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Adsorption of Human Serum Albumin onto PVA-coated Affinity Microporous PTFE Capillary
CHEM. RES. CHINESE UNIVERSITIES
Available online at www.sciencedirect.com
2008,24(2), 154-161 Article ID 1005-9040~2008~-02-154-08
ScienceDirect
Adsorption of Human Serum Albumin onto PVA-coated Affinity Microporous PTFE Capillary JIN Gu', YAO Qi-zhi and ZHANG Lei Department of Chemistry, University of Science and Technology of China, He@ 230026, P. R. China
Abstract Affinity dye-ligand Cibacron Blue F3GA(CB F3GA) was covalently coupled with poly(viny1 alcohol)(PVA) coated on the inner surface of microporous poly(tetra-fluoroethylene)(MPTFE) membranous capillary. The PVA-coated PTFE capillary surface was characterized by XPS and FESEM. The grafting degree of PVA and the amount of CB F3GA immobilized onto the membranous capillary were 23.5 mg/g and 89.6 pmol/g, respectively. These dyed membranous capillaries were chemically and mechanically stable, and could be reproducibly prepared. Human serum albumin(HSA) was selected as model protein. The saturation adsorbance of the dye attached membranous capillary was 85.3 mg HSNg, while the capacity of non-specific adsorption for HSA was less than 0.3 mg/g. Keywords Albumin adsorption; Cibacron blue F3GA; PVA; PTFE; Plasma
1 Introduction Immobilized dye-ligand affinity chromatography has been recognized as an important technique for the separation, purification, and recovery of a wide range of proteins and enzymes. Various dyes were coupled to different bead-based matrices, such as dextran"], agaroseL2],p~lyacrylamide[~], cellulose[41,PVA['], and silica gelK6].However, their use is limited by the compressibility of the soft gels at high flow rates, the high pressure drops for small size beads, and the mass-transfer limitation owing to the internal diffusion. In recent years, the adsorptive membrane has emerged as an alternative to the traditional column chromatography since it brings the solute into close proximity to bound ligands through convective transport, thus reducing the mass-transfer resistance and enabling lower pressure drops and higher flow rate^[^-^]. However, the capacity of membranes is lower than that of column. In addition, membrane fouling is another problem. At present, the preparation and application of a new form of adsorbent is an interesting work. The coating method is the simplest way to modify the membrane surface by introducing hydrophilic and functional polymers. In our previous study, the membranous capillaries were modified by HSA via a radia-
tion-induced graft polymerization technique, and by CB F3GA via a chemical method for bilirubin removal from human In this study, the new sorbent was composed of CB F3GA as dye affinity ligand, and microporous membranous poly(tetrafluoroethylene) (MPTFE) capillary as the carrier matrix. To prevent non-specific interactions between the hydrophobic MPTFE surface and protein molecules, and to attach the ligand (CB F3GA) to the matrix, the capillaries were coated with a hydrophilic layer of PVA after activation. The results of albumin adsorption-desorption studies from aqueous media and human plasma are presented in this article. To our knowledge, a similar method using MPTFE capillaries for HSA adsorption has not been reported earlier.
2 Experimental 2.1
Chemicals
Microporous membranous PTFE capillaries provided by Professor Watanabe, were used for the present study. The capillaries have an internal diameter of 2 mm, and a wall thickness of approximately 0.5 mm. PVA(average Mr 14000, 100% hydrolysed) was purchased from the Chemical Reagent Company of Shanghai, China. CB F3GA and HSA (lyophilized, fraction V) were purchased from Sigma. The blood
*Corresponding author. E-mail: fanruiqing@l63 .com Received May 28,2007; accepted September 5,2007. Supported by the National Natural Science Foundation of China(No.29405038). Copyright 02008, Jilin University. Published by Elsevier Limited. All rights reserved.
JIN GUet al. 155 water. samples were obtained from a healthy human donor. Reagents such as terephthaldehyde, sodium chloride, The PVA adsorbed onto the capillaries was desodium carbonate, hydrochloride acid, etc. were of termined by measuring the initial and final concentraanalytical reagent grade(purchased from Chemical tions of PVA within the adsorption medium, according Reagent Company of Shanghai, China). The fresh pito the KI-12 method, spectrophotometrically at 690 nm[l 31 ranha solution was prepared in concentrated sulfuric acid and hydrogen peroxide(vo1ume ratio 1:1). 2.3.3 immobilization of Cibacron Blue F3GA Briefly, 0.6 g of CB F3GA was dissolved in 20 2.2 Apparatus mL of water; then, after the PVA-coated capillaries were immersed into it for 60 min at 60 "C, 1 g of NaCl A flow injection system (Model FI-2100, Beijing was added. Subsequently, 0.5 g of Na2C03was added Haiguang Instrument Co., China) was used for the to adjust the pH value (to about pH 10) of the solution. feeding of solution. The concentration of protein in The reaction then took place in the following 4 h. The the samples was determined by a Model 7060 autocapillaries were washed with distilled water and memated analyzer(Hitachi, Japan, total protein reagent thanol several times until all unbound dye was reKit: Cat. No. TP7143 and Albumin reagent Kit: Cat. moved. The dye immobilized capillaries were stored No. AB7162, Randox Laboratories Ltd., UK). Fibriin a phosphate solution(pH 7.0) containing 0.02% nogen Kit was obtained from Stago(Cat. Ref. No. (mass fraction) sodium a i d e at 4 "C to prevent mi00608 and 00625, Diagnostica Stago, Asnierescrobial contamination. sur-Seine, France). The dye content of the capillaries was determined 2.3 Cibacron Blue F3GA-attached MPTFE spectrophotometrically by first hydrolyzing the capillaries in an aqueous solution of 12 mol/L hydrochloric 2.3.I Activation of Unmodified MPTFE Capillaries acid, at 80 "C, for 30 min. The solution was then diMPTFE capillaries were first activated by a fresh luted to 6 mol/L with distilled water, and neutralized piranha solution"21. The capillaries were incubated in with an aqueous solution of 6 mol/L NaOH. The con20 mL of fresh piranha solution at room temperature centration was then determined spectrophotometricalfor 40 min. After that, the capillaries were washed ly at its maximum absorbance wavelength(i,,,=6 10 several times with distilled water. nm)[I41. 2.3.2 Coating ofMPTFE Capillaries with PVA The leakage of CB F3GA from the capillaries in IWTFE capillaries were coated with PVA by a a buffer solution with pH 7.3 and that of human plastwo-step procedure["]. In the first step, PVA was dema was followed by treating the capillaries with posited on the surface of the capillary by a simple adaqueous solution or fresh human plasma sample for 24 sorption process carried out in an aqueous medium. h at room temperature. The CB F3GA released after The initial PVA concentration was 40 mg/mL. The this treatment was measured in the liquid phase specsolution(MPTFE capillaries added) was stirred for 2 h trophotometrically at 630 nm"']. with a magnetic stirrer at 200 rimin and room tempe2.3.4 Characterization of PVA-coated PTFE Capilrature. lary Surface In the second step, PVA molecules adsorbed on The unmodified PTFE capillary consists of the MPTFE capillaries were chemically cross-linked 68.35% fluorine and 31.65% carbon. The peak at to provide a stable PVA coating on the surface. After 689.05 eV is attributed to FI, whereas the peaks at adsorption of PVA from the solution, the final acid 291.1 1 and 292.45 eV are attributed to the C in -CF2 concentration of the medium was adjusted to 0.1 No.2
