Anal. Chem. 1994,66, 2584-2589
Easily Regenerable Affinity Chromatographic Zirconia-Based Support with Concanavalin A as a Model Ligand Mlchael H. Glavanovlch' and Peter W. Carr Department of Chemistry and Institute for Advanced Studies in Bioprocess Technology, University of Minnesota, 207 Pleasant St. S.E., Minneapolis, Minnesota 55455-043 I
The goal of this work was to develop a generic approach for producing affinity chromatographic columns which can be regenerated. Concanavalin A (Con A) was immobilized adsorptively by an in situ method onto a zirconium dioxide (zirconia) chromatographic support and used to resolve chromophorically labeled monosaccharides. The Con A was then removed from the zirconia by flushing with base. The same column was regenerated by applying a fresh aliquot of Con A. This cycle was repeated several times to demonstrate consistency in the loading capacity and the stability of the underlying zirconia support. Finally we used glutaraldehyde to cross-link the Con A to increase the long-term stability of the column. Hydrolyzing the protein with acid allowed it to be removed under alkaline conditions and the column regenerated simply by adding more Con A followed by glutaraldehyde cross-linking. Currently, commercially available affinity chromatographic columns of immobilized biopolymers are not regenerable. Proteins, enzymes, and antibodies can be covalently bound to any of a number of supports, thereby anchoring the moleculemore or less permanently to the underlying Despite their high cost, these columns are designed to be used and then discarded when their chromatographic performance degrades, due to gradual loss in the biopolymer's activity. Many researchers have found that the equilibrium binding constant for a surface-bound protein is different from that obtained in solution.1v8-11The number of bonds between the biopolymer and the support greatly affects its kinetic behavior and the specific surface activity due to distortion of the threedimensional structure of the protein.12J3 Walters and others found that they could increase the specific activity by reducing the number of bonds between the ligand and the support. Coulet et al.14found no decrease in enzymatic activity upon ' Present address: TheUpjohn Co., 7256-300-109,7000PortageRd., Kalamazw, MI 49001. (1) Carr, P. W.; Bowers, L. D. Immobilized Enzymes in Analytical and Clinical Chemistry; Wiley-Interscience: New York, 1980. (2) Gray, G . R. Anal. Chem. 1980.52, 9R. (3) Walters, R. R. Anal. Chem. 1985, 57, 1099A. (4) Parikh, I.; Cuatrecasas, P. Chem. Eng. News 1985, Aug. 26, 17. (5) Clonis, Y. D.; Lowe, C. R. J. Chromatogr. 1991, 540, 103. (6) Narayanan, S.R.; Knochs, S.,Jr.; Crane, L. J. J. Chromatogr. 1990,503,93. (7) Nakamura, K.; Toyoda,K.; Kato,Y.;Shimura, K.;Kasai,K-I.J. Chromatogr. 1989,478, 159.
(8) Mucller, A. J.; Carr, P. W. J. Chromatogr. 1984, 284, 33. (9) Mueller, A. J.; Carr, P. W. J. Chromatogr. 1986, 357, 11. (10) Wade, J. L.; Bergold, A. F.; Carr, P. W. Anal. Chem. 1987, 59, 1286. (1 1) Lee,Y.; Wun K.;Tsao,G. in J. J. InImmobilizedEnzyme Technology;Weetall, J. J., Suziki, S.,Eds.; Plenum: New York, 1974; p 129. (12) Landgrcbe, M. E.; Wu, D.; Walters, R. R. Anal. Chem. 1986, 58, 1607. (13) Wu D.; Waltcrs, R. R. J . Chromatogr. 1988, 458, 169. (14) Coulet, P. R.; Carlsson, J.; Porath, J. Biotecchnol. Bioeng. 1981, 23, 663.
