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Adsorption of globular proteins on polymeric microfiltration membranes
DESALINATION ELSEVIER
Desalination 104 (1996) 99-105
Adsorption of globular proteins on polymeric microfiltrafion membranes K. Konttufi* and M. Vuofisto Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, Kemistintie 1A, 02150 Espoo, Finland, Tel. +358-0-4511, Fax. +358-0-4512580
Abstract
Adsorption of a globular model proteins lysozyme, cytochrome c, myoglobin, haemoglobin and human serum albumin on a polymeric microfiltration membrane (MF-MiUipore, mixed esters of cellulose) was studied using a method based on the measurement of streaming potential. The coverage of adsorbed protein on the membrane pore wall was detected as a change in surface charge density, which is accessible from a simple microscopic theory through the measured values of the streaming potential. Adsorption seemed to obey the Langmuir isotherm but the desorption was at least partially irreversible. The adsorption of chloride ions appeared to be independent of the adsorption of human serum albumin. Furthermore, it was experimentally found that a strongly adsorbing protein can be used to modify the membrane matrix so that the adsorption of other proteins is significantly reduced.
Keywords:
Protein adsorption; Streaming potential; Surface charge density; Microfiltration membrane; Ion adsorption
1. Introduction A convective diffusion process in porous membranes has been extensively studied both for the separation of small cations [1] and proteins [2] and for the determination of diffusion coefficients and effective charge numbers of polyelectrolytes [3, 4]. In all these studies the porous membrane was
Presented at the 7th International Symposium on Synthetic Membranes in Science and Industry, Tfibingen, Germany, August 29 - September 1, 1994.
*Corresponding author,
considered to be non-charged. However, the adsorption of charged solute species creates charge d e n s i t y on the s u r f a c e o f the membrane pores. The effects giving rise to this adsorption of ions have recently been discussed [5-7]. These studies have revealed that especially at low electrolyte concentration the adsorption of small ions has a significant effect on the transport across the membrane. T h e c o n t r i b u t i o n of e a s i l y a d s o r b i n g polyelectrolytes, like proteins, to the transport processes needs even more attention. R e c e n t l y , a large n u m b e r o f studies concerning adsorption of proteins on various solid surfaces have been presented. The aim
0011-9164/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved. PH S0011-9164(96)00031-8
1 O0
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
of such studies has been to get information relevant to protein purification, various biomedical applications (e.g. drug delivery), preparation of biosensors, etc. A wide variety of experimental methods have been used including solution depletion [8-12], in situ ellipsometry [13], ultra- and microffitration e x p e r i m e n t s [12, 13], c o n t a c t angle measurements [15], reflectometry [16, 17], microparticle electrophoresis [18-20], and measurement of streaming potential [16, 17, 211. The utilization of the measurement of streaming potential in the studies of adsorption is based on the idea that adsorption of any charged solute on the membrane matrix can be detected as a change in the surface charge density (q2) of the membrane pore. The change in charge density can be used as a direct measure for the amount of adsorbed protein provided that ion adsorption due to the base electrolyte remains constant. Furthermore, q2 is a crucial parameter when the effect of adsorbed charges in a convective diffusion process is estimated [6, 7]. However, the measurement of q2 is nonselective, because only the net change in charge density due to adsorption can be obtained. The measurement thus does not tell us how many adsorbed species are present, The m e t h o d of s t r e a m i n g potential measurement has been proven to be a convenient one for the determination of surface charge densities of porous membranes within a wide range of solution concentrations up to 0.1 M [5]. When the pore radius of a membrane is substantially (at least ten times) larger than the Debye length of the double layer formed by adsorbed charges, the measured streaming potential is practically independent of the pore size. This condition in our case, with a membrane having a nominal pore diameter of 0.22 ~tm, is fulfilled down to a electrolyte concentrations of about 1 mM. Moreover, no information about the membrane constant (A/l, where A is the effective area and 1 is the effective thickness of the membrane) or the porosity of the
membrane is needed for the determination of qe. In this paper, results for the adsorption of several proteins on a MF-Millipore membrane using streaming potential measurements are presented. The effects of protein and base electrolyte concentration, kinetics of adsorption and desorption as well as sequential adsorption of two proteins are the cases elucidated in this study.