mol/L by adding HCI. A 10-mg amount of terephthaldehyde was dissolved in 10 mL of water and the solution was added to the previous medium. Then, stirring was stopped, the temperature was increased to 80 "C, and cross-linking was completed in 2 h. The capillaries were washed several times with hot distilled water. The PVA-coated capillaries were stored under distilled
and -CF3(Fig. 1). When compared to the unmodified PTFE capillary, the percentage of F(only 1.79%) is obviously lower while the percentages of 0 and C are obviously higher. The results show that PVA is sucessfully coated onto the PTFE surface. Fig.2 depicts the FESEM photograph of the unmodified and modified PTFE capillaries. The unmodi-
CHEM. RES. CHINESE UNIVERSITIES
156
vo1.24
(B)
(A)
I
I
I
I
I
I
I
I
I
I
I
Fig.2 FESEM photographs of unmodified and modified PTFE capillaries (A) Unmodified capillary; (B) partially magnified unmodified capillary; (C) PVA-coated capillary.
2.4 On-line Adsorption and Analytical System
To study the dynamic adsorption of albumin, we adopted a kind of FIA system with double channels"']. The affinity MPTFE capillaries(5 cm long MPTFE capillary was used) were connected to the flow injection system directly. The affinity capillary was equipped with a water bath apparatus for controlling temperature. The sample solution containing albumin with different concentrations(i.e., HSA solution in 0.01 molL Tris-0.01 mol/L NaCl, at pH 5 or plasma) was transferred to MPTFE and deposited on it when six valves were fixed at the position of sampling. Then, pumping was stopped, and the six valves were changed to the position of eluting; the pump was started and the eluent(0.01 mol/L Tris-0.5 mol/L NaSCN, pH 8) and developer(arsenaz0 K) were transferred at the same time to elute the adsorbed albumin. The eluted solution was sent to the flow cell to on-line determine the adsorbed albumin at 611 nm after it was reacted adequately by a K-R(a kind of knitted PTFE capillary). The reason for choosing arsenazo K as a developer is that it can react with albumin rapidly, and has the same sensitivity of determination for protein. It is very convenient to determine the adsorption
capacity of albumin using this kind of system. Moreover, the system is fit for dynamic adsorption studying of albumin and clinic analysis. 2.5 Human Serum Albumin Adsorption from Aqueous Solution
The amount of adsorbed HSA(or adsorption capacity) from the solution was described by the following equation:
T=@O-p) VJm (1) where, T is the amount of HSA adsorbed onto the unit mass of the capillary(mg/g polymer); po and p are the concentrations of HSA in the initial(before adsorption) and the final solution(after adsorption)(mg/mL), respectively; V is the volume of the human plasma(mL); and m is the mass of the capillary(g). The concentration of albumin in solution(aqueous solution or plasma) was determined by a Hitachi 7060 automated analyzer. The amount of HSA adsorbed onto the unit mass of the capillary can also be determined by a tailored FIA on-line system"01. In a typical flow injection system, 50 mL of the aqueous solution of HSA was recirculated through the modified capillary for 2 h. The temperature was kept at 25 "C. The HSA adsorption capacity onto the mo-
No.2
JIN GUet al.
dified capillary was determined by measuring the initial and final concentrations of HSA within the Hitachi 7060 automated analyzer. In addition, the flow rate, the pH, and the ionic strengths of the aqueous protein solution(HSA initial concentration 5.0 mg/mL) were investigated as mentioned above. The HSA desorption experiments were performed in a buffer solution containing 1.0 moVL NaSCN at pH 8.0. Briefly, 25 mL of the NaSCN solution was recirculated through the HSA-containing adsorbed affinity capillary for 30 min. The desorption ratio was calculated via the following expression: DR(%)=[(DxlOO)/A] x 100 (2) where, DR is the desorption ratio(%); D and A are the amounts of albumin desorbed from and adsorbed on the affinity capillaries, respectively.
157
The purity of HSA was calculated via the following expression:
P(%)=D"x 1 OOIDp (3) where, P is the purity of HSA(%); DH and Dp are the amounts of human serum albumin desorbed and the total protein desorbed from the affinity capillaries, respectively.
3 Results and Discussion 3.1 Surface Modification of PTFE Membranous Capillary
The reactive equation of activating the PTFE with the piranha solution is as follows:
2.6 Human Serum Albumin Adsorption from Human Plasma
Human serum albumin adsorption studies were carried out in the same flow-injection system. Blood samples were centrifuged at 600 g for 20 min and room temperature to separate the plasma. The initial human plasma of the donor contained 42.3 mg/mL HSA as determined by the Hitathi 7060 automated analyzer. In a typical continuous flow-injection system, 25 mL of the plasma freshly separated from the human blood was recirculated through the CB F3GA affinity capillaries for 2 h. The amount of human serum albumin adsorbed was obtained by measuring the decrease in the HSA concentration in the plasma by the same assay. Phosphate-buffered saline(PBS, pH 7.4, NaCl 0.9%) was used for the dilution of human plasma. In this experiment, the flow-rate of the human plasma was 0.5 mL/min. The temperature was kept at 25 "C. The same desorption procedure was applied for desorption of adsorbed as mentioned above. To observe the performance of affinity capillaries in this group of experiment, the purity of HSA was also studied. The total protein concentration was measured by a Total Protein Reagent Kit and the HSA concentration was determined by an Albumin reagent Kit. Chronometric determination of fibrinogen according to the VonClauss method was performed using a Fibrinogen Kit"61. The y-Globulin concentration was determined from the difference. The initial human plasma contains albumin(42.3 mg/mL), fibrinogen(2.9 mg/mL), and y-Globulin( 18.7 mg/mL).
