INTERACTION OF SWP WITH BOVINE SERUM ALBUMIN (BSA)

Introduction

Serum albumin is one of the most widely studied proteins and is the most abundant protein in plasma with a typical concentration of 5g/100ml. Various researchers have studied the structure and properties of serum albumin and its interaction with other proteins in order to understand how serum albumin affects the functionality of foods in which they have been included as well as novel applications. The latter reason led to the study of the interaction between soluble wheat protein and bovine serum albumin. The structure and properties of SWP are described in chapters 2, 3 and 4. The following sections describe the structure and properties of BSA.

Bovine serum albumin (BSA)

Albumin is generally regarded to mean serum albumin or plasma albumin (care should be taken to distinguish albumen, which refers to egg white, from albumin or serum albumin). The word albumin is also used to describe a protein or a group of proteins defined by solubility in water for example the albumin fraction of wheat (chapter 2). Albumin is the most abundant protein in the circulatory system and contributes 80% to colloid osmotic blood pressure (Carter and Ho, 1994). It has now been determined that serum albumin is chiefly responsible for the maintenance of blood pH (Figge et al., 1991). In mammals albumin is synthesized initially as preproalbumin by the liver. After removal of the signal peptide, the resultant proalbumin is further processed by removal of the six-residue propeptide from the new N-terminus. The albumin released into circulation possesses a half-life of 19 days (Waldmann, 1977).

Structure of BSA

The substantial information on serum albumin has led to some contradictory results and discussions. Based largely on hydrodynamic experiments (Hughes, 1954; Squire et al., 1968; Wright and Thompson, 1975) and low-angle X-ray scattering (Bloomfield, 1966), serum albumin was postulated to be an oblate ellipsoid with dimensions of 140 × 40Å (Figure 5.1). Experiments have continued to support these dimensions ( Bendedouch and Chen, 1983; Feng et al. , 1988). Brown and Shockley (1982), compiled a diverse variety of data and constructed a model of albumin as having the shape of a cigar.

Figure 5.1: Classical preception of the structure of serum albumin (Peters, 1985).

However, studies using 1H NMR indicated that an oblate elipsoid structure of albumin was unlikely; rather a heart-shaped structure was proposed (Bos et al., 1989). This was in agreement with X-ray crystallographic data (Carter et al ., 1989). Previous studies indicated that the secondary structure contained about 68% - 50% alpha-helix and 16% -18% beta-sheet (Sjoholm and Ljungstedt, 1973; Reed et al., 1975; Foster, 1977). In contrast according to X-ray crystallography, there is no beta-sheet in the structure of native serum albumin (Carter et al., 1989). Riley and Arndt (1952, 1953) suggested that thermally denatured bovine serum albumin has probably the same fundamental type of folding of the polypeptide chains as the native one, which is 55% alpha-helix and 45% random conformation from X-ray scattering studies. Harmsen and Braam (1969), using infra-red and ORD spectra concluded that alkali or heat denaturation caused a partial loss of alpha-helical structure with no formation of beta-sheet but from their infra-red spectra, a shoulder appeared for BSA heated above 72°C at 1620 cm-1 indicative of beta-sheet formation. Clark et al. (1981b) testing Astbury's theory (Astbury et al., 1935), that gels formed from heat or chemically denatured proteins arose from the interaction of regions of beta-sheets, carried out tests on BSA using infra-red and Raman spectra. A shoulder was visible on their infra-red spectra at 1620 cm-1 for BSA heated to 75°C and above. The Raman spectra showed an increase at 1235 cm-1 and a decrease at 940 cm-1 for the BSA gel, indicating a drop in alpha-helix content with formation of beta-sheet. Lin and Koenig (1976) investigated heat, acid and alkali denaturation of BSA by Raman spectroscopy and found that heating to 70°C or a change in pH to below 5 or above 10 caused an increase of the 1246 cm-1 band and a decrease of the 938 cm-1 band. The interpretation was similar to those of Clark et al., (1981b) that is a decrease in the alpha-helix content accompanied by an increase in beta-sheet.

Amino acid composition

Albumins are characterized by a low content of tryptophan and methionine and a high content of cystine and the charged amino acids, aspartic and glutamic acids, lysine, and arginine. The glycine and isoleucine content of BSA are lower than in the average protein (Peters, 1985) (Table 5.1).

