- Email: [email protected]
Protein adsorption on silica molecular sieves
Zeolites and Related Materials: Trends, Targets and Challenges Proceedings of 4th International FEZA Conference A. Gédéon, P. Massiani and F. Babonneau (Editors) © 2008 Elsevier B.V. All rights reserved.
611
Protein adsorption on silica molecular sieves Nina A. Eltekova,a Anton Yu. Eltekovb a
Institute Physical Chemistry and Electrochemistry RAS, Leninsky pr. 31, Moscow 119991, Russia b Stranski Laboratory, Technical University Berlin, Strasse des 17 Juni 124, Berlin 10623, Germany
Abstract The adsorption of albumin macromolecules on silica molecular sieves with different pore sizes has been tested in aqueous solutions with neutral pH by batch method at two temperatures. The sizes of protein aggregate in the bulk solution and the temperature effect on the adsorption behavior of protein macromolecules have been obtained from experimental data, suggesting that size-exclusive adsorption takes place. The confinement effect of porous network and its influence on adsorption of BSA at solidliquid interface has been discussed. Keywords: adsorption, BSA, silica, interface
1. Introduction Bovine serum albumin (BSA) attracts much attention of scientists due to its wide application as a model substance for studying the physical and biological aspects of the adsorption of a protein at solid surface and a series of the studies have been made at the hydrophilic silica-water open interface [1 – 10]. BSA is also one of a number of proteins which use to development of biochemical sensors, where the controlling of total adsorbed amount plays a crucial role [7, 8]. Here we present our results obtained from adsorption experiment of BSA on several silica adsorbents with different pore sizes at two temperatures. In our previous study we found the influence of molecular masses and structure of albumin and -globulin macromolecules on adsorption in porous media [4, 6]. In present work we extended these measurements to examine the influence of temperature and confinement effect on BSA adsorption.
2. Experimental 2.1. Chemicals The sample of BSA with molecular mass 67 000 was free of fatty acid and was used as supplied (Serva, Germany). The BSA molecule has an ellipsoid shape of 4 x 12 nm in a folded form [2]. Its isoelectric point is about 4.7. Deionized water was purified by a Milli-Q water purification system (Millipore, Bedford, USA) and was used as a solvent. Ten samples of domestic silica adsorbents with different pore diameters: silica gels KSM, KSK2, SO95, porous glasses PS (VNIINP, Nizhniy Novgorod, Russia) and silochrom S80 (Luminofor, Stavropol, Russia) were used as molecular sieves. Silica sieves were characterized by nitrogen adsorption at 77 K using Gemini 2375 volumetric analyzer (Micrometrics, USA). The BET specific surface area and pore size determined by BJH method for silicas were summarized in Table 1. All adsorbents preliminary were dried in a vacuum camera at 373 K for 120 min.
612
N.A. Eltekova and A.Yu. Eltekov
Table 1. Basic parameters of silica molecular sieves: specific surface area diameter d p (nm) and pore volume V p (cm3 g-1)
S
(m2 g-1), pore
Silica
KSM
KSK2
PS20
PS30
PS40
S80
PS70
SO95
PS120
PS160
S dp Vp
520
340
74
50
100
100
45
24
30
23
3
14
20
30
41
55
70
80
120
160
0.6
1.2
0.7
1.2
1.6
1.3
1.5
0.7
1.5
1.5
2.2. Batch Method For adsorption study 0.1-0.5 g of silica and 5 ml of aqueous protein solution at desired concentration were mixed in ampoules. The ampoules were sealed and kept in a thermostat at constant temperature within 24 hours. The concentration of aqueous protein solution before and after adsorption was measured by laboratory liquid interferometer LIR-1 (LOMO, St-Peterburg, Russia). The uncertainty in measurement of adsorption values was 3-5%.
