Anal. Chem. 1909, 6 1 , 1117-1128 (8) Pollack, G. E.; O'Hara, D.; Hollis, 0. L. J . Chromatogr. Sci. 1984, 2 2 , (9) (10) (11) (12) (13) (14) (15) (16) (17)
343. McNair, H. M.; Ogden, N. W.; Hensley. J. L. Am. Lab. 1985, 34. deZeeun, J.; deNijs, R. C. M. Chrompack Top. 1985, 12, 1. Sievers. R. E.; Giiiis. J. N. Anal. Chem. 1985, 5 7 , 1572-1577. Maroulis, P. J.; Coe, C. G.; Kuznicki, S. M.; Clark, P. J.; Roberts, D. A. US 4,713,362, 1987. Andrawes, F. F.; Gibson, E. K. Anal. Chem. 1980, 5 2 , 846-851. Ettre, L. S.Basic Relationships of Gas Chromatography;Perkin-Elmer Corp.: Norwalk, CT, 1979. Purnell, H. Gas Chromatography;John Wiley & Sons, Inc.: New York, 1962. Littlewood, A. B. Gas Chromatography;Academic Press: New York, 1970. Coe, C. G.; Kuznicki. S. M. US 4,544,378, 1985.
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(18) Barrer, R. M.; Mainwaring, D. E. J. Chem. Soc., Dalton Trans. 1972, 1254. (19) Coe, C. G.; Parris, G. E.; Srinivasan, R.; Auvil, S. R . I n New Developments in Zeolite Science and Techno/ogy-~rocee~ings of the 7th International Zeolite Conference, Tokyo; Iijima, A., Ward, J. W., Murakami, Y., Eds.; Elsevier: New York, 1986; p 1033. (20) Brettell, T. A.: Grob, R. L. Am. Lab. 1985, 17, 19.
RECEIVED for review August 30, 1988. Revised manuscript submitted January 27,1989. Accepted February 14,1989. The authors wish to thank Air Products and Chemicals, Inc., for permission to publish this work.
Improved Resolution of Glycoproteins by Chromatography with Concanavalin A Immobilized on Microparticulate Silica via Temperature-Programmed Elution Alan F. Bergold' and Peter W. Carr*
Department of Chemistry and Institute f o r Advanced S t u d y in Biological Process Technology, University of Minnesota, Minneapolis, Minnesota 55455
The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., Ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyidiimldazoie), as well as the effect of including a monosaccharide In the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.
INTRODUCTION In the decade that has passed since Mosbach (1) introduced the technique of high-performance affinity chromatography (HPAC), our understanding of the factors governing the success or failure of this technique has increased enormously (2). Despite this increased understanding, it is fair to say that Present address: Protein Structure Facility, University of Iowa, Iowa City, IA 52242. 0003-2700/89/0361-1117$01.50/0
HPAC has not mirrored the success of either conventional high-performance liquid chromatography or classical affinity chromatography. Slow desorption kinetics of the surface adsorbed eluite from the immobilized affinity ligand has been identified by many groups (3-5) as the principal rate-limiting factor in HPAC and, therefore, as a controlling factor in achieving narrow zones. In this work we attempt to exploit the column temperature as a means of enhancing the rate of desorption. The elution method used in affinity chromatography depends on the goals of the separation as well as the thermodynamics and kinetics of the biospecific interactions involved. Desorption is typically achieved by changes in pH (6) or ionic strength (7), the use of an inhibitor (8),or a combination of these methods (9). The most common elution scheme for the separation of monosaccharides and glycoproteins when bound to concanavalin A (Con A) is biospecific elution with a competitive inhibitor. The reason for this is 2-fold. First, competitive inhibitors such as a-methyl-D-mannopyranoside (MDM) are readily available and inexpensive. Second, elution with an inhibitor is very mild and selective. Nonbiospecific elution by changes in pH or ionic strength is less commonly used because of the susceptibility of Con A to loss of Ca2+and Mn2+ at low pH (10) and the relative insensitivity of the interaction between Con A and polysaccharides to ionic strength (11). A major disadvantage of inhibitor-based elution schemes is the above mentioned dominant role of surface desorption kinetics on the efficiency of Con A affinity columns (12). We and others have previously pointed out the extreme sluggishness of this process for small monovalent eluities (3-5, 12-14). The desorption of such species from immobilized Con A is very slow (approximately 1 s-l) compared to the rates observed for the desorption of small solutes from a reversed-phase column (>lo0 s-l) (15). As a consequence of the slow kinetics, traditional gradient elution methods are relatively ineffective in sharpening broad, kinetically tailed peaks. Muller has studied the effect of kinetics on the elution of glycoproteins from HPAC columns (12). Under conditions of a linear gradient of 0.1 M MDM, using ovalbumin as the solute, no protein other than the 0 1969 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
