Effects of Spacer Arms on Cibacron Blue 3GA Immobilization and

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Effects of Spacer Arms on Cibacron Blue 3GA Immobilization and Lysozyme Adsorption Using Regenerated Cellulose Membrane Discs Shing-Yi Suen,* Shu-Ying Lin, and Hsin-Cheng Chiu Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan

By using different spacer arms to immobilize Cibacron Blue 3GA onto regenerated cellulose membrane discs, this work investigated their effects on ligand density and corresponding lysozyme adsorption. When EGDGE alone was adopted as a spacer arm, using 0.1 M EGDGE without the formation of liquid film in its reaction with the membrane disc led to higher adsorption capacities. When EGDGE was combined with another reagent with two end functional groups as a spacer arm, EGDGE + 1,4-diaminobutane performed better in both ligand density and adsorption capacity. In addition, the ligand densities using the combinations of EGDGE with another reagent were generally lower than those from EGDGE alone, whereas the ligand utilization percentages were higher. Moreover, adding a spacer arm did not affect the binding strength between lysozyme and Cibacron Blue 3GA, regardless of the type of spacer arms and their length, and the reaction condition. 1. Introduction Adsorption membranes have emerged as a promising separation technique because of their better masstransfer performance, namely having lower pressure drops, higher flow rates, and no intraparticle diffusion compared to traditional column processes.1-4 Besides these advantages, the versatility of designs and applications of adsorptive membranes have also received increased attraction and been investigated in many studies.2-25 Membrane discs are the most frequently utilized membrane type because of their low cost and simple manufacturing and cartridge design. Their adsorption and separation performances in laboratory scale have also been extensively investigated in recent studies.2,15,17,18,21-25 Even though good mass-transfer characteristics have been demonstrated, the application of membrane discs remains problematic. Whether or not membrane discs can provide more or the same adsorption capacity than conventional chromatographic particles remains unclear. If membrane discs cannot do this, this problem, which was not clarified in most studies, would significantly reduce their potential in practical applications. Our previous study26 compared the ligand density and lysozyme adsorption capacity of Cibacron Blue 3GAimmobilized regenerated cellulose membrane discs to commercial gel beads, Blue Sepharose CL-6B. That comparison revealed that, based on the solid volume, the immobilized ligand density of membrane discs was 30-fold greater than the reported ligand density of Blue Sepharose CL-6B, whereas the saturation capacity of lysozyme onto membrane discs was close to that for Blue Sepharose CL-6B. This finding suggested that the adsorption capacity of adsorptive membrane discs could compete with that of gel beads used in traditional chromatography. Moreover, their larger ligand density meant the membrane discs have the potential to offer a higher adsorption capacity than gel beads, but the ligand utilization percentage was strictly limited. In * To whom correspondence should be addressed. Tel: 8864-2852590. Fax: 886-4-2854734. E-mail: [email protected].

other words, a large portion of ligand sites was not utilized and remained unbinding during the adsorption process on the membrane discs. This observation was attributed either to the overcrowded ligand density resulting in binding difficulties for relatively large protein molecules or to the mass-transfer resistance of the liquid film on the solid membrane surface further hindering the protein molecules from reaching the ligand. To increase the ligand accessibility, employment of a spacer arm has been suggested.4 This study investigates how different types of spacer arms and arm lengths affect the immobilization of Cibacron Blue 3GA onto the regenerated cellulose membrane discs. The corresponding lysozyme adsorption capacity and ligand utilization percentage are also studied. These results can further be applied in the design of a dye-affinity membrane technique to optimize separation performance. 2. Experimental Section Materials. The membrane discs used in this work were 25-mm-diameter regenerated cellulose filters from Sartorius AG (Goettingen, Germany), with 80-µm thickness and 0.45-µm pore size. The chemical reagents used as spacer arms were obtained from commercial sources: ethylene glycol diglycidyl ether (EGDGE; TCI, Tokyo, Japan), 1,4-butanediol (Lancaster, Morecambe, England), 4-amino-1-butanol (TCI), 1,2-diaminoethane (Wako Pure Chemical, Osaka, Japan), 1,4-diaminobutane (Lancaster), 1,6-diaminohexane (TCI), and 1,10diaminodecane (TCI). Cibacron Blue 3GA (C9534) and chicken egg white lysozyme (L6876, MW 14300) were purchased from Sigma Chemical (St. Louis, MO). Spacer Arm Coupling. EGDGE as a Spacer Arm. The main spacer arm used in this work is EGDGE, a reagent with two end epoxide groups. EGDGE was chosen as a spacer arm because its epoxide groups are reactive with both the hydroxyl groups on the regenerated cellulose membrane surface and the ligand Cibacron Blue 3GA. The EGDGE reaction with the regenerated cellulose membrane is depicted in Figure 1, and the procedures are described as follows.

10.1021/ie990421t CCC: $19.00 © 2000 American Chemical Society Published on Web 12/31/1999

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Figure 1. Schematic diagram for the reactions of the regenerated cellulose membrane disc with different spacer arm reagents.