2. Experimental The adsorption of five globular proteins, lysozyme (Lys), cytochrome c (Cyt), myoglobin (Myo), h a e m o g l o b i n (Hgb) and human serum albumin (HSA), on MFMillipore membrane (hydrophilic, mixture of cellulose nitrate and cellulose acetate)with the nominal pore diameter 0.22 ~tm were studied. The 20% HSA solution was supplied by the Finnish Red Cross Transfusion Service and the other proteins were supplied by Sigma Chemical Co. (Cyt: C-2506, Lys: L-6876, Hgb: H-2500, Myo: M-0630). The base electrolyte used was KC1 (Merck, p.a.). The pH was set at 6.7 with 0.2 mM phosphate buffer (KH2PO4 and KOH, J.T. Baker, A.C.S). All solutions were prepared using Milli-Q water (Waters) and were sterilized using Sartorius Minisart NML disposable filter units. The apparatus described in reference [5] was also used in this study. The cell was designed so that proteins could be injected directly into the solution in the cell without fouling the electrodes. The experimental procedure was as follows. The electrolyte solution was pumped through the membrane with sinusoidally changing rate (frequency = 1 mHz), which created a symmetrical bidirectional v o l u m e flow across the membrane. The potential and pressure differences (AU and Ap) were recorded continuously. After protein injection, the streaming potential (Usp = A U / A p ) was recorded during each run until a steady value was attained. The concentration of protein
l01
K. Kontturi and M. Vuoristo /Desalination 104 (1996) 99-105
corresponding to this adsorption equilibrium was then determined by means of a UV-VIS spectrophotometer, The accuracy and the sensitivity of the measurement of streaming potential can be significantly i n c r e a s e d by u s i n g a c o n t i n u o u s l y c h a n g i n g flow rate. The potential difference corresponding to zero pressure difference, which is needed for determination of Usp, is measured several times during the experiment. Thus, the streaming potential can be accurately separated from other changes in potential difference (e.g. drift in offset voltage between the electrodes). Furthermore, this procedure enables us to verify in each experiment the linear relationship between AU and Ap. This is essential in determination of the slope (Usp) needed for calculation of q2. Four sets of measurements were carried out in order to study the following cases: 1) Kinetics of adsorption and desorption of proteins in 0 . 0 1 M KC1 solution, 2) The effect of concentration of proteins in 0.01 M KC1 solution, 3) The effect of ionic strength on the adsorption of HSA, and 4) The sequential adsorption and desorption of cytochrome c and HSA in 0.01 M KC1.
experiment and throughout the desorption period the 0.01 M KC1 solution was kept free of protein whereas during the adsorption period the concentration of protein was 300 mg/1. It is evident from Fig. 1 that the adsorption equilibrium for all proteins studied was reached within about 300 seconds. During desorption the proteins behaved differently. Part of the adsorbed lysozyme and haemoglobin appears to desorb rather rapidly so that a new equilibrium is reached, while HSA does not desorb at all and cytochrome c is desorbed at a low rate. It is clearly seen that the adsorption of the studied proteins on the MF-Millipore membrane is a completely or at least partially irreversible process.
q2/ Asrn'2 0.0o6 ~=3oo o.oo4 / ~ q ~ a * ~ 0.oo2- -
--
c-~ ~ •
0 -,, []
~.. ~
--z_~,---
,?g
-0.002 ~ ~A 3. R e s u l t s a n d d i s c u s s i o n
Every m e a s u r e m e n t was started by determining the streaming potential for each membrane equilibrated for 24 hours in the base electrolyte solution. From the values obtained the surface charge density q 2 w a s d e t e r m i n e d a c c o r d i n g to the m e t h o d described in reference [5].
3.1. Kinetics of adsorption In the first set of measurements, the streaming potential of the membrane was monitored during an adsorption period which lasted 2,000 s followed by a desorption period for 1,500 s. In the beginning of each
-o.0o4
-o.0o6
~"~'"~'~'~~~ ' lo0o' '
2000
'
~'e'¢'~'~ 3obo
t/s Fig 1. The surface charge densities determined after introducing protein to the solution (t <2,000 s) followed by desorption in protein free solution (t >2,000 s). c is concentration of protein in mgfl. The proteins are denoted with: Cyt (*), HSA (filled triangle), Hgb (empty square), and Lys (-).
3.2. The effect of the concentration of protein In the second set of measurements, the adsorption of c y t o c h r o m e c, albumin,
102
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
haemoglobin and myoglobin was studied as a function o f p r o t e i n c o n c e n t r a t i o n . The concentration of proteins used in the study varied from 0 to 350 mg/1. The measurements were carried out by increasing the protein concentration starting always from a proteinfree solution. The streaming potential of a MF-Millipore m e m b r a n e equilibrated with 0.01 M KC1 was about Usp° = 65+10 nV/Pa, which corresponds to a surface charge density of 2.8_+0.4 mAs/m 2. This charge density has been proven to be due to the adsorption of chloride ions [5]. The results of the measurements in Fig. 2 were fitted to a Langmuir-type adsorption isotherm: 0 q2 - q2 =
m [ kC ] q2 _ 1 + k C -
(1)
where C = concentration of protein k = a constant q2o = the surface charge density of the membrane before protein adsorption q2m = the maximum change in the surface charge d e n s i t y , i.e. the c h a n g e corresponding to a surface coverage of unity. on-q~/Asm2
~ 0.004 ( / -
,
,
o.0o2 0~ A z~ J.