3. Results and Discussion 3.1. Constant of adsorption equilibrium The isotherms of BSA adsorption from aqueous solutions on porous glasses with different pore sizes were shown on Fig. 1-A. The isotherm equation for the description of equilibrium in liquid phase physical adsorption systems was used in following form [4]
nσ =
nm β (k − 1)a(1 − a) 1 + ( βk − 1)a
where n
σ
(1)
is the excess adsorption value (by Gibbs), n m is limited total amount of
adsorbed protein, β is displacement coefficient, k is the constant of adsorption equilibrium and a is the protein activity in water solutions. The Henry constant of isotherm equation for BSA adsorption from solutions has the following expression
K H = nm β (k − 1)
(2)
This expression is corrected at the condition that the activity a → 0 and that so the activity value a << βk that the product a ( βk − 1) → 0 . The values of n m and K H are more sensitive to the pore size of porous glasses in comparison with the values of
βk
as evident from Table 2.
613
Protein adsorption on silica molecular sieves
Table 2. Constants of Eq. (1) and Henry constant K H of Eq. (2) for BSA adsorption Silica
n m , mg m-2
βk
K H , ml m-2
PS160
1.15
1300
1.47
PS30
0.72
1100
0.85
PS20
0.18
1100
0.21
The degradation of values n m and K H with the narrowing of pores in molecular sieves is due to the great reducing of the amount of accessible pores for BSA and hence the reducing of accessible surface area of silicas. 3.2. Effects of pore size and temperature As can be seen from Fig.1-B the isopycns increase with an increase of silica pore diameters from 15 to 80 nm. The initial part of the dependence ( d p < 15 nm) for σ
which n max is close to zero indicates that the protein can not enter into an adsorbent.
A
1,5
B 2
3 1,0
, mg m
-2
n , mg m
-2
1
2
σ
max
σ
1,0
n
0,5
0,5
1 0,0
0,0 0
5
c, mg ml
10 -1
0
50
100
150
dp, nm
Figure 1. . A - The adsorption isotherms of BSA from aqueous solutions on porous glasses PS20 (1), PS30 (2) and PS160 (3) at 293 K. Vertical dot line is the isopycn for BSA adsorption at equilibrium concentration C = 7 mg ml-1. B - The isopycns of BSA adsorption for silica molecular sieves at 293 (1) and 313 K (2). σ
The constancy of the value n max for silica sorbents with d p > 80 nm is due to equal accessibility of adsorbed volume on the silica surface or to the monolayer capacity. Each isopycn forms a plateau due to formation of dense adsorption layer on the hydroxylated surface of silica. This layer may be a nanometer thick. At 293 K the σ
plateau of n max values for BSA is within 1.1-1.2 mg m-2. At 313 K the first plateau of
614
N.A. Eltekova and A.Yu. Eltekov
σ σ values for BSA is within 1.15-1.18 mg m-2, and the second plateau of n max nmax
values is within 1.4-1.5 mg m-2. The step on isopycn at 313 K can be explained in terms of BSA dimer-shaped structures which occur in bulk solution. Obviously, under a high temperature the adsorption of BSA from aqueous solutions by silicas is accompanied by changes in protein aggregates in bulk solution. The increase of temperature results in folded BSA due to increase the role of hydrophobic interactions in adsorption. It is easy to see from the figure, for both temperatures the maximum adsorbed amount grows along increasing of silica pore diameter. This indicates that BSA is adsorbed more effective by wide-porous solids and sieve effect takes place at the narrowing of pore size. However, at dp > 80 nm the maximum adsorbed amount does not significantly influenced by pore structure. Here, the enhanced adsorption of BSA can be achieved by increasing of the temperature. We suggest that the reason of adsorption enhancement at higher temperature is that BSA molecules undergo major dehydration with increasing of the surrounding temperature. At the same time the adsorption of water molecule reduced strongly by increasing temperature. The isopycn shifts to the left with increase of the temperature in conformity with the reducing of BSA aggregate size at high temperature. The reducing of hydrated BSA aggregate size at 313 K promotes theirs penetration into more narrow pores. The step on isopycn at d p = 40 nm demonstrates the possibility of the existence of BSA dimer in the bulk solution. Thus isopycn allows evaluating the fraction of dimeric BSA structure in 10 and 20% at 293 and 313 K, respectively.