One dry 25-mm membrane disc was immersed into a solution of 0.1 M EGDGE and 100 µL of BF3 etherate (as a catalyst) in 5 mL of an isopropanol-water azeotrope (87.8:12.2 w/w). The reaction was first carried out in vacuo at room temperature. After 2 h, the solution was partially removed, and the remaining solution with the membrane was placed into a vacuumed desiccator for a further 15 h. In the above step, the azeotrope solvent was gradually evaporated to reduce the diffusional resistance for EGDGE molecules to reach the hydroxyl groups on the membrane surface. Different solution volumes were retained with the membrane to evaluate an optimal film thickness for a more efficient reaction. The volumes tested were 0 (without a liquid film remaining), 1, and 3 mL. After the reaction, the membrane was rinsed with deionized water to remove the unreacted EGDGE. In addition, the EGDGE concentration was raised an order (1 M) and also decreased to a tenth (0.01 M) to find an optimal EGDGE concentration for the reaction. Notably, even for 0.01 M concentration, the EGDGE amount remained excessive, and so one end epoxide group could remain free for further ligand immobilization. These experiments adopted 0 mL of EGDGE solution volume at the second reaction step because this condition offered better protein adsorption (the results will be shown later). Other experimental procedures are similar to those described above. Reagents with Two End Functional Groups. To compare the effects of using different spacer molecules and spacer lengths, other reagents with two active end groups were also used as spacers. The reagents adopted include diol, diamine, and the molecule with an amine group at one end and a hydroxyl group at the other end. These reagents are all reactive with the ligand Cibacron Blue 3GA. However, neither the amine nor hydroxyl group could react directly with the hydroxyl group on the membrane surface under mild conditions. Therefore, EGDGE was employed as the activation agent before the new spacer molecule was reacted with the membrane disc. The EGDGE-activated membrane disc was formed under the optimal reaction conditions (0.1 M

EGDGE concentration and 0 mL of liquid volume for the second step reaction) and using the experimental procedures described above. Initially, three reagents of four carbon atoms with different kinds of end groups were used as spacer arms: 1,4-butanediol, 1,4-diaminobutane, and 4-amino1-butanol. The experimental procedures were as follows: A single EGDGE-activated membrane disc was immersed into a 10-mL solution of 3% (w/v) new spacer reagent and reacted in vacuo at room temperature. In the reaction with 1,4-butanediol, 0.2 mL of 1 M KOH was added as a catalyst to accelerate the reaction. After 2 h, the reaction temperature was raised to 50 °C and the reaction was conducted in a shaker for 3 days. Finally, the membrane disc was washed with deionized water to remove the unreacted reagent. Figure 1 schematically depicts the reactions. The reaction of excess diamine with EGDGE-activated membrane left a free amine end for further ligand immobilization. The ninhydrin method27 was adopted to check the existence of this free amine group. The EGDGE-aminated membrane disc was immersed in a ninhydrin solution (10 mL, 12% w/v) in acetone. The membrane was observed to be intense purple at 50 °C for 1.5 h, indicating that the amination onto the membrane disc was successfully achieved. As for the hydroxylation onto the EGDGE-activated membrane disc, no qualitative method was adopted for further checking. Additionally, the effects of spacer arm length on the ligand immobilization and protein adsorption were also investigated. To achieve this, different lengths of diamine were adopted. Diamine was chosen because EGDGE + 1,4-diaminobutane achieved the largest protein adsorption capacity in the above experiments with different types of spacers (the results will be shown later). The other three diamines employed were 1,2diaminoethane, 1,6-diaminohexane, and 1,10-diaminodecane. The experimental procedures were as described above. Cibacron Blue 3GA Immobilization. Ten milliliters of 1.65% (w/v) Cibacron Blue 3GA solution was

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Figure 2. Schematic diagram for the immobilization of Cibacron Blue 3GA onto blank regenerated cellulose membrane disc or spacerbound membranes.

reacted with a blank or spacer-bound membrane disc at 60 °C. After 30 min, 10 mL of a 6% (w/v) NaCl solution was added to the mixture and the reaction was continued at the same temperature for another 1 h. Finally, 10 mL of a 2% (w/v) Na2CO3 solution was added and the temperature was raised to 80 °C. After 1 h more, the membrane disc was thoroughly rinsed with deionized water until no blue Cibacron Blue 3GA was eluted. Cibacron Blue 3GA was immobilized onto blank regenerated cellulose membrane disc or EGDGE-new spacer-bound membranes by coupling its triazine group with the active end group on the membrane, for example, with the hydroxyl or amine group. Figure 2 illustrates the corresponding reaction mechanisms. Meanwhile, the coupling site of Cibacron Blue 3GA with the EGDGE-bound membrane was the primary amine on the anthraquinone ring of the dye. This reaction is also depicted in Figure 2. Batch Adsorption Experiment. The interaction of lysozyme with immobilized Cibacron Blue 3GA is mainly

due to ionic and hydrophobic bindings.28 When pH is below 11, lysozyme is positively charged based on its isoelectric point of 11 and would be expected to interact with Cibacron Blue 3GA primarily by cationic binding. Under this condition, the increase in ionic strength of the adsorption buffer would create competition for the adsorption sites between the positive ions of the buffer and the lysozyme molecule.29 Thus, low ionic strength is suggested for the adsorption process to improve the lysozyme adsorption onto the Cibacron Blue 3GAimmobilized membrane. This study used 50 mM TrisHCl, pH 7, with 0.005% NaN3 as the loading buffer. Additionally, the loading buffer with high salt concentration (1 M KCl in Tris-HCl) was used as the elution buffer to remove the adsorbed lysozyme from the membrane. Both buffers were filtered through 0.2-µm Nylon membranes (Lida Manufacturing, Kenosha, WI). The lysozyme solution was prepared with the loading buffer and filtered by 0.45-µm filters (Millex-HV, Millipore, Beford, MA).