Table 5.1: Amino acid composition of BSA (Brown, 1975; Patterson and Geller, 1977; McGillivray et al., 1979; Reed et al., 1980; Hirayama et al., 1990).
Ala 48Cys 35Asp 41Glu 58
Phe 30Gly 17His 16Ile 15
Lys 60Leu 65Met 5Asn 14
Pro 28Gln 21Arg 26Ser 32
Thr 34Val 38Trp 3Tyr 21

Primary, secondary and tertiary structure

Figure 5.2 shows the bovine albumin amino acid sequence. The BSA molecule is made up of three homologous domains (I, II, III) which are divided into nine loops (L1-L9) by 17 disulphide bonds. The loops in each domain are made up of a sequence of large-small-large loops forming a triplet. Each domain in turn is the product of two subdomains (IA, IB, etc.). The primary structure of albumin is unusual among extracellular proteins in possessing a single sulfhydryl (Cys-34) group. In the light of new information i.e., x-ray crystallographic data (Carter and Ho, 1994) albumin structure is predominantly alpha-helical (67%) with the remaining polypeptide occurring in turns and extended or flexible regions between subdomains with no beta-sheets (Figure 5.3). Each of the domains can be divided into 10 helical segments, 1 - 6 for subdomain A and 7 - 10 for subdomain B (Figure 5.4). The motif for subdomain A is shown in Figure 5.5 and for subdomain B in Figure 5.6. Domains I and II and domains II and III are connected through helical extensions of 10 (I) - 1 (II) and 10 (II) - 1 (III), creating the two longest helices in albumin.

Disulphide bonds

In BSA the disulphide bonds are located in the following positions:

(1) 77-86; (2) 99-115; (3) 114-125; (4) 147-192; (5) 191-200; (6) 223-269; (7) 268-276; (8) 288-302; (9) 301-312; (10) 339-384; (11) 383-392; (12) 415-461; (13) 460-471; (14) 484-500; (15) 499-510; (16) 537-582; (17) 581-590.

The conformations of the disulphides are primarily gauche-gauche-gauche and Cß1-S1-S2-Cß2, with torsion angles clustering around ± 80°. The disulphide pairings are located almost exclusively between helical segments.


Figure 5.7: Location of disulphide bonds ( He and Carter, 1992)

None of the disulphide bonds was accessible to reducing agents in the pH range 5-7, but became progressively available as the pH was raised or lowered. Katchalski et al., (1957), therefore, concluded that the disulphides in albumin were protected at neutral pH from reducing agents. This is also apparent in the structure, which shows that the majority of disulphides are well protected and most are not readily accessible to solvent. Blocking of the free sulphydryl, Cys-34, with iodoacetamide, cysteine, or glutathione prevents the occurrence of mixed disulphides in aged albumin, as well as the occurrence of the albumin dimer (Peters, 1985). During unfolding, the conformation of some of the disulphide bonds change from the gauche-gauche-gauche to gauche-gauche-trans forms, as observed by laser Raman studies (Aoki et al., 1982).

Physical-Chemical Properties

The albumin molecule is not uniformly charged within the primary structure. At neutral pH, Peters (1985) calculated a net charge of -10, -8, and 0 for domains I, II, and III for bovine serum albumin. The surface charge distribution is shown in Figure 5.8.

AB

CD

Figure 5.8: Space filling model of serum albumin molecule with basic residues coloured in blue, acidic residues in red, and neutral ones in yellow. (A) Front view, (B) back view, (C) left side, and (D) right side (Carter and Ho, 1994).

Unlike the asymmetric charge distribution on the primary structure, the distribution on the tertiary structure seems fairly uniform.

Viscosity

The viscosity of a protein solution depends on its intrinsic characteristics, such as molecular mass, size, volume, shape, surface charge and ease of deformation (Bull, 1940; Yang, 1961). In addition, viscosity is influenced by environmental factors such as pH, temperature, ionic strength, ion type, shear conditions and heat treatment (Tung, 1978; Lee and Rha, 1979; Hermansson, 1979b). Serum albumin has been reported to have intrinsic viscosity values of 3.7-4.2 ml g-1 ( Peters, 1985; Kuntz and Kauzmann, 1974; Markus et al., 1964; Reynolds et al., 1967). Kolthoff et al., (1958) reported an increase in viscosity with increased cleavage of the disulphide bonds of BSA. The viscosity of solutions of BSA increased linearly with concentration up to 65 mg ml-1 and exponentially at higher concentrations ( Wetzel et al., 1980) consistent with the results of Menjivar and Rha, (1980).

Effects of pH

Serum albumin undergoes reversible conformational isomerization with changes in pH.

			E <-------> F <-------> N <-------> B <-------> A 

pH of transition:	     2.7         4.3	      8		  10
Name:		     Expanded	  Fast	      Normal	   Basic       Aged
% Helix:	        35	   45		55	    48		48

Figure 5.9: Relationship of isomeric forms of bovine serum albumin (Foster, 1977).