unretained fraction eluted from the column. Only when the MDM concentration was rapidly stepped from 0 to 0.1 M was a small peak observed. It was postulated from these experiments that the desorption of protein was so slow that there was no zone narrowing during gradient development, as is commonly observed in the chromatography of small molecules. “Pulsed” elution with the above inhibitor was just as ineffective in eluting glycoproteins bound t o the Con A matrix. In order t o overcome these limitations, it is necessary t o employ some unusual strategies to achieve elution of proteins in narrow peaks. The most effective ploy used in conjunction with a competitive inhibitor is t o load the sample protein on the column, and after nonbinding species have emerged from the column, fill the column with inhibitor, stop the flow, and allow the solute time to desorb from the stationary phase. In the case of ovalbumin bound t o a n affinity matrix, approximately 8 min was required for quantitative recovery of the protein. IJnfortunately, this method results in the elution of all forms of the bound protein and resolution of the various binding subfractions is lost. The ideal eluent would affect both the kinetics and thermodynamics of the biospecific interaction simultaneously. The need for this type of elution method was the impetus for our investigation into the use of programmed temperature rlution. Despite the fact that temperature can have a large effect on both the thermodynamics and kinetics of protein interactions (16, i 7 , there has been very little work reported on the use of temperature as a n elution parameter and, more importantly, temperature gradients, to bring about fractionation in HPAC. In comparison to gas chromatography, retention in liquid chromatography is less sensitive to temperature. ‘This is certainly true for small molecule chromatography (18, 19); however, the enthalpy of protein-protein interactions can be as large as the heat of vaporization of small molecules. Another major impediment to the use of temperature as an elution method in LC is the general dogma that for “regular” systems, a n increase in temperature results in decreased separation factors (a),and therefore, solvent gradients are the only logical choice. The term “regular” means that a plot of AHo versus log k’is linear, and that elution order is unchanged as the separation temperature is varied (19). While it is true that isothermal temperature changes will generally only exert detrimental effects on resolution for these systems, temperature gradients can improve the separation because band spacing is kept constant for all solutes in the mixture (20.21). This effect was shown rather convincingly by Bowermaster and McNair who were able t o separate a mixture of’ 10 alkyl benzenes with complete base-line resolution in 15-30 min. By use of microbore columns, the thermal response time to a step change was reduced to less than 1 min, as compared to more than 2 min for a conventional bore column. A number of other reports of temperature programming have appeared (22-25), but the widespread application of the technique has not been realized, possibly because of the lack of commercial instrumentation capable of performing the task in comparison to readily available solvent gradient instruments Most of the arguments against temperature programming in LC are directed toward systems that exhibit nearly ideal behavior; that is, there are no chemical kinetic limitations in the system. For affinity chromatography, this is not the case and the real utility uf this elution method reveals itself. Harvey and co-workers were the first to demonstrate the effect of temperature in affinity chromatography (26). They showed that the binding of yeast alcohol dehydrogenase t o immobilized adenosine monophosphate decreased substantially a t higher temperatures. In their study, a n upper temperature of 40 ‘ C n as used to avoid melting the agarose support. In
HPAC, this will not be a problem since silica is much more stable to temperature than are soft gels. Previous studies from this laboratory have shown that dissociation rate constants of small solutes on Con A are very sensitive to temperature ( 5 ) . The apparent activation energy obtained for the dissociation of p-nitrophenylmannoside from immobilized Con A is about 12 kcal/mol. Others have observed that increasing the temperature to between 30 and 50 “C caused the dissociation of a glycogen-Con A complex (28). Temperatures in excess of 50 O C denature Con A (28),but our observations indicate that immobilization stabilizes Con A sufficiently to allow temperatures of u p t o 80 “C to be used for short periods of time without major and immediate loss of activity.