-0.002 -0.004
o
I
10o
,, • i
200 c/mg/L
i
300
400
Fig. 2. The effect of protein concentration on the surface charge density of a MF-Millipore membrane in 0.01 M KC1 solution at pH 6.7. The proteins are denoted with: Cyt (*), HSA (filled triangle), Hgb (empty square), and Myo (empty triangle),
The isotherm (1) describes accurately the changes determined in the surface charge densities for the cases where cytochrome c, albumin or haemoglobin was the adsorbing protein. This is seen from Fig. 2 (solid lines). The values of the parameters in Eq. 1 are given in Table 1. However, it must be realized that the obtained Langmuir isotherms are not valid for the desorption. This fact clearly demonstrates the complicated nature of polyelectrolyte adsorption/desorption phenomena and indicates the a b s e n c e of a kinetic equilibrium as assumed in the derivation of the Langmuir isotherm.
Table 1 Parameters determined for a isotherm for cytochrome c (Cyt),Langmuir-typehuman serumads°rpti°nalbumin (HSA) and haemoglobin (Hgb) in 0.01 M KCI at pH 6.7. Protein Cyt
HSA Hgb
q2 m (mAs/m2) 3.96
-2.61 5.70
k (1/mg) 0.0854
0.1460 0.1848
It is evident from Figs. 1 and 2 that h a e m o g l o b i n is c l e a r l y c a t i o n i c and m y o g l o b i n is slightly anionic at pH 6.8. However, according to the isoelectric pHs of these proteins [22], 6.8 and 7.0, respectively, they should have behaved as non-charged and slightly cationic species, respectively. This discrepancy obviously results from an ion binding phenomenon, which changes the effective charge number of the protein when small ions are present in the solution, and from the fact that the isoelectric pH is usually determined in absence of electrolytes using isoelectric focusing. The effect of pH on the protein adsorption was not studied because the membrane matrix was rather sensitive to pH changes. In addition, the changes in charge density of the membrane w e r e n o t reversible when the pH w a s returned to its initial value. The results for myoglobin were not reproducible enough to
K. Kontturi and M. Vuoristo /Desalination 104 (1996) 99-105
be fitted to an adsorption isotherm. Obviously this is due to the fact that the charge number of myoglobin is small and highly sensitive to the environmental conditions close to its isoelectric pH.
-q2 / Asm -2
3.3. The effect of ionic strength on protein adsorption
o.oo2
In the third set of experiments, the effect of ionic strength on adsorption of H S A was studied. The concentration of H S A was 300 mg/1 and the concentration o f KC1 was changed from 0.2 m M to 100 mM. The surface charge densities were fitted to a Freundiich-type adsorption isotherm, q2 = b • cn, where c is the concentration of base electrolyte. The result of the fit is shown in Fig. 3. The values for the exponent n is 0.351 and for the factor b it is 0.00136. The exponent n is about the same as determined for the MF-Millipore membrane in proteinfree KC1 solution as indicated in Fig. 3 [5]. In other words, the adsorption of chloride ions on this type of membrane appears to be independent of the adsorption of H S A in the c o n c e n t r a t i o n r a n g e studied. This fact together with the numerical value of q2 indicates that the adsorbed ions and proteins are not completely covering the pore walls, i.e., no monolayer is formed and part of the m e m b r a n e matrix is still a c c e s s i b l e to electrolyte solution. Furthermore, it is obvious from the constant adsorption of p r o t e i n in s u c h a w i d e e l e c t r o l y t e concentration region that the electrostatic interactions do not have a dominant role in protein adsorption.
0.001
103
0.010
1
0.005
[] ..........
0.003
0.0005 0.0003 0.0002
.-" 0.1
,,
1
10
c(KC1)/mM Fig. 3. The effect of KC1 concentration on the adsorption of HSA on a MF-Millipore membrane solid line (filled t r i a n g l e ) c o m p a r e d with adsorption of chloride ions in absence of proteins (filled square). The lines represent the F r e u n d l i c h - t y p e isotherm for chloride ion adsorption.
q2 / Asrfi2 o.o01 / 0 HSA~) o ~'~ "~ -| -o.o011
i
Cyt il
0
HSA~yt ! ] i
,,~-~ " i
-0.002. | '
-o.oo3~
I
-o.o04!
, ~
~.oo5
~
i~ i
~
t
~
3.4. Sequential adsorption of two proteins -0.006
The effect of sequential adsorption and desorption periods with two proteins, H S A and cytochrome c, were studied in the fourth set of m e a s u r e m e n t s . One e x p e r i m e n t consisted of a series of successive measurements in a solution containing 200 mg/l albumin or 200 rag/1 cytochrome c or
100
0
i
, '
,
, I !
' t
I l
! t I
i
1000 2000 3000 4000 5000 6000 70bo 80bo 9000 t/s
Fig. 4. The successive adsorption and desorption of two proteins. The solutions in contact with the membrane are denoted by HSA: solution with human serum albumin, Cyt: solution with cytochrome c, and 0: protein free solution. The concentration of protein in both solutions was 200 mg/1.