For the batch adsorption experiment, a Cibacron Blue 3GA-immobilized dry membrane disc was placed in a lysozyme solution (2 mL) of a certain concentration and incubated at room temperature for 12 h. The amount of protein adsorbed was determined from the difference between the protein concentration initially added and that remaining after the batch incubation. The lysozyme concentration in solution was measured using a UV/vis spectrophotomer (UV-1601, Shimadzu, Auburn, Australia) at 280 nm. The extinction coefficient for lysozyme nm 28 Between batches, the membrane (E1280 mg/mL) is 2.65. was washed with an elution buffer to remove the bound lysozyme. Ligand Density Measurement. After measuring a complete adsorption isotherm, each Cibacron Blue 3GAimmobilized membrane was dissolved in 7.5 N HCl at 150 °C for 2 days. The solution was then neutralized to pH 7 using 7.5 N NaOH. The dye concentration in the neutralized solution was measured by UV/vis at 620 nm. The extinction coefficient for Cibacron Blue 3GA at 620 nm is 8682 M-1 cm-1 (reported by Pharmacia Biotech AB, Uppsala, Sweden). 3. Results and Discussion Cibacron Blue 3GA Immobilization. In this work, EGDGE (at different reaction conditions) or EGDGE combined with another reagent with two end functional groups was reacted with the regenerated cellulose membrane disc as a spacer arm. The spacer-bound membranes were then reacted with the ligand Cibacron Blue 3GA. Blue displayed on the outer membrane surface indicated the achievement of successful ligand immobilizations from these various spacer arms. Moreover, the ligand density of each membrane was measured quantitatively for determination of the immobilization degree. The results will be shown and analyzed later. Theoretical Models for Equilibrium Adsorption. For biomolecule adsorption, the Langmuir model is the most frequently adopted single-solute isotherm model because of its simplicity and convenience. The Langmuir model is formulated based on the ideal assumptions of homogeneous adsorption such that the relationship between the adsorbed solute concentration (q) and the solute concentration in solution at equilibrium (c) is

q)

qmc Kd + c

(1)

where qm is the saturation capacity of solute and Kd is the dissociation equilibrium constant. Alternatively, the Langmuir isotherm model can also be presented as

q qm 1 ) - q c Kd Kd

(2)

which corresponds to a negative-slope linear relation in the plot of q/c vs q usually referred to Scatchard analysis. Because the Langmuir model is designed for homogeneous adsorption, considerable deviation from the negative-slope straight line in the Scatchard plot indicates the existence of heterogeneous adsorption. Heterogeneous Scatchard plots generally include concave upward, concave downward, and linear upward.30

A concave-up Scatchard curve often implies either the presence of two or more independent heterogeneous binding sites or negative cooperativity in the binding process. On the other hand, a concave-down curve indicates positive cooperativity between the interacting sites, and a positive-slope linear plot represents an invariant positive cooperativity. This work is only concerned with the model describing the concave-down Scatchard curve because most of the Scatchard plots for the experimental adsorption data resemble this tendency (the results will be shown later). Accordingly, the Suen model30 is adopted:

{

c e K /q 0 q ) q - K /c c > Ks/qs s s s s

(3)

where qs denotes the saturation capacity of the solute and Ks is the dissociation equilibrium constant. The corresponding equation for the Scatchard plot of this model is

q qm 1 ) q - q2 c Ks Ks

(4)

The following analyses of adsorption isotherm behaviors and adsorption heterogeneity adopt both the Langmuir and Suen models. Effects of Spacer Arms on the Adsorption Properties. To investigate the influence of nonspecific binding, a blank membrane disc and the spacer-bound membranes without Cibacron Blue 3GA immobilized were used for lysozyme adsorption at room temperature. The adsorption capacities for these membranes were found to be near zero, which verifies negligible nonspecific binding. Therefore, the following analyses do not consider nonspecific binding. The following adsorption results were obtained in just one trial. However, several adsorption experiments were conducted in a repeated manner to confirm reproducibility. The data were reproducible within a 5% error range (data are not shown here). EGDGE as a Spacer Arm. The reaction of EGDGE with the regenerated cellulose membrane disc comprised two steps: complete immersion in solution for 2 h, followed by continuation of the process on the membrane surface either with (1 or 3 liquid volume) or without a liquid film (0-mL volume) of a EGDGE solution for 15 h. The EGDGE-bound membranes were reacted with Cibacron Blue 3GA, and lysozyme was then adsorbed onto these membranes. Figure 3 presents the results and also plots the adsorption isotherm using a Cibacron Blue 3GA-immobilized membrane without any spacer arm. Figure 3 also plots the fitted results using the Langmuir and Suen models, while Table 1 lists the fitted parameter values. In Figure 3 and Table 1, using 0 mL of a EGDGE solution in the reaction resulted in the largest adsorption capacity of lysozyme which is greater than that without spacer arm, whereas the use of 1 or 3 mL reduced adsorption capacities. This observation indicates that while using EGDGE as a spacer arm could improve protein adsorption onto dye-affinity membrane, a negligible liquid film was required for EGDGE to react with the membrane. The existence of a liquid film, such as 1 or 3 mL of liquid remaining on a 25-mm membrane