U
The adsorption capacity of albumin onto the PVA-coated affinity microporous PTFE capillary is only (0.408+0.028) albumin molecules/mm2when the activated PTFE is coupled indirectly with dye"21. However, the experimental results show that the maximum adsorption capacity of HSA onto the PVA-coated affinity microporous PTFE capillary is 85.3 mg/g polymer when the PVA is grafted onto the activated PTFE membranous capillary. The relation between the concentration of PVA and the amount of PVA immobilized onto the PTFE membranous capillary is displayed in Table 1. Table 1 Aspect and amount of PVA immobilized on the PTFE membranous capillary Concentration of PVA/(mgmL-')
Immobilized mass of PVA/(mg.mL?)
5
Undetected
10
0.6
20
5.9
40
23.5
50
*
Appearance
Uneven Homogenized partly Homogenous Uneven
~~
* Membranous capillary is easily plugged up because of flocculation of PVA.
In this study, MPTFE capillary was selected as the carrier matrix. MPTFE capillary is a new type of functional material, which has a fairly large pore size on the surface and a large porosity as described in our previous articles'"]. The MPTFE capillary is used in the field of analy~is"~'.As already known, the modification of PTFE is performed by the methods of irradiation(ie., pradial, laser, and plasma)['8-201;however, these methods require special equipments and
158
CHEM. RES. CHINESE UNIVERSITIES
destroy the surface structure completely. Now, it can result in a sufficient number of free functional groups without damaging the structural integrity of PTFE polymer using wet chemical methods, Lohbach[12]first modified PTFE vessels with the fresh piranha solution; Watanabe"" modified the PTFE capillary with a NaOH solution. However, the surface of PTFE remains hydrophobic and the active site is quite low. In this study, the PVA was coupled to the surface of PTFE, which can serve as an intermediate for further attachment of CB F3GA to an immobilized molecule. Subsequently, CB F3GA was covalently coupled to the MPTFE capillary. The average density of CB F3GA attached on the capillary is 89.8 ymollg polymer. It is reported that CB F3GA has no adverse effect on biochemical systems[211. Its specific structure(hence their specific affinities) and the dye contents are responsible for its adsorption capacity. CB F3GA has a structure similar to that of bilirubin, and can adsorb more tightly HSA. The studies of CB F3GA leakage from the modified membranous capillaries showed that there was no leakage in any media studied, which indicated that the washing procedure was quite satisfactory for the removal of non-specific adsorbed dye molecules from the membranous capillaries. 3.2 Adsorption from Aqueous Solution
Effects of Flow-rate on Adsorption With the increase of the flow-rate from 0.5 to 2.5 mL/min, the adsorption capacity decreased significantly from 81.2 to 23.6 HSA m d g polymer. This change may be due to the decrease of the residence time in the capillary, which does not provide sufficient time for HSA to interact with the adsorbent or may be due to the change of HSA topography. As already known, the specific interaction of proteins with immobilized small molecules often varies with the change in the protein surface, chemical, and physical variables. Hence, the orientation effect is an important factor for the interaction between small molecules and bio-macromolecules(HSA). Thus, low adsorption capacities were observed at high flow-rates. At flow-rates lower than 0.5 mL/min, some technical problems were encountered in our experiments; therefore, we carried out all other adsorption tests at a flow-rate of 0.5 mL/min. 3.2.2 Effects ofpH Fig.3 provides the effect of pH(4-8) on HSA 3.2.I
Vo1.24
90 80 -
4%
70-
60 -
#-p-
'' / -o a~ 4 0 -
30-
20
L-.
JIN GUet al. 159 ment with the results reported in the literature [23]. tached capillaries significantly increase the capacity of This may be explained by the formation of more HSA adsorbed(up to 85.3 mg/g polymer) because of compact structures of the albumin molecules at high the specific interactions between albumin and CB ionic strengths. More ions may also be attached to F3GA molecules. albumin molecules at high ionic strengths. This causes (I firther stabilization of the protein molecules(higher a solubility), which may lead to lower adsorption of albumin on the affinity capillaries. 3.2.4 Adsorption Rate Fig.4 shows the adsorption rate curves obtained by the decrease of the concentration of HSA within the protein solution with time. As seen, the relatively p(HSA)/(mgmiL-') faster adsorption rates were observed at the beginning Fig.5 Effects of the initial HSA concentration on of the adsorption process, and then, the adsorption HSA adsorption equilibriums were achieved gradually in about 60 min. Flow rate: 0.5 mL/min; pH 5 ; ionic strength: O.Ol(adjusted with NaCI); This may be due to the decrease in the HSA concentemperature:25 ' C ; total solution volume: 50 mL. u. CB F3GA modified PTFE capillary; b. unmodified PTFE capillary. tration with time by adsorption. The driving force decrease(i.e., the concentration difference between the 3.3 HSAAdsorption from Human Plasma aqueous solution and affinity) results in a drop of the The results of HSA adsorption from human adsorption rate when the HSA concentration in the plasma are summarized in Table 2. There is low admobile phase decreases. In addition, the adsorption sorption of HSA on the plain capillaries(the amount of rate increases with increasing HSA concentration. HSA adsorbed is 2.3 mg/g polymer), and a higher adThis may be due to a high driving force, which is the of HSA on the membranous capillary(up to sorption HSA concentration difference between the mobile 85.3 mg/g polymer) was obtained when the affinity phase(i.e., the protein solution) and the stationary capillaries were used. phase(i.e., the affinity capillaries), in the case of high Table 2 HSA adsorption from human HSA concentration. No.2
--.-.-.
6
90
plasma (healthy donor)
-
Q
E 80-
*,
2
f
Y
<
7060 5040 30-
Adsorptioin timdmin
Fig.4 Effects of time on the HSA adsorption at different concentrations Flow rate 0.5 mUmin; pH 5; ionic strengh: O.Ol(adjusted with NaCI); temperature 25 "C; total solution volume. 50 mL. p(Albumin)/(mgmL~'): a. 5.0;b. 4.0;c. 2.0;d. 1.0;e. 0.5.
