Studies in Surface Science and Catalysis 142 R. Aiello, G. Giordano and F. Testa (Editors) 9 2002 Elsevier Science B.V. All rights reserved.
Mesoporous molecular sieves for albumin A.Y.Eltekov and N.A.Eltekova Institute Physical Chemistry Russian Academy of Sciences, Leninsky prospect 31, 119991 Moscow, Russia In presented work we studied the adsorption of bovine serum albumin (BSA) from aqueous solutions by silica gels, silochrom and porous glasses at 293 and 313 K in order to estimate the sizes of self- organized structures of protein macromolecules in bulk solution and also the temperature effect on the adsorption behavior of protein macromolecules. 1. INTRODUCTION Adsorption study of biopolymers and proteins by porous silicas is of considerable interest for possibility to estimate the characteristics of porous structure of silicas and to determine the self- organized structure ofbiopolymers and proteins in bulk solution [1, 2]. The characteristics of the adsorption of biopolymers from aqueous solutions and the adsorption of synthetic polymers such as polystyrenes, polyolefins, polyacrylates are crucial dependent on the type of the monomer unit of the macromolecular chain and the temperature. Unlike the adsorption of synthetic nonelectrolyte polymers, the adsorption of biopolymers and proteins strongly depends on the pH value and the ionic strength of the aqueous solution [2 - 7]. Many biopolymers, including proteins, form ternary and quaternary self- organized aggregates in aqueous solutions [2, 4, 6, 8]. 2. THEORY
The equation for the description of equilibrium in liquid phase physical adsorption systems was used in following form  nff = nmJ3(k-1)a(1-a)/[ l+(j3k- 1)a]
where na is excess adsorption value (by Gibbs) in mg/m2, nm - is limited value of total content for adsorbed protein, 13 is displacement coefficient, k is the constant of adsorption equilibrium and a is the protein activity in water solutions. The equation (1) differs from Semenchenko equation  by 13coefficient. The Henry constant equation
1538 K H = nO/a
(2) for extremely dilute solution (when a ~ 0) has following form K H = nml3(k- 1)
(3) Practically, the excess (by Gibbs) adsorption na was calculated by the formula n a = (C o - C) V / m S
(4) where C O and C are the initial and equilibrium concentrations of the aqueous protein solution, respectively, V is the volume of the aqueous protein solution and m and S are the mass and specific surface area of silica sorbent. 3. EXPERIMENTAL In this work we studied the adsorption of bovine serum albumin (BSA) from aqueous solutions by silica gels, silochrom and porous glasses. The experiments were performed at 293 and 313 K in order to estimate the sizes of self-organized structures of protein macromolecules in bulk solutions and also the temperature effect on the adsorption behavior of protein macromolecules.
3.1. Samples We used globular protein - bovine serum albumin (BSA) (Serva, Germany) without additional treatment. Twice distilled water was used as the solvent. Table 1 summarizes the characteristics of protein. Table 1 Characteristics of BSA macromolecules: molecular mass M (Da), globule size d (nm), isoeletric point IP (pH) Protein
The samples of silica gel KSM, KSK2 and SO95 (GOB VNIINP, Nizhnii Novgorod, Russia), silochrom $80 (Luminofor, Stavropol, Russia) and porous glasses PS (GOB VNIINP, Nizhnii Novgorod, Russia) were used as adsorbents. The samples of PS 20 and PS30 were prepared at the laboratory headed by S.P.Zhdanov in the Institute of Silicate Chemistry RAN in St.Petersburg. The characteristics of the silica samples are given in Table 2.