104
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
neither of them. In the beginning of the experiment, a m e m b r a n e w h i c h was in adsorption e q u i l i b r i u m with a solution containing 200 mg/1 cytochrome c was moved to a protein free solution. The result of this experiment is shown in Fig. 4. After adsorption and partial desorption of c y t o c h r o m e c, the m e m b r a n e s e e m e d to adsorb HSA like a membrane which has not been treated with c y t o c h r o m e c. This behaviour may be due to a fast exchange process [16], i.e. adsorbed cytochrome c is replaced quantitatively by the more easily adsorbing albumin. After the membrane has been in contact with albumin the nature of the adsorption of c y t o c h r o m e c changes: a significant rise in the charge density is still obtained when cytochrome c is introduced but fast desorption is taking place when the membrane is moved to protein-free solution. This indicates that a more strongly adsorbing protein can be used to modify the surface so that adsorption of other proteins is notably reduced. In addition, desorption of other
in this study appeared to be an irreversible process. The interactions b e t w e e n proteins and membrane matrix are not limited by simple electrostatic forces. This result is suggested both by the adsorption of the negatively charged H S A on the negatively charged surface, and by the independence of the chloride ion and HSA adsorption. The adsorption of cytochrome c is reduced s i g n i f i c a n t l y after the s u r f a c e of the membrane is treated with HSA. Thus, the loss of expensive proteins due to adsorption can be minimized by modifying the surface with s o m e easily adsorbing and i n e x p e n s i v e protein, e.g. HSA.
proteins is p r o m o t e d by pre-coating the membrane with a strongly adsorbing protein. It must be realized that the above conclusions are based on a method, which is nonselective,
[2]
References [1]
[3]
4. Conclusions The method based on the measurement of streaming potential can be efficiently used in studying protein a d s o r p t i o n on p o r o u s membranes. In situ information on the charged state of the membrane surface can be obtained as a f u n c t i o n of solution c o m p o s i t i o n . The a p p l i c a b i l i t y o f this procedure is limited to situations where pH is not too close to the isoelectric point of the protein, The adsorption of the proteins studied appeared to obey a Langmuir-type adsorption isotherm. This result is interesting from a theoretical point of view because this isotherm is based on a kinetic equilibrium between adsorption and d e s o r p t i o n processes. However, the adsorption of the proteins used
[4]
K. Kontturi and L.M. Westerberg, Countercurrent electrolysis in a cell where porous membranes have been connected in series with ion-exchange membranes. Part 2. Experimental verification, Sep. Sci. Technol. 23 (1988) 235. A.-K. Kontturi, K. Kontturi, A. Savonen, M. Vuoristo, and D.J. Schiffrin, Electrical migration in convective electrophoresis: Modelling and membrane separation of bovum serum albumin (BSA) and cytocbrome c, J. Chem. Soc. Faraday Trans. 89/1 (1993) 99-106. A.-K. Kontturi, K. Kontturi, P. Niinikoski, A. Savonen, and M. Vuoristo, The effective charge number and diffusion coefficient of cationic cytochrome c in aqueous solution, Acta Chem. Scand. 46 (1992) 348-353. A.-K. Kontturi and K. Kontturi, A method for determination of ionic diffusion coefficients of polydisperse polyelectrolyte, Colloid Interface Sci. 12o (1987) 256. A.-K. Kontturi and K. Kontturi, Determination of effective charge number of polydisperse polyelectrolyte, Colloid Interface Sci. 124 (1988) 328. A.-K. Kontturi and K. Kontturi, Determination of effective charge number of polydisperse polyelectrolyte: An improved method, Acta Polytechnica Scand. 178 (1987) 143. A.-K. Kontturi, Determination of diffusion coefficients and effective charge numbers of lignosulphonate, Chem. Soc. Faraday Trans. 84/1 (1988) 4033. A.-K. Kontturi, Diffusion coefficients and effective charge numbers of lignosulphonate.
K. Kontturi and M. Vuoristo /Desalination 104 (1996) 99-105
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Influence of temperature, Chem. Soc. Faraday Trans. 84/1 (1988) 4043. K. Kontturi, A. Savonen, and M. Vuoristo, Study of adsorption and ion exchange properties of some porous membranes, Acta Chem. Scand. 48 (1994) 1-11. A.-K. Kontturi, K. Kontturi, S. M a f , J.A. Manzanares, P. Niinikoski, and M. Vuoristo, Convective diffusion in porous membranes with adsorbed charges, Langmuir 10 (1994)949-954. K. Kontturi, S. M a f , J.A. Manzanares, J. Pellicer, and M. Vuoristo, A new method for determining transport numbers of charged membranes from c o n v e c t i v e diffusion experiment, J. Electroanal. Chem., in press. R.M. Cornelius, P.W. Wojciechowski, and J.L. Brash, Measurement of protein adsorption kinetics by an in situ, "real-time" solution depletion technique, J. Colloid Interface Sci. 150 (1992) 121. J.M. Peula and F.J. de las Nieves, Adsorption of monomeric bovine serum albumin on sulfonated polystyrene model colloids; 1. Adsorption isotherms and effect of surface charge density, Colloids Surfaces 77 (1993) 199. I~. Kiss, Temperature dependence of bovine serum albumin adsorption onto a poly-(ethylene oxide)-grafted surface, Colloids Surfaces 76 (1993) 135. W. Norde and A.C.I. Anusiem, Adsorption, desorption and re-adsorption of proteins on solid surfaces, Colloids Surfaces 66 (1992) 73. M.K. Ko, J.J. Pellegrino, R. Nassimbene, and P. Marko, Characterization of the adsorptionfouling layer using globular proteins on ultrafiltration membranes, J. Membr. Sci. 76 (1993) 101. E. Iritani, S. Tachi, and T. Murase, Influence of protein adsorption on flow resistance of microfiltration membrane, Colloids Surfaces 89 (1994) 15-22.