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immobilization via metal interactions on a cobalt, zinc, or copper loaded iminodiacetic-agarose column, showing that this type of adsorption is a mild method of immobilization. Recently El Rassi et al.15 extended this finding so as to prepare an affinity column by immobilizing concanavalin A (Con A) to an HPLC column via copper metal interactions. They found that this column could be used to separate various carbohydrate derivatives as well as some glycoproteins. In addition, the Con A could be removed by introducing ions into the mobile phase which compete for the copper sites. Later the Con A could be reapplied. We believe this is the first case of a regenerable affinity column. Porous microparticulate zirconia has many excellent qualities for use as a chromatographic support.16 Zirconia is chemically stable over the pH range 1-14, mechanically stable under high pressure, thermally stable, available in particle sizes of 5 pm in diameter with pore diameters of 100-1000 A and reasonably high surface area. The zirconia surface is, however, chemically very complex. Previous research has indicated that the surface chemistry of zirconia in aqueous media is dominated by a large number of hard Lewis acid sites which have a very high affinity for oxyanions and other hard Lewis base^.'^-^^ This characteristic of strong adsorption of hard Lewis acids is exploited here to prepare Con A HPLC columns. The process of immobilizing a protein on zirconia by adsorption is markedly simpler than the immobilization of biopolymersonsilica. The silica surfacemust first be activated, which can involve several chemical steps, then the ligand is introduced to the support for binding, and finally the particles are packed into a column. Through the use of zirconia, all of the activation steps are bypassed and the ligand, i.e. the biopolymer, can be loaded in situ onto a prepacked HPLC column containing the zirconia. The ligand is held to the zirconia surface simply by adsorption interactions based primarily on the Lewis acid-base interactions. These interactions are sufficiently strong to maintain the stability of the (15) El Rassi, 2.;Truei, Y.; Maa, Y.-F.; Horvath, Cs. Anal. Biochem. 1988,169, 172. (16) Carr, P. W.; Blackwell, J. A.; Weber, T.P.; Schafer. W. A.; Rigncy, M. P. ; In Chromatography in Biotechnology; Horvath, Cs.,Ettre, L. S.,Us.ACS Symposium Series 529, American Chemical Society: Washington, DC, 1993, p 146. (17) Rigney, M. P.; Funkenbusch, E. F.; Carr, P. W. J. Chromatogr. 1990, 499, 291. (18) Rigncy, M. P.; Weber, T.P.; Carr, P. W. J . Chromatogr. 1989, 484, 273. (19) Blackwell, J. A.; Carr, P. W. J . Liq. Chromarogr. 1991, 14, 2875. (20) Blackwell, J. A.; Carr, P. W. J . Liq. Chromatogr. 1992, 15, 727. (21) Blackwell, J. A.; Carr, P. W. Anal. Chem. 1992, 64, 853. (22) Blackwell, J. A.; Carr, P. W. Anal. Chem. 1992, 64, 863. (23) Schafer, W. A.; Carr,P. W.; Funkenbusch, E. F.;Parson, K. A. J. Chromarogr. 1991, 587, 137. (24) Kawahara, M.; Nakamura, H.; Nakajima, T. Anal. Sci. 1989,5,485. (25) Kawahara, M.; Nakamura, H.; Nakajima, T. J . Chromatogr. 1990,515,149. 0003-2700/94/0366-2584$04.50/0
0 1994 American Chemical Society
adsorbed ligand but are labile enough to allow easy removal by passing an alkaline solution through the column. The alkaline treatment restores the column to its original, unmodified state without damaging the zirconia, leaving it ready to accept a new layer of biopolymer. Con A is a lectin obtained from jack bean and has an affinity for glucoside and mannoside residues. The molecular dimensions of the Con A are 4 nm X 4 nm X 8 nm. It has been used in the resolution of glycoproteins, polysaccharide purification, and the study and characterization of cell surface^.^^^^^ Using zirconia as the underlying support will allow the surface to be covered in a fashion which is consistent with a monolayer of Con A. Borchert et a1.28examined several methods of binding Con A to silica and found that the amount of Con A immobilized was not easily reproduced. On zirconia this is not a problem, since the protein is adsorbing to a surface. We show that the support is nearly completely covered. Once immobilized in situ on zirconia, the stability of the Con A column was examined. Additionally, the Con A was crosslinked with glutaraldehyde and its stability and regenerability examined.