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Table 1. Fitted Parameters Using Different Isotherm Modelsa Langmuir model

a

Suen model

spacer arm

qm

Kd

R

qs

Ks

R

without spacer EGDGE (0 mL, 0.1 M) EGDGE (1 mL, 0.1 M) EGDGE (3 mL, 0.1 M) EGDGE (0 mL, 0.01 M) EGDGE (0 mL, 1 M) EGDGE + 1,4-butanediol EGDGE + 4-amino-1-butanol EGDGE + 1,2-diaminoethane EGDGE + 1,4-diaminobutane EGDGE + 1,6-diaminohexane EGDGE + 1,10-diaminodecane

1.8 ( 0.1 2.2 ( 0.3 1.6 ( 0.2 1.6 ( 0.2 1.8 ( 0.3 2.0 ( 0.3 1.7 ( 0.1 1.7 ( 0.1 2.0 ( 0.1 2.1 ( 0.1 1.7 ( 0.1 1.6 ( 0.1

0.02 ( 0.01 0.07 ( 0.04 0.07 ( 0.06 0.07 ( 0.05 0.15 ( 0.08 0.11 ( 0.06 0.05 ( 0.01 0.03 ( 0.01 0.05 ( 0.01 0.04 ( 0.01 0.05 ( 0.02 0.06 ( 0.01

0.96 0.86 0.78 0.81 0.95 0.94 0.99 0.98 0.98 0.98 0.94 0.99

1.7 ( 0.1 1.9 ( 0.3 1.4 ( 0.2 1.4 ( 0.2 1.6 ( 0.04 1.8 ( 0.1 1.4 ( 0.1 1.5 ( 0.1 1.8 ( 0.1 1.8 ( 0.1 1.7 ( 0.1 1.4 ( 0.1

80 ( 8 40 ( 20 60 ( 30 40 ( 20 14 ( 1 20 ( 3 80 ( 20 80 ( 10 50 ( 7 80 ( 20 40 ( 5 70 ( 10

0.98 0.71 0.60 0.72 0.99 0.97 0.92 0.96 0.96 0.91 0.97 0.93

qm: mg/disc. Kd: mg/mL. qs: mg/disc. Ks: (mg/mL)(mg/disc). R: correlation coefficient.

Figure 3. Adsorption isotherms of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using EGDGE as a spacer arm. Different liquid volumes used for EGDGE reaction: b, 0 mL; 2, 1 mL; 1, 3 mL. Comparison case: 9, without spacer. Model results: s, Langmuir; - - -, Suen.

disc, retarded the spacer arm reaction and hence decreased protein adsorption. Results obtained from the dissociation equilibrium constant in Table 1 reveal different trends from the two adopted models. The Kd values from the Langmuir model fitting were the same for the three volumes of liquid and were all greater than those without the spacer arm. On the other hand, the Ks values from the Suen model fitting were almost identical for the three liquid volumes, but all were smaller than those without

the spacer arm. Notably, however, the definitions of the dissociation equilibrium constant for these two models are not the same. The Langmuir model defines the dissociation equilibrium constant as the first-order dissociation rate constant divided by the second-order association rate constant, whereas the Suen model defines it as the zeroth-order dissociation rate constant divided by the second-order association rate constant.30 Therefore, determining which trend is more accurate would be extremely difficult. Moreover, the differences may result from experimental errors because the values of the dissociation equilibrium constant under different conditions were on the same order of magnitude. Consequently, it may be more reasonable to conclude that the affinity strength (inverse of the dissociation equilibrium constant) between immobilized Cibacron Blue 3GA and lysozyme did not differ significantly in all of these cases. In other words, using EGDGE as a spacer arm basically did not affect the affinity strength. From the results in Figure 3 and Table 1, the Langmuir model seemed a little more suitable than the Suen model. To further examine the applicability of the isotherm model and to analyze the adsorption heterogeneity, Figure 4 displays the Scatchard plots for the experimental data in Figure 3. Figure 4 also includes the corresponding Scatchard plots for the two isotherm models using the parameters in Table 1. All of the data in Figure 4 resemble concave-down curves and thus were closer to the Suen model. This phenomenon indicates that lysozyme adsorption onto Cibacron Blue 3GAimmobilized membrane discs was heterogeneous, regardless of whether the membrane was directly used without any spacer arm or it was modified with EGDGE as a spacer arm. Again, it is worthwhile to mention that the binding between lysozyme and Cibacron Blue 3GA was primarily a consequence of hydrophobic and ionic interactions. Therefore, different interactions may occur because of various steric ligand sites. Possibly, a single binding force dominated the adsorption and thus increased the likelihood of homogeneous adsorpotion making the Scatchard plots closer to the negative-slope lines. On the other hand, both of the two binding interactions may be significant and vary binding accessibility in different adsorption sites to induce heterogeneous adsorption. Another possibility for heterogeneous adsorption is multilayer adsorption. In this work, lysozyme aggregation in solution within the experimental concentration range was analyzed by HPLC, and no aggregation phenomenon was found. Therefore, by assuming no change in the conformation of the adsorbed lysozyme


Figure 5. Adsorption isotherms of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using EGDGE as a spacer arm. Different EGDGE concentrations used for reaction: 9, 0.01 M; b, 0.1 M; 2, 1 M. Model results: s, Langmuir; - - -, Suen.