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
105
V. K_risdhasima, P. Vinaraphong, and J. McGuire, Adsorption kinetics and elutability of a-lactoglobulin, and bovine serum albumin at hydrophobic and hydrophilic interfaces, J. Colloid Interface Sci. 161 (1993) 325. R. Miller, S. Treppo, A. Voigt, W. Zingg, and A.W. Neumann, Contact angle kinetics of human albumin solutions at solid surfaces, Colloids Surfaces 69 (1993) 203. H. Shirahama, J. Lyklema, and W. Norde, Comparative protein adsorption in model systems, J. Colloid Interface Sci. 139 (1990) 177. A.V. Elgersma, R.L.J. Zsom, J. Lyklema, and W. Norde, Kinetics of single and competitive protein adsorption studied by reflectometry and streaming potential measurement, Colloids Surfaces 65 (1992)17. E. 13sterberg, K. Bergstr6m, K. Holmberg, J.A. Riggs, J.M. Van Alstine, T.P. Schuman. N.L. Burns, and J.M. Harris, Comparison of polysaccharide and poly(ethylene glycol) coatings for reduction of protein adsorption on polystyrene surfaces, Colloids Surfaces 77 (1993) 159. K. Kandori, Y. Yamamoto, H. Saito, and T. Ishikawa, Adsorption of bovine serum albumin on synthesized non-stoichiometric strontium hydroxyapatites, Colloids Surfaces 80 (1993) 287. M.K.Y. Rao, P. Somasundaran, K.M. Schilling, B. Carson, and K.P. Ananthapadmanabhan, Bacterial adhesion onto apatite minerals electrokinetic aspects, Colloids Surfaces 79 (1993) 293. W. Norde and E. Rouwendal, Streaming potential measurement as a tool to study protein adsorption kinetics, I. Colloid Interface Sci. 139 (1990) 169. A.L. Lehninger (Ed.), Biochemistry, 2nd ed., Worth Publishers Inc., New York, 1975, p. 162.
Desalination 104 (1996) 99-105
Adsorption of globular proteins on polymeric microfiltrafion membranes K. Konttufi* and M. Vuofisto Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, Kemistintie 1A, 02150 Espoo, Finland, Tel. +358-0-4511, Fax. +358-0-4512580
Abstract
Adsorption of a globular model proteins lysozyme, cytochrome c, myoglobin, haemoglobin and human serum albumin on a polymeric microfiltration membrane (MF-MiUipore, mixed esters of cellulose) was studied using a method based on the measurement of streaming potential. The coverage of adsorbed protein on the membrane pore wall was detected as a change in surface charge density, which is accessible from a simple microscopic theory through the measured values of the streaming potential. Adsorption seemed to obey the Langmuir isotherm but the desorption was at least partially irreversible. The adsorption of chloride ions appeared to be independent of the adsorption of human serum albumin. Furthermore, it was experimentally found that a strongly adsorbing protein can be used to modify the membrane matrix so that the adsorption of other proteins is significantly reduced.
Keywords:
Protein adsorption; Streaming potential; Surface charge density; Microfiltration membrane; Ion adsorption
1. Introduction A convective diffusion process in porous membranes has been extensively studied both for the separation of small cations [1] and proteins [2] and for the determination of diffusion coefficients and effective charge numbers of polyelectrolytes [3, 4]. In all these studies the porous membrane was
Presented at the 7th International Symposium on Synthetic Membranes in Science and Industry, Tfibingen, Germany, August 29 - September 1, 1994.