EXPERIMENTAL SECTION Reagents. Concanavalin A (Type V), p-nitrophenyl-aD-mannopyranoside (pNp-man), p-nitrophenyl-a-D-glucopyranoside (pNpglu), p-nitrophenyl-&bgalactopyranoside( p N p gal), a-methyl-D-mannopyranoside (MDM), and N-morpholinoethanesulfonic acid monohydrate (MES) were purchased from Sigma (St. Louis, MO). Acetic acid (99+% gold label) was obtained from Aldrich (Milwaukee, WI). Manganous chloride tetrahydrate and sodium chloride were obtained from Baker (Phillipsburg, NJ). Calcium chloride dihydrate and sodium hydroxide (50% by weight) were obtained from Fisher Scientific (Fairlawn, NJ). Hydrochloric acid was obtained from Mallinkrodt (Paris, KY). All chemicals were reagent grade or better. Zirconium dioxide particles (zirconia, pore diameter 540 A, 12.7 m2/g, and mean particle diameter 8 pm) were obtained from 3M (St. Paul, MN). Solutions of 0.5 M hydrochloricacid and 0.5 M sodium hydroxide were prepared by adding the appropriate amount of concentrated hydrochloric acid or 50% by weight aqueous solution of sodium hydroxide to HPLC water. Water used in these studies was prepared by passing house deionized water through a Barnstead Nanopure system equipped with an organic free cartridge and a 0.2-pm final filter. All water was boiled and cooled prior to use to remove dissolved gasses, hereafter referred to as HPLC water. Particle Pretreatment. To maintain the consistency in the surface chemistry between batches of zirconia, the newly received particles were subjected to a series of washes. The particles were rinsed for 1 h at room temperature with each of the following solutions: 250 mL of 0.5 M hydrochloric acid, 250 mL of HPLC water, 250 mL of 0.5 M sodium hydroxide, and finally 250 mL of HPLC water. (26) Nicolson, G.L. In Concanaualin A as a Tool; Bittiger, H., Schnebli, H. P., Eds.;Wiley: New York, 1976; p 3. (27) Reckc, G. N., Jr.; Beckcr, J. W.; Cunningham, B. A,; Wang, J. L.;Yahara, I.; Edelman, G.M. In Concanaualin A; Chowdhury, T . K., Weiss, A. K., Eds.; Plenum: New York, 1975; p 13. (28) Borchert, A.; Larsson, P.-0.; Mosbach, K. J. Chromatogr. 1982, 244, 49.
Apparatus, Column Packing, and Chromatographic Conditions. The analytical columns were cut and polished to a 1 cm length from 0.64 cm o.d., 0.46 cm i.d. Precision Bore 3 16 stainless-steel tubing (Supelco, Bellefonte, PA). The column end fittings (Walters columns29) were made locally and used with 2-pm Kel-F encased frits (Alltech, Deerfield, IL). The columns were packed with zirconia particles at 4500 psi from an isopropyl alcohol suspension by the upward stirred slurry method. The HPLC system was comprised of a high-pressure pump equipped with a pulse dampener (Altex), an injector valve (7120, Rheodyne) equipped with either a S-pL, 1-mL, or 10-mL loop, and a UV-visible detector operating at 280 nm (LC-15, Perkin-Elmer). Thermostating was accomplished by using a thermostated water circulator (Haake, Model FE) and a glass column flow jacket. Three buffers wereused for the workdescribed here. Buffer A consisted of 0.15 M sodium sulfate, 0.025 M MES, 0.001 M manganese chloride, and 0.001 M calcium chloride at pH 6.0. Buffer B consisted of 0.15 M sodium sulfate, 0.1 M acetic acid, 0.001 M manganese chloride, and 0.001 M calcium chloride at pH 4.5. Buffer C contained 0.45 M sodium chloride, 0.1 M acetic acid, 0.00 1 M manganese chloride, and 0.001 M calcium chloride, pH 4.5. The buffers were prepared by dissolving the acid and sodium salt in 1 L of HPLC water, adjusting the pH with sodium hydroxide solution, and then adding the other salts. All buffers were filtered through a 0.45-pm filter before use. In Situ Adsorptive Immobilization of Concanavalin A. A solution of Con A was prepared by weighing a known amount (fO.O1 mg) of Con A (approximately 50 mg) into a 10-mL volumetric flask and then filling to the mark with buffer B. This solution was then filtered through a 0.45-pm filter. The column was equilibrated with the acetate buffer by passing 30 mL of the buffer through the column at 1.O mL/ min. The flow rate was then slowed to 0.2 mL/min and a 1.O-mL injection loop was filled with the Con A solution. The solution was injected onto the column and the absorbance monitored at 280 nm. Two injections of the Con A solution were made, the initial injection and a second injection 15-30 min later. After breakthrough was observed, the flow to the column