Figure 4. Scatchard plots for adsorption of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using EGDGE as a spacer arm. Different liquid volumes used for EGDGE reaction: b, 0 mL; 2, 1 mL; 1, 3 mL. Comparison case: 9, without spacer. Model results: s, Langmuir; - - -, Suen.

molecule to attract and/or react with other native lysozyme molecules, this work does not consider multilayer lysozyme adsorption. Having determined that 0 mL was the optimal liquid volume for the EGDGE reaction, this work also investigated the optimal EGDGE reaction concentration. The concentrations tested were 0.01, 0.1, and 1 M. Figures 5 and 6 display the results of lysozyme adsorption isotherms and Scatchard plots under these reaction conditions, and Table 1 lists the fitted model parameters. Figure 5 clearly shows that the largest lysozyme adsorption capacity occurred with 0.1 M EGDGE concentration, indicating this is the optimal reaction concentration. In the Scatchard plot of Figure 6, the Suen model agreed with the experimental data better than

the Langmuir model. This phenomenon verifies again that the lysozyme adsorption onto the dye-affinity membrane disc using EGDGE as a spacer arm remained heterogeneous regardless of the reaction conditions. Furthermore, the maximum point on the concave-down Scatchard curves in Figures 4 and 6 was located around 1 mg/disc capacity. According to Table 1, for both of the Langmuir and Suen models, the value of the saturation capacity for the 0.01 M case was very close to that without a spacer arm, whereas the corresponding values for 0.1 and 1 M were greater. This observation implies that the use of EGDGE as a spacer arm with negligible liquid film for reaction could improve the adsorption capacity of a dyeimmobilized membrane disc using higher EGDGE concentrations. As for the dissociation equilibrium constant, given the possible experimental errors, the dissociation equilibrium constant (and consequently the affinity strength) may not be influenced by the reaction condition of EGDGE concentration. Different Types and Lengths of Spacer Arms. This work used different spacer molecules with two end functional groups to investigate their effects. The reagents employed included 1,4-butanediol, 1,4-diaminobutane, and 4-amino-1-butanol, all with four carbon atoms. Because these molecules could not directly react with the hydroxyl groups of the membrane disc, EGDGE was used as the activation agent such that the actual spacer arm contained EGDGE and the new molecule. Figure 7 presents the lysozyme adsorption isotherms to the Cibacron Blue 3GA-immobilized membranes modified with these different spacers, and Figure 8 displays the corresponding Scatchard plots. Table 1 lists the fitted parameter values using the Langmuir and Suen models.

484


Figure 7. Adsorption isotherms of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using the combination of EGDGE and another reagent with two active end groups as a spacer arm. Reagents used: 9, 1,4-butanediol; b, 1,4-diaminobutane; 2, 4-amino-1-butanol. Model results: s, Langmuir; - - -, Suen.

Figure 6. Scatchard plots for adsorption of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using EGDGE as a spacer arm. Different EGDGE concentrations used for reaction: 9, 0.01 M; b, 0.1 M; 2, 1 M. Model results: s, Langmuir; - - -, Suen.

In summing up the observations from Figure 7 and the results in Table 1, the spacer arm using EGDGE + 1,4-diaminobutane resulted in a higher capacity than that without the spacer arm, whereas the capacities for the other two spacer types were lower. Consequently, this study considers diamine to be a better choice of spacer arm. As to the model fitting, both models gave fair agreement with the experimental data, as shown in Table 1. However, most Scatchard curves in Figure 8 resemble the negative-slope straight line, which implies homogeneous adsorption. Additionally, the values of the dissociation equilibrium constant for different spacer types were almost identical for both of the Langmuir and Suen models. This finding suggests that the type of spacer arm did not influence the binding strength between the immobilized Cibacron Blue 3GA and lysozyme. The effect of spacer arm length on the protein adsorption was also studied in this work. Because of the above observation that the use of diamine resulted in better adsorption capacity, diamines with varying atom numbers were employed to evaluate the effect of the spacer length. The molecules adopted were 1,2-diaminoethane, 1,4-diaminobutane, 1,6-diaminohexane, and 1,10-diaminodecane. Again, EGDGE was used for activation prior to reacting the membrane disc with diamine. The experimental and model results of lysozyme adsorption

are presented in Figures 9 and 10 and Table 1. The results for 1,4-diaminobutane were taken directly from Figures 7 and 8. According to Figure 9 and Table 1, 1,4-diaminobutane provided the largest adsorption capacity, followed by 1,2-diaminoethane, 1,6-diaminohexane, and 1,10-diaminodecane. Recalling that the actual spacer arm was the combination of EGDGE (a 10-atom molecule) and diamine, the actual spacer lengths for 1,2-diaminoethane, 1,4-diaminobutane, 1,6-diaminohexane, and 1,10-diaminodecane should be 14, 16, 18, and 22 atoms long, respectively. Thus, the use of a 16-atom spacer arm improved the adsorption capacity. In Table 1, the values of the dissociation equilibrium constant for these spacer arms of varying length were close, indicating that the binding strength between lysozyme and dye was hardly influenced by the spacer arm length. In Figure 10, some of the Scatchard plots resemble a concave-down curve, while the others are close to the negative-slope straight line. Consequently, no definite tendency of adsorption homogeneity or heterogeneity was observed for use of EGDGE + diamine as a spacer arm. Effects of Spacer Arms on the Ligand Density and Utilization Percentage. To quantitatively investigate the degree of immobilization, the ligand density for each Cibacron Blue 3GA-immobilized membrane disc was measured. The results are summarized in Table 2. Table 2 also lists the ligand utilization percentage based on monolayer adsorption. Where EGDGE was used as a spacer arm with different reaction liquid volumes, 0 mL had the highest density, followed by 3 and 1 mL. Moreover, even the


Figure 8. Scatchard plots for adsorption of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using the combination of EGDGE and another reagent with two active end groups as a spacer arm. Reagents used: 9, 1,4-butanediol; b, 1,4-diaminobutane; 2, 4-amino-1-butanol. Model results: s, Langmuir; - - -, Suen.