*Corresponding author,
considered to be non-charged. However, the adsorption of charged solute species creates charge d e n s i t y on the s u r f a c e o f the membrane pores. The effects giving rise to this adsorption of ions have recently been discussed [5-7]. These studies have revealed that especially at low electrolyte concentration the adsorption of small ions has a significant effect on the transport across the membrane. T h e c o n t r i b u t i o n of e a s i l y a d s o r b i n g polyelectrolytes, like proteins, to the transport processes needs even more attention. R e c e n t l y , a large n u m b e r o f studies concerning adsorption of proteins on various solid surfaces have been presented. The aim
0011-9164/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved. PH S0011-9164(96)00031-8
1 O0
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
of such studies has been to get information relevant to protein purification, various biomedical applications (e.g. drug delivery), preparation of biosensors, etc. A wide variety of experimental methods have been used including solution depletion [8-12], in situ ellipsometry [13], ultra- and microffitration e x p e r i m e n t s [12, 13], c o n t a c t angle measurements [15], reflectometry [16, 17], microparticle electrophoresis [18-20], and measurement of streaming potential [16, 17, 211. The utilization of the measurement of streaming potential in the studies of adsorption is based on the idea that adsorption of any charged solute on the membrane matrix can be detected as a change in the surface charge density (q2) of the membrane pore. The change in charge density can be used as a direct measure for the amount of adsorbed protein provided that ion adsorption due to the base electrolyte remains constant. Furthermore, q2 is a crucial parameter when the effect of adsorbed charges in a convective diffusion process is estimated [6, 7]. However, the measurement of q2 is nonselective, because only the net change in charge density due to adsorption can be obtained. The measurement thus does not tell us how many adsorbed species are present, The m e t h o d of s t r e a m i n g potential measurement has been proven to be a convenient one for the determination of surface charge densities of porous membranes within a wide range of solution concentrations up to 0.1 M [5]. When the pore radius of a membrane is substantially (at least ten times) larger than the Debye length of the double layer formed by adsorbed charges, the measured streaming potential is practically independent of the pore size. This condition in our case, with a membrane having a nominal pore diameter of 0.22 ~tm, is fulfilled down to a electrolyte concentrations of about 1 mM. Moreover, no information about the membrane constant (A/l, where A is the effective area and 1 is the effective thickness of the membrane) or the porosity of the
membrane is needed for the determination of qe. In this paper, results for the adsorption of several proteins on a MF-Millipore membrane using streaming potential measurements are presented. The effects of protein and base electrolyte concentration, kinetics of adsorption and desorption as well as sequential adsorption of two proteins are the cases elucidated in this study.
2. Experimental The adsorption of five globular proteins, lysozyme (Lys), cytochrome c (Cyt), myoglobin (Myo), h a e m o g l o b i n (Hgb) and human serum albumin (HSA), on MFMillipore membrane (hydrophilic, mixture of cellulose nitrate and cellulose acetate)with the nominal pore diameter 0.22 ~tm were studied. The 20% HSA solution was supplied by the Finnish Red Cross Transfusion Service and the other proteins were supplied by Sigma Chemical Co. (Cyt: C-2506, Lys: L-6876, Hgb: H-2500, Myo: M-0630). The base electrolyte used was KC1 (Merck, p.a.). The pH was set at 6.7 with 0.2 mM phosphate buffer (KH2PO4 and KOH, J.T. Baker, A.C.S). All solutions were prepared using Milli-Q water (Waters) and were sterilized using Sartorius Minisart NML disposable filter units. The apparatus described in reference [5] was also used in this study. The cell was designed so that proteins could be injected directly into the solution in the cell without fouling the electrodes. The experimental procedure was as follows. The electrolyte solution was pumped through the membrane with sinusoidally changing rate (frequency = 1 mHz), which created a symmetrical bidirectional v o l u m e flow across the membrane. The potential and pressure differences (AU and Ap) were recorded continuously. After protein injection, the streaming potential (Usp = A U / A p ) was recorded during each run until a steady value was attained. The concentration of protein
l01
K. Kontturi and M. Vuoristo /Desalination 104 (1996) 99-105
corresponding to this adsorption equilibrium was then determined by means of a UV-VIS spectrophotometer, The accuracy and the sensitivity of the measurement of streaming potential can be significantly i n c r e a s e d by u s i n g a c o n t i n u o u s l y c h a n g i n g flow rate. The potential difference corresponding to zero pressure difference, which is needed for determination of Usp, is measured several times during the experiment. Thus, the streaming potential can be accurately separated from other changes in potential difference (e.g. drift in offset voltage between the electrodes). Furthermore, this procedure enables us to verify in each experiment the linear relationship between AU and Ap. This is essential in determination of the slope (Usp) needed for calculation of q2. Four sets of measurements were carried out in order to study the following cases: 1) Kinetics of adsorption and desorption of proteins in 0 . 0 1 M KC1 solution, 2) The effect of concentration of proteins in 0.01 M KC1 solution, 3) The effect of ionic strength on the adsorption of HSA, and 4) The sequential adsorption and desorption of cytochrome c and HSA in 0.01 M KC1.
experiment and throughout the desorption period the 0.01 M KC1 solution was kept free of protein whereas during the adsorption period the concentration of protein was 300 mg/1. It is evident from Fig. 1 that the adsorption equilibrium for all proteins studied was reached within about 300 seconds. During desorption the proteins behaved differently. Part of the adsorbed lysozyme and haemoglobin appears to desorb rather rapidly so that a new equilibrium is reached, while HSA does not desorb at all and cytochrome c is desorbed at a low rate. It is clearly seen that the adsorption of the studied proteins on the MF-Millipore membrane is a completely or at least partially irreversible process.
q2/ Asrn'2 0.0o6 ~=3oo o.oo4 / ~ q ~ a * ~ 0.oo2- -
--
c-~ ~ •
0 -,, []
~.. ~
--z_~,---
,?g
-0.002 ~ ~A 3. R e s u l t s a n d d i s c u s s i o n
Every m e a s u r e m e n t was started by determining the streaming potential for each membrane equilibrated for 24 hours in the base electrolyte solution. From the values obtained the surface charge density q 2 w a s d e t e r m i n e d a c c o r d i n g to the m e t h o d described in reference [5].