was stopped for 20 min. The column was then equilibrated with buffer A for at least 60 min at a flow rate of 1.OmL/min to flush out any unbound protein. This protein phase was used as is, without further modification. Chromatography of pNitropheny1 Sugars. The chromatography ofp-nitrophenyl-labeled sugars was performed using buffer A as the mobile phase at a flow rate of 1.0 mL/min. The concentration of the labeled sugars used in this work were as follows: 50 pM pNp-gal, 100 pM pNp-glu, 1 mM pNp-man. Desorption of Concanavalin A under Alkaline Conditions. A 1.0-cm guard column packed with zirconia was installed prior to the analytical column when caustic solutions were injected onto the HPLC. This column was sacrificial and protected the analytical column from any particulates which may be introduced into the LC system from the alkaline solutions. The system was equilibrated with 60 mL of HPLC water at a flow rate of 1.0 mL/min and a 10-mL injection (29) Walters, R. R. Anal. Chem. 1983, 55, 592.
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loop filled with 0.1 M sodium hydroxide and 0.02 M potassium bromide. Sodium hydroxide was used as the displacing agent, and potassium bromide was used to moderate the buildup of charge on the zirconia surface. We experienced a large increase in back-pressure when the columns were flushed with sodium hydroxide alone. This solution was allowed to pass through the column, and the absorbance was monitored at 280 nm. Adsorptive Immobilization of Con A with Glutaraldehyde Cross-Link. Two methods were used to prepare glutaraldehyde-cross-linked Con A. The first involved adsorption of Con A from solution. A 0.48-g portion of zirconia was suspended in 10 mL of buffer C and sonicated under vacuum in a 50-mL Erlenmeyer flask. A 10.14 mL sample of a 1.83 mg/mL Con A, 3.02 mM MDM solution in buffer C was transferred to the zirconia slurry. This mixture was swirled for 1 min every ten min six times. After 1 h, the absorbance of the supernatant was measured and compared to a calibration curve made using known concentrations of Con A in a quartz UV-vis cell at 277.9 nm (A, for Con A). The supernatant was then decanted, and the zirconia particles were rinsed twice with 20-mL portions of HPLC water. Twenty milliliters of buffer C were added, and the zirconia was resuspended by swirling. Finally, 8 mL of 25% glutaraldehyde was added and the mixture swirled. The mixture was allowed to stand at room temperature for 1.5 h and was swirled for 1 min every 15 min. After this process, 0.1 g of MDM was added to the mixture, and the resulting mixture swirled again for 1 min. This mixture was allowed to sit overnight at 4 "C. The supernatant was then decanted, and 50 mL of buffer C with 0.1 M sodium cyanoborohydride and 0.0021 M MDM were added. This slurry was allowed to react for 3.5 h at room temperature with brief swirling every half hour. Afterward the supernatant was decanted and the zirconia washed three times with 30 mL each of buffer C. Approximately 10.01 mg of Concanavalin A adsorbed to 0.48 g of zirconia; 0.22 g of zirconia is necessary to pack a 10 mm by 4.6 mm i.d. column for a total of 4.59 mg of Con A in the column. The zirconia was placed into the packing cell and packed into a 1.0 cm X 0.46 cm column at 3000 psi. The slurry solvent was buffer C with 0.02 M MDM, and the reservoir solvent was HPLC water. The column was stored at 4 "C in buffer C when not in use. In Situ Adsorption and Cross-Linking. The second method for cross-linking Con A began by adsorbing it onto zirconia in situ as described under the adsorptive method of immobilization. Following adsorption of Con A, the column was removed from the LC. A 1.0-mL aliquot of a solution consisting of 2.5 mL of buffer C, 1 mL of 25% aqueous glutaraldehyde, and 0.025 g of MDM was passed manually through the column with a 1-mL syringe. This procedure took 10 s. The column was allowed to sit as such for 1.5 h, after which an additional 0.2 mL was passed through the column. The ends were then capped, and the column was stored overnight at 4 OC. The next day thecolumn was flushed with buffer A to remove any unreacted glutaraldehyde. The stabilityoftheproteinaceousstationaryphasewas thenstudied. When not in use, all columns were stored in buffer A at 4 "C with their ends capped. 2588
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1 0.08 0.u.
