetate, the elastic modulus was considerably reduced. Iodoacetate blocks all the Cys-34 residues and prevents them from reacting as follows:

Cys-CH2-SH + ICH2COO-  ------------->  Cys-CH2-S-CH2COO- + HI
Cysteine Iodoacetate S-Carboxymethylcysteine Hydrogen iodide

The foregoing shows that the most important force in BSA gelation was the reaction of Cys-34. When all the disulphides bonds were reduced by mercaptoethanol and then allowed to reform through disulphide interchange reaction, a substantial 6 fold increase in the elastic modulus was observed (Table 5.3). This reaction completely destabilised the BSA structure and a network was formed through covalent polymerization similar to rubber elasticity. A temperature sweep of 12% (w/w) BSA solution or SWP treated with beta-mercaptoethanol showed a considerable 1000 fold increase in the elasticity of BSA as compared to SWP. The G' for BSA was independent of temperature, so it is possible that the thermoelastic inversion point had been reached. This is the point at which force will be essentially independent of temperature and thermal expansion and entropy contraction balance. SWP on the other hand gradually increased its elasticity up to 60°C and then started melting with increasing temperature (Figure 5.21).