1539 Table 2 Characteristics of silicas: specific surface area S (m2/g), pore diameter dp (nm), pore volume Vp (cm3/g) Silica
The adsorption experimental procedure was described in detail elsewhere [8, 9]. For adsorption study 0.1 - 0.5 g of silica sample (preliminary dried in a vacuum camera at 373 K for 2 h) and 5 ml of aqueous protein solution of a desired concentration were mixed in 10 ml ampoules. The ampoules were sealed and kept in a thermostat at a constant temperature until the adsorption equilibrium was established. The concentration of aqueous protein solution before and after adsorption was measured on an laboratory liquid interferometer. 4. RESULTS AND DISCUSSION
There are four levels of self- organization of protein macromolecules. The primary protein structure is determined by the chemical bonding (via covalent peptide bonds) of the amino acids in the protein molecule. The secondary (helical) structure is controlled by the self- organization of the polypeptide network under the action of hydrogen bonding between the peptide groups. The ternary and quaternary structures are governed by the configurations of the linear and helical sections of the protein chains (this type of selforganization is governed by the intermolecular interaction between the linear and helical sections of the protein chain). 4.1. Concentration effect
Table 3 shows the dependences (isotherms) of BSA adsorption values (n6) for three samples of porous glass on equilibrium concentration of BSA in aqueous solutions (C). The
1540 adsorption values refer to unit surface area that permits the estimation of the effect of the specific surface area of the sorbents. Table 3 Experimental values of BSA adsorption (n~ mg/m2) on silica sorbents from aqueous solutions at 293 K Silica
BSA concentration (C), m~/ml 1
Note that the adsorption values are largely determined by the size of pores in the porous glass and by self- organized structure ofBSA macromolecules in aqueous solution. The excess adsorption of BSA from aqueous solutions on the negatively charged surface of three porous glasses is positive and increases with the increasing of the pore size in the adsorbents. Sieve effects were observed for the interaction of high molecular polystyrene molecules on mesoporous silicas [2, 8, 9]. The adsorption values of BSA on all three porous glasses in the range of equilibrium concentration from 3 to 6 mg/ml are practically independed of the equilibrium concentration. This suggests that the accessible part of the surface of the porous glass is saturated with protein. The adsorption of proteins from dilute aqueous solutions on the surface of solids has been examined in detail in many works [ 10 - 18]. It was found that electrostatic forces essentially influence the adsorption interaction between charged protein macromolecules and a charged adsorbent surface. Protein macromolecules, as a whole, are hydrophilic in aqueous media; however, they contain hydrophobic sites. Electric charged at the surface are neutralized by counterions from the ambient aqueous solution, resulting in the formation of an electric double layer. When a protein macromolecule approaches the surface, the electric double layers can overlap with each other. As a result, the electric charges are reversibly redistributed, and ions from the bulk solutions penetrate into the adsorption protein layer. The transport of ions is accompanied by variations in the thermodynamic functions. It was experimentally found  that a moderate number of ions from the solution were sufficient to neutralize the charge at the points of contact of a protein molecule and the adsorbent surface. During adsorption, the degrees of hydration of the protein macromolecule and the adsorbent surface may change [10 - 13]. As a result, the hydrophilic parts of the macromolecule and the sorbent surface retain their initial states, whereas the hydrophobic sites are dehydrated, thereby promoting the hydrophobic interaction between the protein and the adsorbent surface. The process of dehydration results in a significant decrease in the Gibbs free energy of the adsorption system.
4.2. Adsorption constant Table 4 shows the comparison of BSA adsorption parameters from equation (1) and (2) for three porous glasses. Table 4 BSA adsorption parameters: limited adsorption value n m (mg/m2), constant of adsorption equilibrium 13k and Henry constant K H (ml/m2) Adsorbent
The results in table 4 show good correlation the adsorption parameter n m with the experimental adsorption values for all three porous glasses (see table 3). Note that the values of adsorption equilibrium constant 13k are close for all three porous glasses. Hence the constant of adsorption equilibrium for BSA-water-porous glass systems does not strongly depend on a pore size of adsorbents. On the contrary the values of K H are more sensitive to the pore structure of adsorbents and change in the same manner. The migration of protein macromolecules from the bulk solution to the liquid-solid interface can be accompanied by a rearrangement of its structure [14, 15]. In a bulk solution, or far from the region of action of adsorption forces, hydrophobic parts of the protein are hidden in the interior of the macromolecule. Intramolecular hydrophobic interactions keep the secondary structure of the protein in the form of helices and layers. Near the adsorbent surface, the hydrophobic sections of the macromolecule tend to approach hydrophobic sites at the adsorbent surface. When the intramolecular hydrophobic interactions in a protein macromolecule are replaced by intermolecular hydrophobic interactions, the secondary protein structure may change with an increase in the conformation entropy of the protein molecule. Using Fourier-transform IR spectroscopy [ 10] and evanescent fluorescence spectroscopy [ 11 ] has been found that the adsorption of a protein was accompanied by changes in its secondary structure.