3.2.5 Effects of the HSA Initial Concentration Fig.5 shows the non-specific and specific adsorptions of HSA onto the plain capillaries and dye immobilized capillaries, respectively. As seen from Fig.5, the amount of HSA adsorbed on the unmodified capillaries is quite low, which is about 2.3 mg/g polymer. With increasing HSA concentration in solution, the capacity of HSA adsorbed increases. CB F3GA at-
HSA conwnbatiod (mgmL-')
Amount of protein adsorbed/(mgg-' polymer) Fibrinogen y-Globulin HSA
2.6 5.3
0.50
0.62
0.80
1.25
12.3
10.5
1.15
1.85
82.6
56.8
21.2
1.56
2.31
98.5
42.3
2.05
2.88
125.8
As presented in Table 2, the adsorption of HSA onto affinity capillaries in plasma was higher(about 1.5 folds) than those obtained in aqueous solution. This may be due to the conformational structure of HSA molecules in human plasma being considerably more suitable for specific interaction with the affinity capillaries. In addition, the high HSA concentration(42.3 mg/mL) may also contribute to this high adsorption capacity on affinity capillaries. There is a competitive adsorption in human plasma, and therefore, other protein molecules may also be adsorbed. The experiment of competitive protein adsorption was also carried out in these studies. As seen from Table 2, the adsorptions of other plasma proteins(i.e., fibrinogen and y-Globulin) on the affinity capillaries are low.
CHEM. RES. CHINESE UNIVERSITIES
160
It should be noted that HSA is the most abundant protein in plasma. It generally constitutes more than half of the total proteins in plasma. The low adsorptions of fibrinogen and y-Globulin are owing to the high concentration of HSA. This maybe due to the hydrophilic surface of PVA-coated MPTFE capillary. 3.4 Desorption of HSA
Desorption of HSA was carried out in a 1.0 mol/L NaSCN solution at pH 8. In a typical desorption of HSA, 25 mL of eluant was recirculated through the HSA adsorbed affinity capillaries for 30 min, at 25 "C. As presented in Tables 3 and 4, more than 92% of the adsorbed albumin molecules could be easily desorbed from the affinity capillaries. When the NaSCN is used, the specific interaction forces(such as electrostatic and hydrophobic forces) between the albumin molecules and the dye molecules are broken, which forces the desorption of albumin molecules from the solid matrix. In addition, SCN- ions bind albumin molecules easily, which also affects the conformational structure of the adsorbed albumin molecules. Furthermore, the electrostatic interactions between the adsorbed protein and CB F3GA can be easily decreased because of the conformational changes of albumin molecules. Table 3 Desorption of albumin from affinity capillaries in aqueous solution p(Aqueous solution)/(memL3
05 10 20 40 50
HSA adsorbed/ (mamL-')
Desorption ratio (%)
28 9 56 6 70 4 80 3 85 3
96 6 95 8 95 2 96 2 94 3
Table 4 Desorption of albumin from affinity capillaries in human plasma p(Human plasma)/ (mgmL-') 2.6 5.3 10.5 21.2 42.3
HSA adsorbed/ (mgg-') 56.8 72.3 82.1 98.6 125.8
Desorption ratio (%)
PurltY(%)
94.5 94.3 92.8 93.5 93.2
93.6 92.8 93.5 94.3 91.9
3.5 Reusability
The reusability is one of the important advantages of this novel sorbent. To show the reusability of the affinity capillaries, the adsorption-desorption cycle of HSA was repeated ten times with the same capillaries. There was no remarkable reduction in the adsorption capacity of the affinity capillaries. The adsorption capacity of HSA on the affinity capillaries decreased
Vo1.24
only 3.9% after ten cycles. 3.6
Estimate of Adsorption Capacities
Note that different sorbents with different adsorption capacities were reported in the literature for albumin adsorption. McCreath et al.'241showed an equilibrium adsorption capacity of 9.7 mg.g-' HSA.mL-' with the PVA coated perfluoropolymer containing anion exchange and cation exchange groups. Zeng et al.[14]reported an adsorption capacity of 10.2 mg HSA/g with Cibacron Blue F3GA attached polyethersulfone supported chitosan sorbent. Li et al.'251used Cibacron Blue F3GA-attached poly (ethylene imine)-coated titania to achieved an adsorption capacity of 4.4 mg HSA/g. Denizli et al.'26,271 obtained adsorption capacities of 3 5-2 1 5 mg HSA/g polymer. Nash and ChaseL2*] presented adsorption capacities of l l .7-27 mg HSA/g. The maximum HSA adsorption achieved with the sorbent system developed in this study was 85.3 mg/g polymer, which is reasonably comparable with the related literature.
4 Conclusions Mechanically stable, microporous PTFE membrane capillary was modified by PVA. The capillaries carrying the PVA were incorporated with Cibacron Blue F3GA. The dye/PVA attached capillaries have a high adsorption capacity, a shorter time of saturation adsorption and eluting, and the desorption ratio(%) is not less than 95%. The adsorption under different pH values and ionic strengths and the desorption with different eluants have revealed the high selectivity of Cibacron Blue F3GA membranous capillary for HSA. The interaction of HSA with immobilized CB F3GA can be categorized into specific interactions and non-specific interactions. Acknowledgement We are very grateful to Professor Watanabe(University of Science and Technology of Tokyo) for providing the microporous PTFE membranous capillary. References [ l ] Quadri F., Dean P. D. G., Biochem. J., 1980,191,53 [2] Lowe C. R., Hans H., Spibey N., et al., Anal. Biochem., 1980, 103, 23 [3] Bohme H. J., Kopperschlager G., Schulz J., ef al., J . Chromarogr., 1972,209,61
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No.2 [4] Dean P. D. G., Watson D. H., J. Chromatogr., 1979,165,301
[ 5 ] Denizli A,, Tuncel A,, Kozluca A,, et al., Sep. Sci. Technol., 1997,32,