3.1. Kinetics of adsorption In the first set of measurements, the streaming potential of the membrane was monitored during an adsorption period which lasted 2,000 s followed by a desorption period for 1,500 s. In the beginning of each
-o.0o4
-o.0o6
~"~'"~'~'~~~ ' lo0o' '
2000
'
~'e'¢'~'~ 3obo
t/s Fig 1. The surface charge densities determined after introducing protein to the solution (t <2,000 s) followed by desorption in protein free solution (t >2,000 s). c is concentration of protein in mgfl. The proteins are denoted with: Cyt (*), HSA (filled triangle), Hgb (empty square), and Lys (-).
3.2. The effect of the concentration of protein In the second set of measurements, the adsorption of c y t o c h r o m e c, albumin,
102
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
haemoglobin and myoglobin was studied as a function o f p r o t e i n c o n c e n t r a t i o n . The concentration of proteins used in the study varied from 0 to 350 mg/1. The measurements were carried out by increasing the protein concentration starting always from a proteinfree solution. The streaming potential of a MF-Millipore m e m b r a n e equilibrated with 0.01 M KC1 was about Usp° = 65+10 nV/Pa, which corresponds to a surface charge density of 2.8_+0.4 mAs/m 2. This charge density has been proven to be due to the adsorption of chloride ions [5]. The results of the measurements in Fig. 2 were fitted to a Langmuir-type adsorption isotherm: 0 q2 - q2 =
m [ kC ] q2 _ 1 + k C -
(1)
where C = concentration of protein k = a constant q2o = the surface charge density of the membrane before protein adsorption q2m = the maximum change in the surface charge d e n s i t y , i.e. the c h a n g e corresponding to a surface coverage of unity. on-q~/Asm2
~ 0.004 ( / -
,
,
o.0o2 0~ A z~ J.
-0.002 -0.004
o
I
10o
,, • i
200 c/mg/L
i
300
400
Fig. 2. The effect of protein concentration on the surface charge density of a MF-Millipore membrane in 0.01 M KC1 solution at pH 6.7. The proteins are denoted with: Cyt (*), HSA (filled triangle), Hgb (empty square), and Myo (empty triangle),
The isotherm (1) describes accurately the changes determined in the surface charge densities for the cases where cytochrome c, albumin or haemoglobin was the adsorbing protein. This is seen from Fig. 2 (solid lines). The values of the parameters in Eq. 1 are given in Table 1. However, it must be realized that the obtained Langmuir isotherms are not valid for the desorption. This fact clearly demonstrates the complicated nature of polyelectrolyte adsorption/desorption phenomena and indicates the a b s e n c e of a kinetic equilibrium as assumed in the derivation of the Langmuir isotherm.
Table 1 Parameters determined for a isotherm for cytochrome c (Cyt),Langmuir-typehuman serumads°rpti°nalbumin (HSA) and haemoglobin (Hgb) in 0.01 M KCI at pH 6.7. Protein Cyt
HSA Hgb
q2 m (mAs/m2) 3.96
-2.61 5.70
k (1/mg) 0.0854
0.1460 0.1848
It is evident from Figs. 1 and 2 that h a e m o g l o b i n is c l e a r l y c a t i o n i c and m y o g l o b i n is slightly anionic at pH 6.8. However, according to the isoelectric pHs of these proteins [22], 6.8 and 7.0, respectively, they should have behaved as non-charged and slightly cationic species, respectively. This discrepancy obviously results from an ion binding phenomenon, which changes the effective charge number of the protein when small ions are present in the solution, and from the fact that the isoelectric pH is usually determined in absence of electrolytes using isoelectric focusing. The effect of pH on the protein adsorption was not studied because the membrane matrix was rather sensitive to pH changes. In addition, the changes in charge density of the membrane w e r e n o t reversible when the pH w a s returned to its initial value. The results for myoglobin were not reproducible enough to
K. Kontturi and M. Vuoristo /Desalination 104 (1996) 99-105
be fitted to an adsorption isotherm. Obviously this is due to the fact that the charge number of myoglobin is small and highly sensitive to the environmental conditions close to its isoelectric pH.