The N-F transition involves the unfolding of domain III ( Geisow and Beaven, 1977; Khan, 1986). The F form is characterized by a dramatic increase in viscosity, much lower solubility, and a significant loss in helical content ( Foster, 1960). At pH values lower than 4, albumin undergoes another expansion with a loss of the intra-domain helices (10) of domain I which is connected to helix (1) of domain II, and that of helix (10) of domain II connected to helix (1) of domain III (Figure 5.10). This expanded form is known as the (E) form which has an increased intrinsic viscosity, and a rise in the hydrodynamic axial ratio from about 4 to 9 (Harrington et al., 1956). At pH 9, albumin changes conformation to the basic form (B). If solutions of albumin are maintained at pH 9 and low ionic strength at 3°C for 3 to 4 days, another isomerization occurs which is known as the (A) form.

N formF form

E form

Figure 5.10: Ribbon diagram of serum albumin in its N form, and in its proposed F and E forms (Carter and Ho, 1994)

Effect of Heat

Serum albumin when heat-treated, goes through two structural stages. The first stage is reversible whilst the second stage is irreversible but does not necessarily result in a complete destruction of the ordered structure (Kuznetsow et al., 1975; Lin and Koenig, 1976; Oakes, 1976). Heating up to 65°C can be regarded as the first stage, with subsequent heating above that as the second stage (Wetzel et al., 1980). The onset temperature of conformation change as found by DSC was 58.1°C (Poole et al., 1987) and the temperature of denaturation 62°C (Ruegg et al., 1977). Results from CD and IR spectroscopy indicated that beta-sheets were formed when albumin was heated above 65°C ( Wetzel et al., 1980), 70°C (Lin and Koenig, 1976; Clark et al., 1981b). The beta-sheet formed was more pronounced on cooling and was concentration dependent. Wetzel et al., (1980) found a shoulder in the beta-sheet band at a concentration of 50mg/ml at 70°C, 1.4mg/ml at 80°C but not for 0.5mg/ml at 75°C. Because beta-sheet structures were not indicated in the dilute solution this suggests that the beta-sheets are intermolecular.

The sedimentation coefficient of albumin at neutral pH is around 4 - 4.5 S. If albumin samples are heated to more than 60°C and cooled down again, apart from the native protein, a faster sedimenting but heterogenous fraction with sedimentation coefficients of 26 - 36 S are found. The formation of these high molecular weight complexes was temperature and concentration dependent (Wetzel et al., 1980). Spin labelling Cys-34 (Hull et al., 1975; Wetzel et al., 1980) and fluorescence investigations proved Cys-34 to be located in a pocket. X-ray crystallography indicated that Cys-34 is located in a crevice on the surface of the protein and that the reactive sulphur is protected by several residues (Figure 5.11). Cys-34 has been found to be the most reactive sulfhydryl with a pKSH of 5 compared with 8.5 and 8.9 for cysteine and glutathione (Pedersen and Jacobsen, 1980). Blocking of Cys-34 with cysteine, glutathione, or other chemicals such as N-iodosuccinimide stabilised albumin against dimer formation (Peters, 1985). Electron spin resonance measurements indicated that the pocket around Cys-34 unfolded during thermal denaturation (Wetzel et al., 1980). Therefore, dimer formation during heating is probably due to disulphide bonding.

Thus, it may be concluded that, in the reversible structural stage, some of the alpha-helices are transformed to random coils. If the side chains of neighbouring residues of two peptide strands point in opposite directions and are perpendicular to a plane so that hydrogen bonds can form between the the strands, then IR, CD, and Raman spectroscopy will see them as beta-sheets. This means that aggregates are formed through the hydrogen bonding of beta-sheets between monomers. The beta-sheets formed are most likely to be antiparallel, since they are bound to be on the surface of the monomers. Parallel sheets are less twisted than antiparallel and are always buried. Antiparallel sheets can withstand greater distortions (twisting and beta-bulges) and greater exposure to solvent. As the temperature is increased past the reversible stage, unfolding of the pocket exposing Cys-34 takes place giving easy access to the formation of disulphide bridges. Since disulphide bridges are covalent bonds, this stage is irreversible.

Figure 5.11: Stereo ball-stick model of serum albumin structure at the region around residue Cys-34. Red, oxygen; yellow, carbon; blue, nitrogen; green, sulphur. (Carter and Ho, 1994)

Richardson and Ross-Murphy (1981) put forward a hypothesis from their model of two intersecting lines, that the two processes, unfolding and aggregation can be distinguished. Below 57°C, unfolding was rate-determining whereas above that temperature aggregation was rate-determining.