103 161 Lowe C. R., Small D. A. P., Atkinson A,, L . Biochem., 1981,13,33 [7] Hou K. C., Mandaro R. M., Biotechniques, 1986,4,358 [ 8 ] Thommes J., Kula M., Biotechnol. Prop,, 1995, 11, 357
[9] Roper D., Lightfoot E., J. Chromatogr., 1!)95, 702, 3
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[19] Svorcik V., Rockova K., Ratajova E., Nuclear Instruments cmd Methods in Physics Research Section B: Beam Interactions with Mate-
rials anddtom, 2004,217, 307
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[20] Tu C. Y., Chen C. P., Wang Y. C., European Po4vmer Journal, 2004,
40,1541 [21] Weber B., Willeford K., Moe J., Biochem. Biophys. Res. C’ommun., 1979,86,252
[lo] Zhang L., Jin G., Chinese ChemicalLetters, 2005, 16, 1495
[22] Denizli A., Salih B., $enel S., J. Appl. folym. Sci., 1998, 68, 657
[ l l ] Zhang L., Jin G., J. Chromatog. B, 2005, 821, 112
[23] Chen W. Y., Wu C. F., Liu C. C., J. Colloid Interface Sci., 1996. 180, 135
[ 121 Lohbach C., Bakowsky U., Kncuer C., et nl., Chem. Commun., 2002,
2568 [I31 Tuncel A,, Denizli A,, Purvis D., et al., J. Chromatog. A, 1993, 634, 161
[24] MeCreath G. E., Owen R. D., Nash D. C., J. Chromatogr. A , 1997, 773, 73
[14] Zeng X. F., Ruckenstein E., J. Membr. Sci., 1996, 227,271
[25] Li Y., Spencer H. G . , Shalaby W., et al., Polymers of Biologicul and Biomedical Signijkance, ACS, Washington DC, 1994, 297
[IS] Ruckenstein E., Zeng X. F., J. Membr. Sci., 1998,142, 13 [ 161 National Committee for Clinical Laboratory Standards(USA),
[26] Denizli A,, K6kturk G., Yavuz H., React. Funcf. Polym., 1999, 40, 195
NCCLS, Pennsylvania, 1999,99
[17] lburaim A,, ltagaki M., Watanabe K., Bunseki Kagaku, 2001,50,739 [I81 Lunkwitz K , Lappan U., Cheler S. U., Journal ojFluorine Chemi.stry, 2004, 125, 863
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Available online at www.sciencedirect.com
2008,24(2), 154-161 Article ID 1005-9040~2008~-02-154-08
ScienceDirect
Adsorption of Human Serum Albumin onto PVA-coated Affinity Microporous PTFE Capillary JIN Gu', YAO Qi-zhi and ZHANG Lei Department of Chemistry, University of Science and Technology of China, He@ 230026, P. R. China
Abstract Affinity dye-ligand Cibacron Blue F3GA(CB F3GA) was covalently coupled with poly(viny1 alcohol)(PVA) coated on the inner surface of microporous poly(tetra-fluoroethylene)(MPTFE) membranous capillary. The PVA-coated PTFE capillary surface was characterized by XPS and FESEM. The grafting degree of PVA and the amount of CB F3GA immobilized onto the membranous capillary were 23.5 mg/g and 89.6 pmol/g, respectively. These dyed membranous capillaries were chemically and mechanically stable, and could be reproducibly prepared. Human serum albumin(HSA) was selected as model protein. The saturation adsorbance of the dye attached membranous capillary was 85.3 mg HSNg, while the capacity of non-specific adsorption for HSA was less than 0.3 mg/g. Keywords Albumin adsorption; Cibacron blue F3GA; PVA; PTFE; Plasma
1 Introduction Immobilized dye-ligand affinity chromatography has been recognized as an important technique for the separation, purification, and recovery of a wide range of proteins and enzymes. Various dyes were coupled to different bead-based matrices, such as dextran"], agaroseL2],p~lyacrylamide[~], cellulose[41,PVA['], and silica gelK6].However, their use is limited by the compressibility of the soft gels at high flow rates, the high pressure drops for small size beads, and the mass-transfer limitation owing to the internal diffusion. In recent years, the adsorptive membrane has emerged as an alternative to the traditional column chromatography since it brings the solute into close proximity to bound ligands through convective transport, thus reducing the mass-transfer resistance and enabling lower pressure drops and higher flow rate^[^-^]. However, the capacity of membranes is lower than that of column. In addition, membrane fouling is another problem. At present, the preparation and application of a new form of adsorbent is an interesting work. The coating method is the simplest way to modify the membrane surface by introducing hydrophilic and functional polymers. In our previous study, the membranous capillaries were modified by HSA via a radia-
tion-induced graft polymerization technique, and by CB F3GA via a chemical method for bilirubin removal from human In this study, the new sorbent was composed of CB F3GA as dye affinity ligand, and microporous membranous poly(tetrafluoroethylene) (MPTFE) capillary as the carrier matrix. To prevent non-specific interactions between the hydrophobic MPTFE surface and protein molecules, and to attach the ligand (CB F3GA) to the matrix, the capillaries were coated with a hydrophilic layer of PVA after activation. The results of albumin adsorption-desorption studies from aqueous media and human plasma are presented in this article. To our knowledge, a similar method using MPTFE capillaries for HSA adsorption has not been reported earlier.
2 Experimental 2.1
Chemicals
Microporous membranous PTFE capillaries provided by Professor Watanabe, were used for the present study. The capillaries have an internal diameter of 2 mm, and a wall thickness of approximately 0.5 mm. PVA(average Mr 14000, 100% hydrolysed) was purchased from the Chemical Reagent Company of Shanghai, China. CB F3GA and HSA (lyophilized, fraction V) were purchased from Sigma. The blood
*Corresponding author. E-mail: fanruiqing@l63 .com Received May 28,2007; accepted September 5,2007. Supported by the National Natural Science Foundation of China(No.29405038). Copyright 02008, Jilin University. Published by Elsevier Limited. All rights reserved.