4. Conclusion The interaction process of diluted solution of BSA with porous silica samples at 293 and 313 K has been satisfactorily described by the adsorption equilibrium isotherm equation for the monolayer model. The decrease of the calculated values of limited amount of adsorbed BSA with the diminishing of pore diameter of silica samples has indicated the molecular sieves effect development. The isopycn for BSA adsorption on silica pore diameters has allowed estimating of BSA aggregates in bulk solution at 293 and 313 K. The analysis of obtained results indicates that the temperature increase in the adsorption system apparently promotes the reducing of protein aggregate sizes and the relaxing of H-bond in the hydrate cover of protein aggregates.
References [1] [2] [3] [4] [5] [6] [7]
W. Norde and J. Lyklema, J. Colloid Interface Sci. 66 (1978) 277. S. Kondo, E. Amano and M. Kurimoto, Pure Appl. Chem. 61 (1989) 1897. T. J. Su, J. R. Lu, R. K. Thomas, Z. F.Cui and J. Penfold, J. Phys. Chem. B 102 (1998) 8100. N. A. Eltekova and A. Yu. Eltekov, Rus. J. Phys. Chem. A 74 (2000) 958. C. E.Giacomelli and W. Norde, J. Colloid Interface Sci. 233 (2001) 234. A. Yu. Eltekov and N. A. Eltekova, Rus. J. Phys. Chem. A 77 (2003) 1134. C. W. Suh, M.Y. Kim, J. B. Choo, J. K. Kim, H. K. Kim and E. K. Lee, J. Biotechnol. 112 (2004) 267. [8] S. J. McClellan and E. I. Franses, Colloids Surf. A: Physicochem. Eng. Asp. 260 (2005) 265. [9] H. Larsericsdotter, S. Oscarsson and J. Buijs, J. Colloid Interface Sci. 289 (2005) 26. [10] K. C. Kwok, K. M.Yeung and N. H. Cheung, Langmuir 23 (2007) 1948.
611
Protein adsorption on silica molecular sieves Nina A. Eltekova,a Anton Yu. Eltekovb a
Institute Physical Chemistry and Electrochemistry RAS, Leninsky pr. 31, Moscow 119991, Russia b Stranski Laboratory, Technical University Berlin, Strasse des 17 Juni 124, Berlin 10623, Germany
Abstract The adsorption of albumin macromolecules on silica molecular sieves with different pore sizes has been tested in aqueous solutions with neutral pH by batch method at two temperatures. The sizes of protein aggregate in the bulk solution and the temperature effect on the adsorption behavior of protein macromolecules have been obtained from experimental data, suggesting that size-exclusive adsorption takes place. The confinement effect of porous network and its influence on adsorption of BSA at solidliquid interface has been discussed. Keywords: adsorption, BSA, silica, interface
1. Introduction Bovine serum albumin (BSA) attracts much attention of scientists due to its wide application as a model substance for studying the physical and biological aspects of the adsorption of a protein at solid surface and a series of the studies have been made at the hydrophilic silica-water open interface [1 – 10]. BSA is also one of a number of proteins which use to development of biochemical sensors, where the controlling of total adsorbed amount plays a crucial role [7, 8]. Here we present our results obtained from adsorption experiment of BSA on several silica adsorbents with different pore sizes at two temperatures. In our previous study we found the influence of molecular masses and structure of albumin and -globulin macromolecules on adsorption in porous media [4, 6]. In present work we extended these measurements to examine the influence of temperature and confinement effect on BSA adsorption.