Functional properties of BSA

Foaming

Foaming may be defined as the creation and stabilization of gas bubbles in a liquid. Proteins diffuse to the air-water interface and reduce surface tension. At the interface they partially unfold and associate to produce an intermolecular cohesive film with some degree of elasticity. Foam expansion and stability improved when BSA interacted with basic proteins like lysozyme and clupeine (Poole et al., 1984) due to cross-links formed between BSA and lysozyme at the interface. BSA on its own performs best near its isoelectric point when electrostatic repulsion is at its minimum. When it interacts with lysozyme, the greatest expansion and stability was found between pH 8 and 9, which is between the isoelectric point of BSA (4.7) and lysozyme (10.7) when the proteins are oppositely charged. Clupeine with a pI at 12 was found to be more effective than lysozyme. Lipids inhibit foaming by displacing protein molecules from the air-liquid interface and by disrupting the integrity of the protein film (Ross, 1950). This lipid inhibition was counteracted when BSA interacted with clupeine at the air-liquid interface ( Poole et al., 1986).

Gelation properties of BSA

BSA when heated formed soluble aggregates through disulphide and noncovalent bonds. Alpha-Lactalbumin, on the other hand does not form soluble aggregates on its own but interacts with BSA through disulphide interchange to form soluble aggregates (Matsudomi et al., 1993). Soluble aggregates of polymerized molecules were formed during the early stages of heat-induced gelation of proteins, and subsequent polymerization resulted in the formation of a rigid gel network ( Nakamura et al., 1986; Kitabatake et al., 1989). The addition of alpha-lactalbumin to BSA reduced the gelling ability of BSA, as measured by the complex modulus G* (Paulsson et al., 1986).

Gelation according to Ferry (1948) is a two step mechanism. An initiation step involving unfolding or dissociation of the protein molecules, followed by an aggregation step in which association or aggregation reactions occur, resulting in gel formation under appropriate conditions. For the formation of a highly ordered gel, it is essential that the aggregation step proceed at a slower rate than the unfolding step (Hermansson, 1978; 1979a). The denaturation temperature of BSA by differential thermal analysis (DTA) was found to be 64°C (Itoh et al., 1976) and 62°C by differential scanning calorimetry ( Ruegg et al., 1977). The denaturation temperature of BSA increased when it binds to fatty acids (Bernal and Jelen, 1985) but reduced when it interacts with clupeine (Poole et al., 1987). Therefore it appears that the interaction of one biopolymer eg. fatty acid to BSA led to stability of the BSA molecule, whereas another molecule eg. clupeine interacted and facilitated the initiation of unfolding of BSA.

Ligand-binding

Perhaps, the most outstanding property of albumin is its ability to bind reversibly an incredible variety of ligands (Goodman, 1958; Daughaday, 1959; Yates and Urguhart, 1962; Jacobsen, 1969; Klopfenstein, 1969; Burke et al., 1971; Unger, 1972; Westphal and Harding, 1973; Beaven et al., 1974; Jacobsen, 1977; Richardson et al., 1977; Spector and Fletcher, 1978; Brodersen, 1979; Adams and Berman, 1980; Savu et al., 1981; Roda et al., 1982). BSA is the principal carrier of fatty acids that are otherwise insoluble in circulating plasma. It also performs many other functions such as, sequestering oxygen free radicals and inactivating various toxic lipophilic metabolites such as bilirubin (Emerson, 1989). Albumin has a high affinity for fatty acids, hematin, bilirubin and a broad affinity for small negatively charged aromatic compounds. It forms covalent adducts with pyridoxyl phosphate, cysteine, glutathione, and various metals, such as Cu (II), Ni (II), Hg (II), Ag (II), and Au (I). As a multifunctional transport protein, albumin is the key carrier or reservoir of nitric oxide, which has been implicated in a number of important physiological processes, including neurotransmission (Stamler et al., 1992). It also belongs to a multigene family of proteins that include alpha-fetoprotein (AFP) and vitamin D-binding protein (VDP), which is also known as G complement (GC) protein. Although AFP is considered the fetal counterpart of albumin, its binding properties are distinct and it has been suggested that AFP may have higher affinity for some unknown ligands important for fetal development. VDP plays an important role in calcium regulation. ADP and VDP both interact with the class II major histocompatibility complex suggesting that these proteins may play an important role in modulating the immune system (van Oers et al., 1989). In circulating plasma approximately 30% of free sulphydryl, Cys-34, is oxidized by cysteine and glutathione (Peters, 1985).