JIN GUet al. 155 water. samples were obtained from a healthy human donor. Reagents such as terephthaldehyde, sodium chloride, The PVA adsorbed onto the capillaries was desodium carbonate, hydrochloride acid, etc. were of termined by measuring the initial and final concentraanalytical reagent grade(purchased from Chemical tions of PVA within the adsorption medium, according Reagent Company of Shanghai, China). The fresh pito the KI-12 method, spectrophotometrically at 690 nm[l 31 ranha solution was prepared in concentrated sulfuric acid and hydrogen peroxide(vo1ume ratio 1:1). 2.3.3 immobilization of Cibacron Blue F3GA Briefly, 0.6 g of CB F3GA was dissolved in 20 2.2 Apparatus mL of water; then, after the PVA-coated capillaries were immersed into it for 60 min at 60 "C, 1 g of NaCl A flow injection system (Model FI-2100, Beijing was added. Subsequently, 0.5 g of Na2C03was added Haiguang Instrument Co., China) was used for the to adjust the pH value (to about pH 10) of the solution. feeding of solution. The concentration of protein in The reaction then took place in the following 4 h. The the samples was determined by a Model 7060 autocapillaries were washed with distilled water and memated analyzer(Hitachi, Japan, total protein reagent thanol several times until all unbound dye was reKit: Cat. No. TP7143 and Albumin reagent Kit: Cat. moved. The dye immobilized capillaries were stored No. AB7162, Randox Laboratories Ltd., UK). Fibriin a phosphate solution(pH 7.0) containing 0.02% nogen Kit was obtained from Stago(Cat. Ref. No. (mass fraction) sodium a i d e at 4 "C to prevent mi00608 and 00625, Diagnostica Stago, Asnierescrobial contamination. sur-Seine, France). The dye content of the capillaries was determined 2.3 Cibacron Blue F3GA-attached MPTFE spectrophotometrically by first hydrolyzing the capillaries in an aqueous solution of 12 mol/L hydrochloric 2.3.I Activation of Unmodified MPTFE Capillaries acid, at 80 "C, for 30 min. The solution was then diMPTFE capillaries were first activated by a fresh luted to 6 mol/L with distilled water, and neutralized piranha solution"21. The capillaries were incubated in with an aqueous solution of 6 mol/L NaOH. The con20 mL of fresh piranha solution at room temperature centration was then determined spectrophotometricalfor 40 min. After that, the capillaries were washed ly at its maximum absorbance wavelength(i,,,=6 10 several times with distilled water. nm)[I41. 2.3.2 Coating ofMPTFE Capillaries with PVA The leakage of CB F3GA from the capillaries in IWTFE capillaries were coated with PVA by a a buffer solution with pH 7.3 and that of human plastwo-step procedure["]. In the first step, PVA was dema was followed by treating the capillaries with posited on the surface of the capillary by a simple adaqueous solution or fresh human plasma sample for 24 sorption process carried out in an aqueous medium. h at room temperature. The CB F3GA released after The initial PVA concentration was 40 mg/mL. The this treatment was measured in the liquid phase specsolution(MPTFE capillaries added) was stirred for 2 h trophotometrically at 630 nm"']. with a magnetic stirrer at 200 rimin and room tempe2.3.4 Characterization of PVA-coated PTFE Capilrature. lary Surface In the second step, PVA molecules adsorbed on The unmodified PTFE capillary consists of the MPTFE capillaries were chemically cross-linked 68.35% fluorine and 31.65% carbon. The peak at to provide a stable PVA coating on the surface. After 689.05 eV is attributed to FI, whereas the peaks at adsorption of PVA from the solution, the final acid 291.1 1 and 292.45 eV are attributed to the C in -CF2 concentration of the medium was adjusted to 0.1 No.2
mol/L by adding HCI. A 10-mg amount of terephthaldehyde was dissolved in 10 mL of water and the solution was added to the previous medium. Then, stirring was stopped, the temperature was increased to 80 "C, and cross-linking was completed in 2 h. The capillaries were washed several times with hot distilled water. The PVA-coated capillaries were stored under distilled
and -CF3(Fig. 1). When compared to the unmodified PTFE capillary, the percentage of F(only 1.79%) is obviously lower while the percentages of 0 and C are obviously higher. The results show that PVA is sucessfully coated onto the PTFE surface. Fig.2 depicts the FESEM photograph of the unmodified and modified PTFE capillaries. The unmodi-
CHEM. RES. CHINESE UNIVERSITIES
156
vo1.24
(B)
(A)
I
I
I
I
I
I
I
I
I
I
I
Fig.2 FESEM photographs of unmodified and modified PTFE capillaries (A) Unmodified capillary; (B) partially magnified unmodified capillary; (C) PVA-coated capillary.
2.4 On-line Adsorption and Analytical System
To study the dynamic adsorption of albumin, we adopted a kind of FIA system with double channels"']. The affinity MPTFE capillaries(5 cm long MPTFE capillary was used) were connected to the flow injection system directly. The affinity capillary was equipped with a water bath apparatus for controlling temperature. The sample solution containing albumin with different concentrations(i.e., HSA solution in 0.01 molL Tris-0.01 mol/L NaCl, at pH 5 or plasma) was transferred to MPTFE and deposited on it when six valves were fixed at the position of sampling. Then, pumping was stopped, and the six valves were changed to the position of eluting; the pump was started and the eluent(0.01 mol/L Tris-0.5 mol/L NaSCN, pH 8) and developer(arsenaz0 K) were transferred at the same time to elute the adsorbed albumin. The eluted solution was sent to the flow cell to on-line determine the adsorbed albumin at 611 nm after it was reacted adequately by a K-R(a kind of knitted PTFE capillary). The reason for choosing arsenazo K as a developer is that it can react with albumin rapidly, and has the same sensitivity of determination for protein. It is very convenient to determine the adsorption
capacity of albumin using this kind of system. Moreover, the system is fit for dynamic adsorption studying of albumin and clinic analysis. 2.5 Human Serum Albumin Adsorption from Aqueous Solution
The amount of adsorbed HSA(or adsorption capacity) from the solution was described by the following equation:
T=@O-p) VJm (1) where, T is the amount of HSA adsorbed onto the unit mass of the capillary(mg/g polymer); po and p are the concentrations of HSA in the initial(before adsorption) and the final solution(after adsorption)(mg/mL), respectively; V is the volume of the human plasma(mL); and m is the mass of the capillary(g). The concentration of albumin in solution(aqueous solution or plasma) was determined by a Hitachi 7060 automated analyzer. The amount of HSA adsorbed onto the unit mass of the capillary can also be determined by a tailored FIA on-line system"01. In a typical flow injection system, 50 mL of the aqueous solution of HSA was recirculated through the modified capillary for 2 h. The temperature was kept at 25 "C. The HSA adsorption capacity onto the mo-
No.2
JIN GUet al.
dified capillary was determined by measuring the initial and final concentrations of HSA within the Hitachi 7060 automated analyzer. In addition, the flow rate, the pH, and the ionic strengths of the aqueous protein solution(HSA initial concentration 5.0 mg/mL) were investigated as mentioned above. The HSA desorption experiments were performed in a buffer solution containing 1.0 moVL NaSCN at pH 8.0. Briefly, 25 mL of the NaSCN solution was recirculated through the HSA-containing adsorbed affinity capillary for 30 min. The desorption ratio was calculated via the following expression: DR(%)=[(DxlOO)/A] x 100 (2) where, DR is the desorption ratio(%); D and A are the amounts of albumin desorbed from and adsorbed on the affinity capillaries, respectively.
157
The purity of HSA was calculated via the following expression:
P(%)=D"x 1 OOIDp (3) where, P is the purity of HSA(%); DH and Dp are the amounts of human serum albumin desorbed and the total protein desorbed from the affinity capillaries, respectively.