Figure 9. Adsorption isotherms of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using the combination of EGDGE and a diamine as a spacer arm. Diamines used: 9, 1,2-diaminoethane; b, 1,4-diaminobutane; 2, 1,6-diaminohexane; 1, 1,10diaminodecane. Model results: s, Langmuir; - - -, Suen.

lowest density was close to the density without using any spacer arm. This implies that employing EGDGE as a spacer arm could increase the immobilized Cibacron Blue 3GA, a phenomenon which may be attributed to the high reactivity of the epoxide groups of EGDGE with both ligand and membrane. The reactive epoxide ends of EGDGE may have activated more hydroxyl groups of a regenerated cellulose membrane disc for further ligand immobilization. In conclusion, the ideal reaction condition for EGDGE with the membrane for increasing ligand density should be on the solid surface without any liquid film. Regarding the ligand utilization percentage, 1-mL liquid volume rendered a result close to that without spacer arm, whereas 0 and 3 mL reduced the degree of utilization. Importantly, the coupling site of the epoxide group with Cibacron Blue 3GA was on the primary amine of the anthraquinone ring of the dye (see Figure 2), the same site where lysozyme could bind with Cibacron Blue 3GA. Using EGDGE as a spacer arm may have sterically hindered the lysozyme binding (assuming monolayer adsorption), which explains why the amount of immobilized ligand increased but the ligand utilization percentage for lysozyme adsorption decreased. It is also possible that, in the cases of high

ligand density, the sites of immobilized Cibacron Blue 3GA were too crowded to be reached easily by lysozyme molecules and hence the utilization degree was reduced. Regarding the comparison of different concentrations used in the EGDGE reaction, the order of immobilized ligand density, from high to low, was 0.1, 0.01, and 1 M. The density for the 1 M case was less than that without a spacer arm. With a higher concentration of EGDGE, more EGDGE was conjugated onto the membrane disc and hence more Cibacron Blue 3GA could be immobilized. However, for 1 M concentration, the corresponding bound EGDGE sites may be too crowded for the Cibacron Blue 3GA molecule to reach, because of the steric encumbrance of the NH2 group on the anthraquinone ring of the dye (the reaction site). Consequently, the ligand density for the 1 M case was reduced. On the other hand, for the other lower concentration cases, the ligand utilization degree decreased with increasing ligand density. This phenomenon resembles the results from different liquid volumes, the probable reasons for which have been described previously. The data in Table 2 were also used to analyze the effects of spacer arm types on the ligand immobilization. The results show that the use of 4-amino-1-butanol raised the ligand density, whereas 1,4-butanediol and

486


and 1,2-diaminoethane. All of these densities were less than that without a spacer arm. Meanwhile, 1,2diaminoethane had the largest ligand utilization percentage but exhibited the lowest ligand density. Again, this indicates that lower ligand density is associated with higher ligand utilization percentage. In summing up the results in Table 2, when only EGDGE was used as a spacer arm, the ligand density was generally higher than that without a spacer arm; however, the ligand utilization percentage was lower. On the other hand, when EGDGE with another reagent was employed as a spacer arm, the ligand density was generally lower than that without a spacer arm, while the ligand utilization percentage was increased. Therefore, we conclude that the use of EGDGE with another reagent decreased ligand immobilization. However, the decrease in ligand density could be compensated by the increase in the ligand utilization percentage and protein adsorption amount. The evidence thus indicates that adopting a longer spacer arm like the combination of EGDGE with another reagent would improve ligand utilization. According to this study, the molecule with medium ligand density and high protein adsorption capacity was 1,4-diaminobutane. 4. Conclusion

Figure 10. Scatchard plots for adsorption of lysozyme to Cibacron Blue 3GA-immobilized membrane discs using the combination of EGDGE and a diamine as a spacer arm. Diamines used: 9, 1,2diaminoethane; b, 1,4-diaminobutane; 2, 1,6-diaminohexane; 1, 1,10-diaminodecane. Model results: s, Langmuir, - - -, Suen.

1,4-diaminobutane improved the ligand utilization percentage. The higher degree of ligand immobilization for 4-amino-1-butanol was probably related to it having two different end groups, letting it react more easily. Therefore, more reactive groups of EGDGE-activated membane and Cibacron Blue 3GA were reacted with 4-amino-1-butanol. On the other hand, as described previously, a lower ligand density could reduce the degree of crowdedness and hence increase the ratio of sites for protein adsorption. Finally, the data for different spacer arm lengths in Table 2 were compared with the data without any spacer arm. The use oif EGDGE + 1,4-diaminodecane as a spacer arm showed the highest ligand density, followed by 1,6-diaminohexane, 1,10-diaminodecane,