I I
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Figwe 1. Typlcal breakthrough curve of Con A for column loading. Second Injection of 1 mL of 5.0 mg/mL Con A Injected. Flow rate: 0.2 mL/mln of buffer B.
In Situ Removal of Glutaraldehyde-Cross-Linked Con A from Zirconia. Cross-linked Con A was removed in two steps. First, hot 0.5 M nitric acid was used to hydrolyze the protein. For this procedure, a 1-cm zirconia-filled guard column was installed before the analytical column to protect it from any contaminants introduced by theacidicsolution. A waterjacket was fitted over the two columns, and a thermostat-controlled water bath was used to maintain a column temperature of 80 "C. The nitric acid solution was passed through the column for 8 h at a flow rate of 0.5 mL/min. The column was then rinsed by passing pure deionized water through it for 30 min at 0.5 mL/min. Finally, the hydrolyzed protein was desorbed from the column with a solution of sodium hydroxide as described above (see caustic desorption of concanavalin A). Stability Study of Proteinaceous Stationary Phases. The stability of the stationary phases was evaluated by passing several thousand column volumes of mobile phase through the column while the decay in retention of pNp-man, a solute which interacts strongly with Con A, was monitored. Isotherm of pNp-man Interaction with Zirconia-Bound Concanavalin A. A 1 cm X 0.46 cm column was packed with zirconia and then coated with Con A in situ via the adsorptive immobilization method. The binding isotherm of pNp-man to Con A was measured by injecting large volumes of successivelyhigher concentrations of pNp-man onto a column containing adsorptively immobilized Con A at a flow rate of 0.5 mL/min and ambient temperature. Afterward, the Con A was then cross-linked with glutaraldehyde as stated above. The isotherm for pNp-man was again measured on the crosslinked Con A under the same conditions. RESULTS AND DISCUSSION Adsorptive Immobilization of Concanavalin A and Stability Study. Our major initial concerns were whether a protein could be reproducibly loaded, stripped, and reloaded in situ. When Con A was loaded by adsorption and not cross-linked, we found that in three successive cycles 5.8, 5.8, and 6.0 mg of the protein could be loaded onto the same column. The amount of protein loaded was measured via the frontal chromatogram as shown in Figure 1. More than one injection was necessary to observe a breakthrough, because the solution injected contained 5.0 mg of Con A. The columns contained about 0.22 g of zirconia for a total of 2.8 m* of available surface area; 5.8 mg of Con A occupies about 2.3 m2of surface. This represents 82% coverage of the zirconia surface.
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Figure 2. Desorption of Con A under alkaline conditions. Two consecutive injections of 10 mL of 0.1 M sodium hydroxide. Flow rate: 1 mL/min of water.
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column volumes Figure 4. Comparison of the stabiilty of the first adsorbed stationary phase (A) of concanavalin A and that after two regenerative cycles (B) by measuringthe retention of pnitrophenyl-a-bmannopyranosMe. Chromatographic conditions are the same as in Figure 3.