3 Results and Discussion 3.1 Surface Modification of PTFE Membranous Capillary
The reactive equation of activating the PTFE with the piranha solution is as follows:
2.6 Human Serum Albumin Adsorption from Human Plasma
Human serum albumin adsorption studies were carried out in the same flow-injection system. Blood samples were centrifuged at 600 g for 20 min and room temperature to separate the plasma. The initial human plasma of the donor contained 42.3 mg/mL HSA as determined by the Hitathi 7060 automated analyzer. In a typical continuous flow-injection system, 25 mL of the plasma freshly separated from the human blood was recirculated through the CB F3GA affinity capillaries for 2 h. The amount of human serum albumin adsorbed was obtained by measuring the decrease in the HSA concentration in the plasma by the same assay. Phosphate-buffered saline(PBS, pH 7.4, NaCl 0.9%) was used for the dilution of human plasma. In this experiment, the flow-rate of the human plasma was 0.5 mL/min. The temperature was kept at 25 "C. The same desorption procedure was applied for desorption of adsorbed as mentioned above. To observe the performance of affinity capillaries in this group of experiment, the purity of HSA was also studied. The total protein concentration was measured by a Total Protein Reagent Kit and the HSA concentration was determined by an Albumin reagent Kit. Chronometric determination of fibrinogen according to the VonClauss method was performed using a Fibrinogen Kit"61. The y-Globulin concentration was determined from the difference. The initial human plasma contains albumin(42.3 mg/mL), fibrinogen(2.9 mg/mL), and y-Globulin( 18.7 mg/mL).
U
The adsorption capacity of albumin onto the PVA-coated affinity microporous PTFE capillary is only (0.408+0.028) albumin molecules/mm2when the activated PTFE is coupled indirectly with dye"21. However, the experimental results show that the maximum adsorption capacity of HSA onto the PVA-coated affinity microporous PTFE capillary is 85.3 mg/g polymer when the PVA is grafted onto the activated PTFE membranous capillary. The relation between the concentration of PVA and the amount of PVA immobilized onto the PTFE membranous capillary is displayed in Table 1. Table 1 Aspect and amount of PVA immobilized on the PTFE membranous capillary Concentration of PVA/(mgmL-')
Immobilized mass of PVA/(mg.mL?)
5
Undetected
10
0.6
20
5.9
40
23.5
50
*
Appearance
Uneven Homogenized partly Homogenous Uneven
~~
* Membranous capillary is easily plugged up because of flocculation of PVA.
In this study, MPTFE capillary was selected as the carrier matrix. MPTFE capillary is a new type of functional material, which has a fairly large pore size on the surface and a large porosity as described in our previous articles'"]. The MPTFE capillary is used in the field of analy~is"~'.As already known, the modification of PTFE is performed by the methods of irradiation(ie., pradial, laser, and plasma)['8-201;however, these methods require special equipments and
158
CHEM. RES. CHINESE UNIVERSITIES
destroy the surface structure completely. Now, it can result in a sufficient number of free functional groups without damaging the structural integrity of PTFE polymer using wet chemical methods, Lohbach[12]first modified PTFE vessels with the fresh piranha solution; Watanabe"" modified the PTFE capillary with a NaOH solution. However, the surface of PTFE remains hydrophobic and the active site is quite low. In this study, the PVA was coupled to the surface of PTFE, which can serve as an intermediate for further attachment of CB F3GA to an immobilized molecule. Subsequently, CB F3GA was covalently coupled to the MPTFE capillary. The average density of CB F3GA attached on the capillary is 89.8 ymollg polymer. It is reported that CB F3GA has no adverse effect on biochemical systems[211. Its specific structure(hence their specific affinities) and the dye contents are responsible for its adsorption capacity. CB F3GA has a structure similar to that of bilirubin, and can adsorb more tightly HSA. The studies of CB F3GA leakage from the modified membranous capillaries showed that there was no leakage in any media studied, which indicated that the washing procedure was quite satisfactory for the removal of non-specific adsorbed dye molecules from the membranous capillaries. 3.2 Adsorption from Aqueous Solution
Effects of Flow-rate on Adsorption With the increase of the flow-rate from 0.5 to 2.5 mL/min, the adsorption capacity decreased significantly from 81.2 to 23.6 HSA m d g polymer. This change may be due to the decrease of the residence time in the capillary, which does not provide sufficient time for HSA to interact with the adsorbent or may be due to the change of HSA topography. As already known, the specific interaction of proteins with immobilized small molecules often varies with the change in the protein surface, chemical, and physical variables. Hence, the orientation effect is an important factor for the interaction between small molecules and bio-macromolecules(HSA). Thus, low adsorption capacities were observed at high flow-rates. At flow-rates lower than 0.5 mL/min, some technical problems were encountered in our experiments; therefore, we carried out all other adsorption tests at a flow-rate of 0.5 mL/min. 3.2.2 Effects ofpH Fig.3 provides the effect of pH(4-8) on HSA 3.2.I
Vo1.24
90 80 -
4%
70-
60 -
#-p-
'' / -o a~ 4 0 -
30-
20
L-.
JIN GUet al. 159 ment with the results reported in the literature [23]. tached capillaries significantly increase the capacity of This may be explained by the formation of more HSA adsorbed(up to 85.3 mg/g polymer) because of compact structures of the albumin molecules at high the specific interactions between albumin and CB ionic strengths. More ions may also be attached to F3GA molecules. albumin molecules at high ionic strengths. This causes (I firther stabilization of the protein molecules(higher a solubility), which may lead to lower adsorption of albumin on the affinity capillaries. 3.2.4 Adsorption Rate Fig.4 shows the adsorption rate curves obtained by the decrease of the concentration of HSA within the protein solution with time. As seen, the relatively p(HSA)/(mgmiL-') faster adsorption rates were observed at the beginning Fig.5 Effects of the initial HSA concentration on of the adsorption process, and then, the adsorption HSA adsorption equilibriums were achieved gradually in about 60 min. Flow rate: 0.5 mL/min; pH 5 ; ionic strength: O.Ol(adjusted with NaCI); This may be due to the decrease in the HSA concentemperature:25 ' C ; total solution volume: 50 mL. u. CB F3GA modified PTFE capillary; b. unmodified PTFE capillary. tration with time by adsorption. The driving force decrease(i.e., the concentration difference between the 3.3 HSAAdsorption from Human Plasma aqueous solution and affinity) results in a drop of the The results of HSA adsorption from human adsorption rate when the HSA concentration in the plasma are summarized in Table 2. There is low admobile phase decreases. In addition, the adsorption sorption of HSA on the plain capillaries(the amount of rate increases with increasing HSA concentration. HSA adsorbed is 2.3 mg/g polymer), and a higher adThis may be due to a high driving force, which is the of HSA on the membranous capillary(up to sorption HSA concentration difference between the mobile 85.3 mg/g polymer) was obtained when the affinity phase(i.e., the protein solution) and the stationary capillaries were used. phase(i.e., the affinity capillaries), in the case of high Table 2 HSA adsorption from human HSA concentration. No.2
--.-.-.