This study investigated the adsorption behaviors of lysozyme onto Cibacron Blue 3GA-immobilized membrane discs using various spacer arms and various reaction conditions. When EGDGE alone was adopted as a spacer arm, our results indicated that the optimal reaction condition to achieve a high adsorption capacity was with no liquid film on the membrane surface and a 0.1 M concentration. However, although employing EGDGE as a spacer arm could increase the amounts of both immobilized Cibacron Blue 3GA and adsorbed protein, the corresponding overall ligand utilization percentage was lss than that without using a spacer arm. To promote ligand utilization and to study the effects of various spacer arm types and lengths, other reagents with two end functional groups were employed and EGDGE was used as the activation agent. For different spacer types, EGDGE + 1,4-diaminobutane increased the adsorption capacity and 4-amino-1-butanol raised the ligand density. Furthermore, the use of 1,4-butanediol and 1,4-diaminobutane improved the ligand utilization percentage. Consequently, this study considers diamine a better choice of spacer arm. As for the effect of spacer arm length, the use of EGDGE + 1,4-diaminodecane as a spacer arm displayed the highest ligand density and lysozyme adsorption capacity, but 1,2diaminoethane had the largest ligand utilization percentage. In addition, where EGDGE was combined with another reagent as a spacer arm, the ligand densities were lower than those when EGDGE alone was used as a spacer arm, but the ligand utilization percentages were higher. In summing up the above results, we conclude that lower ligand density is associated with higher ligand utilization percentage, because of reduced hindrance from less crowded binding sites. Accordingly, the selection of a suitable spacer molecule is rather important for raising protein adsorption capacity onto the dyeaffinity membrane disc and ligand utilization percentage. Moreover, our results demonstrate that EGDGE + 1,4-diaminobutane is a good choice for maximizing

Ind. Eng. Chem. Res., Vol. 39, No. 2, 2000 487 Table 2. Values of Ligand Density and Utilization Percentage for Different Spacer Arms Langmuir model ligand density

lysozyme saturation capacity

Suen model

spacer arm

mg/disc

µmol/disc

mg/disc

µmol/disc

utilization percentage (%)

without spacer EGDGE (0 mL, 0.1 M) EGDGE (1 mL, 0.1 M) EGDGE (3 mL, 0.1 M) EGDGE (0 mL, 0.01 M) EGDGE (0 mL, 1 M) EGDGE + 1,4-butanediol EGDGE + 4-amino-1-butanol EGDGE + 1,2-diaminoethane EGDGE + 1,4-diaminobutane EGDGE + 1,6-diaminohexane EGDGE + 1,10-diaminodecane

0.37 0.74 0.34 0.50 0.61 0.30 0.28 0.50 0.16 0.33 0.24 0.22

0.47 0.96 0.44 0.64 0.79 0.39 0.35 0.64 0.21 0.42 0.31 0.28

1.8 2.2 1.6 1.6 1.8 2.0 1.7 1.7 2.0 2.1 1.7 1.6

0.12 0.16 0.11 0.11 0.13 0.14 0.12 0.12 0.14 0.15 0.12 0.11

26 17 25 17 16 36 34 19 67 36 39 39

both protein adsorption capacity and ligand utilization degree. On the other hand, results obtained from the dissociation equilibrium constant indicate that the binding strength between lysozyme and Cibacron Blue 3GA remained largely uninfluenced by the addition of a spacer arm under different spacer types and lengths and under different reaction conditions. Literature Cited (1) Brandt, S.; Goffe, R. A.; Kessler, S. B.; O’Connor, J. L.; Zale, S. E. Membrane-based Affinity Technology for Commercial Scale Purifications. Bio/Technology 1988, 6, 779. (2) Briefs, K.-G.; Kula, M.-R. Fast Protein Chromatography on Analytical and Preparative Scale Using Modified Microporous Membranes. Chem. Eng. Sci. 1992, 47, 141. (3) Roper, D. K.; Lightfoot, E. N. Separation of Bimolecules Using Adsorptive Membranes. J. Chromatogr. A 1995, 702, 3. (4) Charcosset, C. Purification of Proteins by Membrane Chromatography. J. Chem. Technol. Biotechnol. 1998, 71, 95. (5) Hou, K. C.; Zaniewski, R.; Roy, S. Protein A Immobilized Affinity Cartridge for Immunoglobulin Purification. Biotechnol. Appl. Biochem. 1991, 13, 257. (6) Kim, M.; Saito, K.; Furusaki, S.; Sato, T.; Sugo, T.; Ishigaki, I. Adsorption and Elution of Bovine γ-Globulin Using an Affinity Membrane Containing Hydrophobic Amino Acids as Ligands. J. Chromatogr. 1991, 585, 45. (7) Iwata, H.; Saito, K.; Furusaki, S.; Sugo, T.; Okamoto, J. Adsorption Characteristics of an Immobilized Metal Affinity Membrane. Biotechnol. Prog. 1991, 7, 412. (8) Langlotz, P.; Kroner, K. H. Surface-modified Membranes as a Matrix for Protein Purification. J. Chromatogr. 1992, 591, 107. (9) Nachman, M.; Azad, A. R. M.; Bailon, P. Membrane-based Receptor Affinity Chromatography. J. Chromatogr. 1992, 597, 155. (10) Nachman, M. Kinetic Aspects of Membrane-based Immunoaffinity Chromatography. J. Chromatogr. 1992, 597, 167. (11) Nachman, M.; Azad, A. R. M.; Bailon, P. Efficient Recovery of Recombinant Proteins Using Membrane-based Immunoaffinity Chromatography (MIC). Biotechnol. Bioeng. 1992, 40, 564. (12) Kugel, K.; Moseley, A.; Harding, G. B.; Klein, E. Microporous Poly(caprolactam) Hollow Fibers for Therapeutic Affinity Adsorption. J. Membr. Sci. 1992, 74, 115. (13) Klein, E.; Eichholz, E.; Yeager, D. H. Affinity Membranes Prepared from Hydrophilic Coatings on Microporous Polysulfone Hollow Fibers. J. Membr. Sci. 1994, 90, 69. (14) Klein, E.; Eichholz, E.; Theimer, F.; Yeager, D. Chitosan Modified Sulfonated Poly(ethersulfone) as a Support for Affinity Separations. J. Membr. Sci. 1994, 95, 199. (15) Liu, H.-C.; Fried, J. R. Breakthrough of Lysozyme through an Affinity Membrane of Cellulose-Cibacron Blue. AIChE J. 1994, 40, 40. (16) Serafica, G. C.; Pimbley, J.; Belfort, G. Protein Fractionation Using Fast Flow Immobilized Metal Chelate Affinity Membranes. Biotechnol. Bioeng. 1994, 43, 21.