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Since the amount of Con A which adsorbed to the support in the second and third cycles was as high as in the first cycle, we concluded that all of the Con A was removed from the zirconia surface by the alkaline wash. A typical desorption profile of Con A under alkaline conditions is shown in Figure 2. In addition, the 0.1 M sodium hydroxide did not significantly alter the zirconia in that the amount of Con A able to adsorb to the surface remained very nearly constant. A typical separation of the test sugars is shown in Figure 3; all three are baseline resolved. Peak profiles for pNp-man on the first adsorbed stationary phase (cycle one) and the third adsorbed phase (cycle three) are virtually identical. In addition, none of the p-nitrophenyl-labeled solutes exhibited nonspecific adsorption on zirconia. When no Con A was present on the support, all solutes eluted in the void volume. The stabilities of the phase-one and phase-three preparations were studied by monitoring the retention factor (k’) of pNp-man as several thousand column volumes of mobile phase were passed through the column. This is the most retained solute of the test group, and its retention is expected to be the most sensitive to changes in the Con A. The results for cycles one and three can be seen in Figure 4A and 4B, respectively.
The k’ is plotted versus the number of column volumes of mobile phase passed through the column, where one column volume equals the void volume of the column (0.2 mL). The decrease in the k’ of pNp-man is quite linear with respect to column volumes of mobile phase. Regressing the data and extrapolating to zero column volumes reveals the initial k’ of pNp-man to be 22.3 f 0.7 and 22.2f 0.7 for cycles one and three, respectively. The very good agreement in the initial k’ values further supports our conclusion that Con A can be reproducibly loaded and stripped from porous zirconia. The rates of decay of k’, however, are quite different for the two preparations (-2.1 X 10-4 f 5 X for cycle one and -5.8 X 10-4 f 4.2 X for cycle three). The rate of decrease in k‘ for phase three is nearly 3 times greater than that for phase one. The decay in k’ is due to the desorption of protein from the zirconia support. This was easily shown by injecting Con A after the stability study experiment was terminated. We found that a substantial amount of Con A was able to adsorb to the column. For example, after 8000 column volumes of mobile phase were passed over phase three, 1.66 mg of Con A adsorbed after a 5.17 mg/mL solution of Con A was injected onto the column. This suggests that at least 1.66 mg of Con A desorbed from the support through the course of the experiment. This represents a 28% loss in Con A, however, the decrease in k’ observed over the course of the experiment was only 21%. Although we currently have no explanation for this small discrepancy, the relationship between the surface site density of Con A and the k’ of pNpman has been shown to be nonlinear.1° The results strongly suggest that the deterioration in k’ is due to desorption of Con A from the zirconia and not due to loss of its biological activity. AnalytlcalChemlstty, Vol. 66, No. 15, August 1, 1994
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Figure 5. Comparison of elution profiles of pnltrophenyCa-0mannopyranoslde from the in vltro immobilization method and the in situ immobilizationmethod. Chromatographicconditionsare the same as in Figure 4. Table 1. Comparison of In VHro and in SHu Prepared Statlonary Phares. amt of Con A
method
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41 114 87
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Adsorptive Immobilization of Con A with Glutaraldehyde Cross-Link, While adsorptiveimmobilization is sufficient for short-term use, it does not provide long term stability of the stationary phase. We therefore decided to see if the loss of Con A could be stopped by cross-linking it with glutaraldehyde. We first tested in vitro cross-linking of the Con A; this was done in a manner similar to that used with silica supports. We also tested an in situ or “on-column” method. In the latter case 5.86 mg Con A adsorbed to the zirconia in the column. Thesedifferences are due to the fact that two separate columns were used, one for each method. With the in vitro procedure the glutaraldehyde cross-link was reduced with sodium cyanoborohydride in a manner similar to the preparation of silica-immobilized protein^.