6
90
plasma (healthy donor)
-
Q
E 80-
*,
2
f
Y
<
7060 5040 30-
Adsorptioin timdmin
Fig.4 Effects of time on the HSA adsorption at different concentrations Flow rate 0.5 mUmin; pH 5; ionic strengh: O.Ol(adjusted with NaCI); temperature 25 "C; total solution volume. 50 mL. p(Albumin)/(mgmL~'): a. 5.0;b. 4.0;c. 2.0;d. 1.0;e. 0.5.
3.2.5 Effects of the HSA Initial Concentration Fig.5 shows the non-specific and specific adsorptions of HSA onto the plain capillaries and dye immobilized capillaries, respectively. As seen from Fig.5, the amount of HSA adsorbed on the unmodified capillaries is quite low, which is about 2.3 mg/g polymer. With increasing HSA concentration in solution, the capacity of HSA adsorbed increases. CB F3GA at-
HSA conwnbatiod (mgmL-')
Amount of protein adsorbed/(mgg-' polymer) Fibrinogen y-Globulin HSA
2.6 5.3
0.50
0.62
0.80
1.25
12.3
10.5
1.15
1.85
82.6
56.8
21.2
1.56
2.31
98.5
42.3
2.05
2.88
125.8
As presented in Table 2, the adsorption of HSA onto affinity capillaries in plasma was higher(about 1.5 folds) than those obtained in aqueous solution. This may be due to the conformational structure of HSA molecules in human plasma being considerably more suitable for specific interaction with the affinity capillaries. In addition, the high HSA concentration(42.3 mg/mL) may also contribute to this high adsorption capacity on affinity capillaries. There is a competitive adsorption in human plasma, and therefore, other protein molecules may also be adsorbed. The experiment of competitive protein adsorption was also carried out in these studies. As seen from Table 2, the adsorptions of other plasma proteins(i.e., fibrinogen and y-Globulin) on the affinity capillaries are low.
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It should be noted that HSA is the most abundant protein in plasma. It generally constitutes more than half of the total proteins in plasma. The low adsorptions of fibrinogen and y-Globulin are owing to the high concentration of HSA. This maybe due to the hydrophilic surface of PVA-coated MPTFE capillary. 3.4 Desorption of HSA
Desorption of HSA was carried out in a 1.0 mol/L NaSCN solution at pH 8. In a typical desorption of HSA, 25 mL of eluant was recirculated through the HSA adsorbed affinity capillaries for 30 min, at 25 "C. As presented in Tables 3 and 4, more than 92% of the adsorbed albumin molecules could be easily desorbed from the affinity capillaries. When the NaSCN is used, the specific interaction forces(such as electrostatic and hydrophobic forces) between the albumin molecules and the dye molecules are broken, which forces the desorption of albumin molecules from the solid matrix. In addition, SCN- ions bind albumin molecules easily, which also affects the conformational structure of the adsorbed albumin molecules. Furthermore, the electrostatic interactions between the adsorbed protein and CB F3GA can be easily decreased because of the conformational changes of albumin molecules. Table 3 Desorption of albumin from affinity capillaries in aqueous solution p(Aqueous solution)/(memL3
05 10 20 40 50
HSA adsorbed/ (mamL-')
Desorption ratio (%)
28 9 56 6 70 4 80 3 85 3
96 6 95 8 95 2 96 2 94 3
Table 4 Desorption of albumin from affinity capillaries in human plasma p(Human plasma)/ (mgmL-') 2.6 5.3 10.5 21.2 42.3
HSA adsorbed/ (mgg-') 56.8 72.3 82.1 98.6 125.8
Desorption ratio (%)
PurltY(%)
94.5 94.3 92.8 93.5 93.2
93.6 92.8 93.5 94.3 91.9
3.5 Reusability
The reusability is one of the important advantages of this novel sorbent. To show the reusability of the affinity capillaries, the adsorption-desorption cycle of HSA was repeated ten times with the same capillaries. There was no remarkable reduction in the adsorption capacity of the affinity capillaries. The adsorption capacity of HSA on the affinity capillaries decreased
Vo1.24
only 3.9% after ten cycles. 3.6
Estimate of Adsorption Capacities
Note that different sorbents with different adsorption capacities were reported in the literature for albumin adsorption. McCreath et al.'241showed an equilibrium adsorption capacity of 9.7 mg.g-' HSA.mL-' with the PVA coated perfluoropolymer containing anion exchange and cation exchange groups. Zeng et al.[14]reported an adsorption capacity of 10.2 mg HSA/g with Cibacron Blue F3GA attached polyethersulfone supported chitosan sorbent. Li et al.'251used Cibacron Blue F3GA-attached poly (ethylene imine)-coated titania to achieved an adsorption capacity of 4.4 mg HSA/g. Denizli et al.'26,271 obtained adsorption capacities of 3 5-2 1 5 mg HSA/g polymer. Nash and ChaseL2*] presented adsorption capacities of l l .7-27 mg HSA/g. The maximum HSA adsorption achieved with the sorbent system developed in this study was 85.3 mg/g polymer, which is reasonably comparable with the related literature.
4 Conclusions Mechanically stable, microporous PTFE membrane capillary was modified by PVA. The capillaries carrying the PVA were incorporated with Cibacron Blue F3GA. The dye/PVA attached capillaries have a high adsorption capacity, a shorter time of saturation adsorption and eluting, and the desorption ratio(%) is not less than 95%. The adsorption under different pH values and ionic strengths and the desorption with different eluants have revealed the high selectivity of Cibacron Blue F3GA membranous capillary for HSA. The interaction of HSA with immobilized CB F3GA can be categorized into specific interactions and non-specific interactions. Acknowledgement We are very grateful to Professor Watanabe(University of Science and Technology of Tokyo) for providing the microporous PTFE membranous capillary. References [ l ] Quadri F., Dean P. D. G., Biochem. J., 1980,191,53 [2] Lowe C. R., Hans H., Spibey N., et al., Anal. Biochem., 1980, 103, 23 [3] Bohme H. J., Kopperschlager G., Schulz J., ef al., J . Chromarogr., 1972,209,61
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