2. Experimental 2.1. Chemicals The sample of BSA with molecular mass 67 000 was free of fatty acid and was used as supplied (Serva, Germany). The BSA molecule has an ellipsoid shape of 4 x 12 nm in a folded form [2]. Its isoelectric point is about 4.7. Deionized water was purified by a Milli-Q water purification system (Millipore, Bedford, USA) and was used as a solvent. Ten samples of domestic silica adsorbents with different pore diameters: silica gels KSM, KSK2, SO95, porous glasses PS (VNIINP, Nizhniy Novgorod, Russia) and silochrom S80 (Luminofor, Stavropol, Russia) were used as molecular sieves. Silica sieves were characterized by nitrogen adsorption at 77 K using Gemini 2375 volumetric analyzer (Micrometrics, USA). The BET specific surface area and pore size determined by BJH method for silicas were summarized in Table 1. All adsorbents preliminary were dried in a vacuum camera at 373 K for 120 min.
612
N.A. Eltekova and A.Yu. Eltekov
Table 1. Basic parameters of silica molecular sieves: specific surface area diameter d p (nm) and pore volume V p (cm3 g-1)
S
(m2 g-1), pore
Silica
KSM
KSK2
PS20
PS30
PS40
S80
PS70
SO95
PS120
PS160
S dp Vp
520
340
74
50
100
100
45
24
30
23
3
14
20
30
41
55
70
80
120
160
0.6
1.2
0.7
1.2
1.6
1.3
1.5
0.7
1.5
1.5
2.2. Batch Method For adsorption study 0.1-0.5 g of silica and 5 ml of aqueous protein solution at desired concentration were mixed in ampoules. The ampoules were sealed and kept in a thermostat at constant temperature within 24 hours. The concentration of aqueous protein solution before and after adsorption was measured by laboratory liquid interferometer LIR-1 (LOMO, St-Peterburg, Russia). The uncertainty in measurement of adsorption values was 3-5%.
3. Results and Discussion 3.1. Constant of adsorption equilibrium The isotherms of BSA adsorption from aqueous solutions on porous glasses with different pore sizes were shown on Fig. 1-A. The isotherm equation for the description of equilibrium in liquid phase physical adsorption systems was used in following form [4]
nσ =
nm β (k − 1)a(1 − a) 1 + ( βk − 1)a
where n
σ
(1)
is the excess adsorption value (by Gibbs), n m is limited total amount of
adsorbed protein, β is displacement coefficient, k is the constant of adsorption equilibrium and a is the protein activity in water solutions. The Henry constant of isotherm equation for BSA adsorption from solutions has the following expression
K H = nm β (k − 1)
(2)
This expression is corrected at the condition that the activity a → 0 and that so the activity value a << βk that the product a ( βk − 1) → 0 . The values of n m and K H are more sensitive to the pore size of porous glasses in comparison with the values of
βk
as evident from Table 2.
613
Protein adsorption on silica molecular sieves
Table 2. Constants of Eq. (1) and Henry constant K H of Eq. (2) for BSA adsorption Silica
n m , mg m-2
βk
K H , ml m-2
PS160
1.15
1300
1.47
PS30
0.72
1100
0.85
PS20
0.18
1100
0.21
The degradation of values n m and K H with the narrowing of pores in molecular sieves is due to the great reducing of the amount of accessible pores for BSA and hence the reducing of accessible surface area of silicas. 3.2. Effects of pore size and temperature As can be seen from Fig.1-B the isopycns increase with an increase of silica pore diameters from 15 to 80 nm. The initial part of the dependence ( d p < 15 nm) for σ
which n max is close to zero indicates that the protein can not enter into an adsorbent.