4.3. Temperature effect Table 5 shows the dependences of the maximum values of the excess adsorption for BSA from aqueous solutions by porous silica samples at 293 and 313 K (isopycns). Table 5 Maximum amounts of BSA adsorption (mg/m2) on silica sorbents from aqueous solutions at 293 and 313 K Silica
K S K 2 PS20 PS30 PS40 $80
PS70 SO95 PS120 PS160
1542 Note that the maximum adsorption values are largely determined by the size of pores in the porous silicas. The maximum values of the excess adsorption of BSA from aqueous solutions increase with an increase of pore size of silica adsorbents. The fact that the excess adsorption of BSA from aqueous solutions on the porous silica KSM at 293 K is negative can be explained by the electrostatic repulsion of protein molecules and by the sieve effect (negatively charged solvated BSA macromolecules, whose average at 293 K size exceeds 12 nm, cannot penetrate into pores of KSM silica sample). Because of this, the pores accumulate water molecules, which leads to an increase in the concentration of BSA macromolecules in the bulk solution. Sieve effects were observed for the interaction of high molecular polystyrene molecules on mesoporous silicas [8, 9]. The fact that the isopycns level off to form a plateau is indicative of the formation of dense adsorption layers at the hydroxylated surface of silica. These layers may be as thick as 1.0 nm. At 293 K and pH 7.0, the maximum adsorption values for BSA on silicas with pores larger than 40 nm in diameter are similar, 1.1 - 1.2 mg/m2. At 313 K and pH 7.0, the maximum adsorption values for BSA on silicas with pores larger than 40 nm in diameter are similar, 1.15 - 1.18 mg/m 2, and the second plateau of maximum adsorption values on silicas with pores larger than 70 nm in diameter are similar, 1.4 - 1.5 mg/m2. Obviously the adsorption of BSA from aqueous solutions by silicas is accompanied by changes in protein self-organized structure in bulk solution under a high temperature. A protein molecule is attached to the adsorption surface by different forces. Since proteins are polyelectrolytes, they carry both positive and negative charges. The positive charges are thought to be placed at the NH+3 groups in the peptide chain, while the negative charges are located at the COO- groups. Because of this the electrostatic interaction primarily manifests itself during the adsorption of the protein at hydrophilic areas. When a protein molecule interacts with a hydrophilic surface, carrying a charge of the same sing, electrostatic repulsion arises. The hydrophobic (dispersion) interaction of proteins is observed during their adsorption on the hydrophobic surface of carbonaceous sorbents or hydrophobized silicas. However, loose protein molecules can be adsorbed on a hydrophilic surface carrying a charge of the same sing as the protein molecule despite the action of electrostatic repulsion [ 16, 17]. 4.4. Pore size effect
When protein macromolecules are sorbed on a porous silica, they are accumulated near pore openings. The rate of diffusion of protein macromolecules in the bulk solution is small, but the rate of their diffusion into pores, whose size is comparable with the diameter of solvated macromolecules, is still lower because of steric hindrances. The time required to attain the state of equilibrium for the adsorption ofBSA macromolecules on silochrom $80 is 4 h, the analogous characteristic for PS20 is above 80 h. The accumulation of protein macromolecules near pore openings may be accompanied by their association. This means that, namely, associates of protein macromolecules penetrate into pores. This phenomenon is not observed for nonporous and macroporous adsorbents. Another factor influencing the dependence of the adsorption value on the pore size is the localization of strongly adsorbed protein macromolecules at openings leading inside
1543 the pore system at the early stage of the interaction between the protein and adsorbent. For example, the localization of protein macromolecules near pore openings results in their narrowing by the thickness of the adsorption layer. A similar effect was observed  for separation of BSA and lysozime on silica gel with pores 28 nm in diameter. As a result BSA is eluted from the column before lysozime, whose molecules are smaller and can freely penetrate into the adsorbent pores. The diffusion ofBSA macromolecules near silica adsorbent grains slows down. The electrostatic charge of the hydrophilic silica surface (zero-charge point at pH 2 - 3 ) is responsible for the repulsion of BSA macromolecules (isoelectric point is pH 4.7 ). However, the rearrangement of the structure of protein macromolecule occurs under the action of hydrophobic forces. Due to the availability of hydrophilic sites, BSA macromolecules are strongly adsorbed near pore openings, resulting in a significant decrease in the pore size ( by the thickness of the adsorption layer comprised of firmly bound BSA macromolecules). Thus, negatively charges BSA macromolecules are sorbed spontaneously (at pH 7.0) on negatively charged areas of the hydrophilic silica surface. The adsorption occurs under the action of two forces: hydrophobic attraction (dispersion forces) and the electrostatic repulsion between likely charged molecules and the adsorbent surface [ 17, 18]. The results of Table 5 suggest that in dilute aqueous solutions (C = 2 - 8 mg/ml) at 293 K BSA macromolecules have self-organized structures which are 25 nm in size and at 313 K BSA macromolecules have self- organized structures which are 25 nm and 65 nm. This suggests that BSA macromolecules exist in aqueous solutions in the form of associates and solvated self- organized structures because X-ray analysis shows that the sizes of these molecules are 7 nm. Thus the results obtained it possible estimate the sizes of BSA macromolecules in dilute aqueous solutions and the thickness the adsorption layers composed of unfolded BSA molecules at the silica surface. 5. CONCLUSION Studying the adsorption of BSA on the surface of uniformly porous silica from aqueous solutions made it possible to determine the influence of the porous structure on the self-organization of adsorption macromolecules. A comparison of the apparent sizes of the protein macromolecules calculated from the adsorption isotherms and the XRD sizes suggests that adsorption layers strongly (irreversibly) bound to the surface near pore openings are formed. These layers hinder or completely block the migration of macromolecules into cavities smaller than 20 nm for BSA. The self-organization of the adsorption layers may be due to hydrate interactions of the hydrophobic groups of the macromolecules and electrostatic interactions leading to the formation of an electric double layer. The effective sizes of the macromolecules calculated without due regard for the thickness of the adsorption layers and electrostatic repulsion may be overestimated by a factor of 2 - 3. The contribution from the association of the protein molecules accumulated near pore openings in the adsorbent was considered.
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