lysozyme saturation capacity mg/disc

µmol/disc

utilization percentage (%)

1.7 1.9 1.4 1.4 1.6 1.8 1.4 1.5 1.8 1.8 1.7 1.4

0.12 0.13 0.10 0.10 0.11 0.13 0.10 0.11 0.13 0.13 0.12 0.10

26 14 23 16 14 33 29 17 62 31 39 36

(17) Guo, W.; Shang, Z.; Yu, Y.; Zhou, L. Membrane Affinity Chromatography of Alkaline Phosphatase. J. Chromatogr. A 1994, 685, 344. (18) Suen, S.-Y.; Etzel, M. R. Sorption Kinetics and Breakthrough Curves for Pepsin and Chymosin Using Pepstatin A Affinity Membranes. J. Chromatogr. A 1994, 686, 179. (19) Bueno, S. M. A.; Haupt, K.; Vijayalakshmi, M. A. Separation of Immunoglobulin G from Human Serum by Pseudobioaffinity Chromatography Using Immobilized L-Histidine in Hollow Fibre Membranes. J. Chromatogr. B 1995, 667, 57. (20) Charcosset, C.; Su, Z.; Karoor, S.; Daun, G.; Colton, C. K. Protein A Immunoaffinity Hollow Fiber Membranes for Immunoglobulin G Purification: Experimental Characterization. Biotechnol. Bioeng. 1995, 48, 415. (21) Klein, E.; Yeager, D.; Seshadri, R.; Baurmeister, U. Affinity Adsorption Devices Prepared from Microporous Poly(amide) Hollow Fibers and Sheet Membranes. J. Membr. Sci. 1997, 129, 31. (22) Denizli, A.; Salih, B.; Arica, M. Y.; Kesenci, K.; Hasirci, V.; Piskin, E. Cibacron Blue F3GA-incorporated Macroporous Poly(2-hydroxyethyl methacrylate) Affinity Membranes for Heavy Metal Removal. J. Chromatogr. A 1997, 758, 217. (23) Denizli, A.; Tanyolac, D.; Salih, B.; Aydinlar, E.; Ozdural, A.; Piskin, E. Adsorption of Heavy-metal Ions on Cibacron Blue F3GA-immobilized Microporous Polyvinylbutyral-based Affinity Membranes. J. Membr. Sci. 1997, 137, 1. (24) Weissenborn, M.; Hutter, B.; Singh, M.; Beeskow, T. C.; Anspach, F. B. A Study of Combined Filtration and Adsorption on Nylon-based Dye-affinity Membranes: Separation of Recombinant L-Alanine Dehydrogenase from Crude Fermentation Broth. Biotechnol. Appl. Biochem. 1997, 25, 159. (25) Gebauer, K. H.; Thommes, J.; Kula, M.-R. Breakthrough Performance of High-capacity Membrane Adsorbers in Protein Chromatography. Chem. Eng. Sci. 1997, 52, 405. (26) Suen, S.-Y.; Chang, Y.-S. Adsorption and Desorption of Lysozyme and Albumin to Cibacron Blue 3GA Using Gel Beads and Membrane Discs. J. Chin. Inst. Chem. Eng. 1998, 29, 307. (27) Stanford, M.; Stein, W. H. A. Modified Ninhydrin Reagent for the Photometric Determination of Amino Acids and Related Compounds. J. Biol. Chem. 1954, 211, 907. (28) Boyer, P. M.; Hsu, J. T. Effects of Ligand Concentration on Protein Adsorption in Dye-ligand Adsorbents. Chem. Eng. Sci. 1992, 47, 241. (29) Hashim, M. A.; Chu, K.-H.; Tsan, P.-S. Effects of Ionic Strength and pH on the Adsorption Equilibria of Lysozyme on Ion Exchangers. J. Chem. Technol. Biotechnol. 1995, 62, 253. (30) Suen, S.-Y. An Isotherm Model Describing Concave-down Scatchard Curve for Heterogeneous Affinity Adsorption. J. Chem. Technol. Biotechnol. 1997, 70, 278.

Received for review June 15, 1999 Revised manuscript received November 1, 1999 Accepted November 10, 1999 IE990421T

Effects of Spacer Arms on Cibacron Blue 3GA Immobilization and

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