A
1,5
B 2
3 1,0
, mg m
-2
n , mg m
-2
1
2
σ
max
σ
1,0
n
0,5
0,5
1 0,0
0,0 0
5
c, mg ml
10 -1
0
50
100
150
dp, nm
Figure 1. . A - The adsorption isotherms of BSA from aqueous solutions on porous glasses PS20 (1), PS30 (2) and PS160 (3) at 293 K. Vertical dot line is the isopycn for BSA adsorption at equilibrium concentration C = 7 mg ml-1. B - The isopycns of BSA adsorption for silica molecular sieves at 293 (1) and 313 K (2). σ
The constancy of the value n max for silica sorbents with d p > 80 nm is due to equal accessibility of adsorbed volume on the silica surface or to the monolayer capacity. Each isopycn forms a plateau due to formation of dense adsorption layer on the hydroxylated surface of silica. This layer may be a nanometer thick. At 293 K the σ
plateau of n max values for BSA is within 1.1-1.2 mg m-2. At 313 K the first plateau of
614
N.A. Eltekova and A.Yu. Eltekov
σ σ values for BSA is within 1.15-1.18 mg m-2, and the second plateau of n max nmax
values is within 1.4-1.5 mg m-2. The step on isopycn at 313 K can be explained in terms of BSA dimer-shaped structures which occur in bulk solution. Obviously, under a high temperature the adsorption of BSA from aqueous solutions by silicas is accompanied by changes in protein aggregates in bulk solution. The increase of temperature results in folded BSA due to increase the role of hydrophobic interactions in adsorption. It is easy to see from the figure, for both temperatures the maximum adsorbed amount grows along increasing of silica pore diameter. This indicates that BSA is adsorbed more effective by wide-porous solids and sieve effect takes place at the narrowing of pore size. However, at dp > 80 nm the maximum adsorbed amount does not significantly influenced by pore structure. Here, the enhanced adsorption of BSA can be achieved by increasing of the temperature. We suggest that the reason of adsorption enhancement at higher temperature is that BSA molecules undergo major dehydration with increasing of the surrounding temperature. At the same time the adsorption of water molecule reduced strongly by increasing temperature. The isopycn shifts to the left with increase of the temperature in conformity with the reducing of BSA aggregate size at high temperature. The reducing of hydrated BSA aggregate size at 313 K promotes theirs penetration into more narrow pores. The step on isopycn at d p = 40 nm demonstrates the possibility of the existence of BSA dimer in the bulk solution. Thus isopycn allows evaluating the fraction of dimeric BSA structure in 10 and 20% at 293 and 313 K, respectively.
4. Conclusion The interaction process of diluted solution of BSA with porous silica samples at 293 and 313 K has been satisfactorily described by the adsorption equilibrium isotherm equation for the monolayer model. The decrease of the calculated values of limited amount of adsorbed BSA with the diminishing of pore diameter of silica samples has indicated the molecular sieves effect development. The isopycn for BSA adsorption on silica pore diameters has allowed estimating of BSA aggregates in bulk solution at 293 and 313 K. The analysis of obtained results indicates that the temperature increase in the adsorption system apparently promotes the reducing of protein aggregate sizes and the relaxing of H-bond in the hydrate cover of protein aggregates.
References [1] [2] [3] [4] [5] [6] [7]
W. Norde and J. Lyklema, J. Colloid Interface Sci. 66 (1978) 277. S. Kondo, E. Amano and M. Kurimoto, Pure Appl. Chem. 61 (1989) 1897. T. J. Su, J. R. Lu, R. K. Thomas, Z. F.Cui and J. Penfold, J. Phys. Chem. B 102 (1998) 8100. N. A. Eltekova and A. Yu. Eltekov, Rus. J. Phys. Chem. A 74 (2000) 958. C. E.Giacomelli and W. Norde, J. Colloid Interface Sci. 233 (2001) 234. A. Yu. Eltekov and N. A. Eltekova, Rus. J. Phys. Chem. A 77 (2003) 1134. C. W. Suh, M.Y. Kim, J. B. Choo, J. K. Kim, H. K. Kim and E. K. Lee, J. Biotechnol. 112 (2004) 267. [8] S. J. McClellan and E. I. Franses, Colloids Surf. A: Physicochem. Eng. Asp. 260 (2005) 265. [9] H. Larsericsdotter, S. Oscarsson and J. Buijs, J. Colloid Interface Sci. 289 (2005) 26. [10] K. C. Kwok, K. M.Yeung and N. H. Cheung, Langmuir 23 (2007) 1948.