^^^ The peak profiles for the solute pNp-man eluting from the two different columns are shown in Figure 5. The profile obtained with the in vitro prepared phase is much wider than that for the in situ prepared phase. The reduced plate heights ( h ) and k’ for the two chromatograms are given in Table 1. The two profiles are distinctly different; the in vitro immobilized affinity support showed a shoulder in the chromatogram of pNp-man. The two columns are so different that attributing the shoulder to one specific procedural difference is not prudent. These differences include the length of time that glutaraldehyde is in contact with the Con A, high-pressure packing of the precoated zirconia, and use of a reducing agent in the preparation of the in vitro support. Despite the poor peak shape, the peak maximum was still used to calculate retention time, and hence k’. In addition to the peak profiles, the rates of decay of k‘ for pNp-man for the two types of supports are different. The in vitro prepared support demonstrated a markedly larger decrease in k’ with the number of column volumes of mobile phase passed through the column, despite the fact that the 2588
AnalyticalChemistry, Vol. 66, No. 15, August 1, 1994
cross-link was reduced and rendered irreversible. The decay in k’ of pNp-man versus the amount of mobile phase passed through the column was -2.2 X 10-4 f 9.1 X for the in vitro prepared column and -2.7 X f 3.3 X for the in situ prepared column. This phenomenon was not studied further and was disregarded in favor of the in situ prepared Con A support, which showed no decrease in k’ when more than 12 000 column volumes of mobile phase were passed through the column. The k’ of pNp-man on this phase was considerably higher than on phases where the Con A is not cross-linked. We suspect that the glutaraldehydecross-linking changed the affinity of Con A for pNp-man, and we are currently investigating this phenomenon. The cross-link made with glutaraldehyde in the in situ method was not reduced because the Schiff base formed upon reaction of glutaraldehyde with protein should be reversible. This should facilitate the removal of the Con A from the zirconia. We made several attempts to do this, albeit unsuccessfully. Ten-milliliter solutions of 0.1 M sodium hydroxide, 1.0 M acetic acid, 1.0 M hydroxylamine, 0.5 M nitric acid, 0.1 M hydrazine hydrochloride, or 15 M ammonia, when passed through the column, did not reverse the crosslink. As a last resort, the amide bonds of the protein were hydrolyzed with hot nitric acid. This is clearly a harsh procedure, but it does demonstrate the remarkable stability of zirconia. After hydrolysis, a 0.1 M sodium hydroxide solution easily removed the peptide fragments. Con A was reapplied to the zirconia and the column regenerated. We found that 6.27 mg of Con A adsorbed to the zirconia in this cycle, 7% more than the previous cycle. The hot nitric acid treatment may have changed the surface chemistry slightly such that more protein is able to adsorb, or it may have increased the surface area available to the protein, thus increasing the loading capacity. Chromatograms of pNpman on the first cross-linked phase and after the regenerative cycle are virtually identical, indicating that the two phases are chromatographically very similar and the initial k’ values for the two stationary phases are very similar, 27.2 and 27.5 for preparative cycles one and two, respectively. Isotherm for pNpman Interactionwith Zirconia-Bound Con A. We measured the binding isotherm for pNp-man on two different Con A stationary phases. The first isotherm was obtained with a column where the Con A was immobilized solely by adsorption. The second isotherm was obtained from the same column after the Con A was cross-linked with glutaraldehyde. From the adsorption isotherms of pNp-man, we obtained an estimate of the number of moles of binding sites in the column (Nactive conA). We represented this as a fraction of the total number of moles of Con A in the column (Ntotal Con A). The data for the binding isotherm of pNp-man to adsorptively immobilized Con A are shown in Figure 6. After the Con A was cross-linked, the number of available binding sites decreased by about 7%. With adsorptive immobilization we observed 76%activity and, after crosslinking, 71% activity, assuming two active sites per molecule of Con A. These values are taken from the last data point, at a concentration of 1.11 mM pNp-man. Since the slope of the isotherm is still positive, we must conclude that the truevalues are higher than those indicated. The difficulty in measuring breakthrough volumes very close to the void volume precluded
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