-q2 / Asm -2
3.3. The effect of ionic strength on protein adsorption
o.oo2
In the third set of experiments, the effect of ionic strength on adsorption of H S A was studied. The concentration of H S A was 300 mg/1 and the concentration o f KC1 was changed from 0.2 m M to 100 mM. The surface charge densities were fitted to a Freundiich-type adsorption isotherm, q2 = b • cn, where c is the concentration of base electrolyte. The result of the fit is shown in Fig. 3. The values for the exponent n is 0.351 and for the factor b it is 0.00136. The exponent n is about the same as determined for the MF-Millipore membrane in proteinfree KC1 solution as indicated in Fig. 3 [5]. In other words, the adsorption of chloride ions on this type of membrane appears to be independent of the adsorption of H S A in the c o n c e n t r a t i o n r a n g e studied. This fact together with the numerical value of q2 indicates that the adsorbed ions and proteins are not completely covering the pore walls, i.e., no monolayer is formed and part of the m e m b r a n e matrix is still a c c e s s i b l e to electrolyte solution. Furthermore, it is obvious from the constant adsorption of p r o t e i n in s u c h a w i d e e l e c t r o l y t e concentration region that the electrostatic interactions do not have a dominant role in protein adsorption.
0.001
103
0.010
1
0.005
[] ..........
0.003
0.0005 0.0003 0.0002
.-" 0.1
,,
1
10
c(KC1)/mM Fig. 3. The effect of KC1 concentration on the adsorption of HSA on a MF-Millipore membrane solid line (filled t r i a n g l e ) c o m p a r e d with adsorption of chloride ions in absence of proteins (filled square). The lines represent the F r e u n d l i c h - t y p e isotherm for chloride ion adsorption.
q2 / Asrfi2 o.o01 / 0 HSA~) o ~'~ "~ -| -o.o011
i
Cyt il
0
HSA~yt ! ] i
,,~-~ " i
-0.002. | '
-o.oo3~
I
-o.o04!
, ~
~.oo5
~
i~ i
~
t
~
3.4. Sequential adsorption of two proteins -0.006
The effect of sequential adsorption and desorption periods with two proteins, H S A and cytochrome c, were studied in the fourth set of m e a s u r e m e n t s . One e x p e r i m e n t consisted of a series of successive measurements in a solution containing 200 mg/l albumin or 200 rag/1 cytochrome c or
100
0
i
, '
,
, I !
' t
I l
! t I
i
1000 2000 3000 4000 5000 6000 70bo 80bo 9000 t/s
Fig. 4. The successive adsorption and desorption of two proteins. The solutions in contact with the membrane are denoted by HSA: solution with human serum albumin, Cyt: solution with cytochrome c, and 0: protein free solution. The concentration of protein in both solutions was 200 mg/1.
104
K. Kontturi and M. Vuoristo / Desalination 104 (1996) 99-105
neither of them. In the beginning of the experiment, a m e m b r a n e w h i c h was in adsorption e q u i l i b r i u m with a solution containing 200 mg/1 cytochrome c was moved to a protein free solution. The result of this experiment is shown in Fig. 4. After adsorption and partial desorption of c y t o c h r o m e c, the m e m b r a n e s e e m e d to adsorb HSA like a membrane which has not been treated with c y t o c h r o m e c. This behaviour may be due to a fast exchange process [16], i.e. adsorbed cytochrome c is replaced quantitatively by the more easily adsorbing albumin. After the membrane has been in contact with albumin the nature of the adsorption of c y t o c h r o m e c changes: a significant rise in the charge density is still obtained when cytochrome c is introduced but fast desorption is taking place when the membrane is moved to protein-free solution. This indicates that a more strongly adsorbing protein can be used to modify the surface so that adsorption of other proteins is notably reduced. In addition, desorption of other
in this study appeared to be an irreversible process. The interactions b e t w e e n proteins and membrane matrix are not limited by simple electrostatic forces. This result is suggested both by the adsorption of the negatively charged H S A on the negatively charged surface, and by the independence of the chloride ion and HSA adsorption. The adsorption of cytochrome c is reduced s i g n i f i c a n t l y after the s u r f a c e of the membrane is treated with HSA. Thus, the loss of expensive proteins due to adsorption can be minimized by modifying the surface with s o m e easily adsorbing and i n e x p e n s i v e protein, e.g. HSA.
proteins is p r o m o t e d by pre-coating the membrane with a strongly adsorbing protein. It must be realized that the above conclusions are based on a method, which is nonselective,
[2]
References [1]
[3]
4. Conclusions The method based on the measurement of streaming potential can be efficiently used in studying protein a d s o r p t i o n on p o r o u s membranes. In situ information on the charged state of the membrane surface can be obtained as a f u n c t i o n of solution c o m p o s i t i o n . The a p p l i c a b i l i t y o f this procedure is limited to situations where pH is not too close to the isoelectric point of the protein, The adsorption of the proteins studied appeared to obey a Langmuir-type adsorption isotherm. This result is interesting from a theoretical point of view because this isotherm is based on a kinetic equilibrium between adsorption and d e s o r p t i o n processes. However, the adsorption of the proteins used
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