EXPERIMENTAL SECTION Reagents. Concanavalin A (Con A, type IV), a-methyl-Dmannose (MDM, grade 111), ovalbumin (OVA, grade VI), horseradish peroxidase (HRP, type VI), guaiacol, and aminoethanol were obtained from Sigma (St. Louis, MO). cy-Methyl-D-mannose was recrystallized from hot methanol to remove some impurities which caused base-line disturbances during the programmed elution studies. Nucleosil500-10 (pore diameter 500 A, particle diameter 10 pm) was purchased from Alltech Associates (Deerfield, I11) and (7-glycidoxypropy1)trimethoxysilanewas obtained from Petrarch Systems, Inc. (Levittown, PA). 1,l’-Carbonyldiimidaole (CDI) was obtained from Aldrich (Milwaukee, WI). All other chemicals were reagent grade and were used as received. Apparatus. The chromatographic system used in the affinity experiments was comprised of a Hewlett-Packard 1084B solvent delivery system, a Rheodyne 7120 injector fitted with either a 10or 100-pL loop, a Rheodyne 7040 switching valve, a Perkin-Elmer LC-15 UV monitor with a 10-pL flow cell, and a Hewlett-Packard 3390 integrator. The affinity column was placed in the loop of a switching valve to enable stopped flow application of the sample on the column. The valve also served as a low dead volume heat sink to minimize disturbance of the base line by heat transfer from the mobile phase to the detector. Temperature gradients were generated by using a modified version of a Model CH-1488 dual zone temperature controller and a Model 90032 preheater module from Systec, Inc. (Minneapolis, MN). This is an isothermal device that is easily modified and interfaced to an Apple 11+ computer through the digital to analog (D/A) converter of an ADALAB card (Interactive Microware, Inc., State College, PA). A U-cool refrigeration unit (Neslab, Portsmouth, NH) was used to lower the temperature below ambient and to rapidly cool the column at the end of a run. Anion exchange chromatography was carried out on a Waters HPLC consisting of two Model 501 pumps, a Model 660 gradient controller, a Model 710B WISP, a Lambda Max 481 variable wavelength detector, and a 740 data station. Isoelectric focusing was done with an LKB multiphor I1 horizontal electrophoresis cell (LKB, Gaithersburg, MD), a Bio-Rad Model 3000xi power supply, and a Neslab Exacal circulating temperature controller with a flow-through refrigeration unit. Enzyme assays were done on a Beckman DU Model 65 spectrophotometer using the kinetics package for data reduction. Methods. Prior to immobilization, the lectin was purified by using the method of Cunningham et al. by incubation a t 37 O C in 1% (w/v) NH4HC03for 12-16 h (29). Diol silica was prepared from Nucleosil 500-10 and y-glycidoxysilane by a method developed in this laboratory (30). Con A was then coupled to the derivatized support by the CDI method (31). The diol silica (0.6 g) was activated by reacting it a t room temperature with 0.2 g of CDI in 2.4 mL of anhydrous 1,4-dioxane for 30 min in a stoppered flask. The activated support was then washed in a medium porosity scintered glass filter with 100 mL of 1,4-dioxane and then 100 mL of 0.1 M borate buffer (pH 8.5) containing 10 mM MgCI,, 1 mM CaC12,0.5 M NaCl, and 1 mM MDM. After all buffer had been removed, the slightly moist cake of silica was added to 16 mL of purified Con A which had been dialyzed against the above borate buffer for 24 h at room temperature. The silica and Con A were allowed to react for 24 h a t 4 O C with gentle agitation. A t the end of this time, 50 pL of aminoethanol was added to remove activated hydroxyls and the mixture reacted for
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
another 24 h a t 4 "C. The amount of Con A bound to the silica was determined from the change in absorbance of the initial Con A solution at 280 nm. The amount of lectin found in the various washing steps was also taken into account. For most of the work described here, two columns were used one with 87 and a second that had 93 mg of Con A bound per gram of silica. Column Packing and Chromatographic Conditions for Affinity Chromatography. The Con A affinity columns were slurry packed a t 2500 psi using HPLC grade water for both the slurry and packing solvents. Water was used as the solvent. The column blanks used were 5 cm long by 2.1 mm i.d. with an outside diameter of ' / g in. The Systec heater module consists of a thin copper film heating element and a length of in. HPLC tubing (27-pL volume) through which the mobile phase passes on its way to the column. The tubing is encased in a thermally conductive epoxy potting material. The heating unit is a hollow cylinder which is 1.5 cm i.d. by 2.5 cm 0.d. by 3.5 cm in length. In order to improve the conduction of heat directly between the heater and the chromatographic column, the column was wrapped with copper wire so that it fit snugly into the heater. The entire assembly, that is the heater and the column, was encased in 1 in. i.d. plastic pipe insulation to minimize convective heat losses. In order to rapidly return the system to an initial temperature and to work at temperatures below ambient, a compressed air line and cold finger of a Neslab refrigeration unit were also situated within the pipe insulation. When cooling is needed, air is passed over the cooling coil and then over the column-preheater assembly. This arrangement allows a lower temperature limit of 5 "C to be achieved. The mobile phase used for all affinity separations was 0.02 M sodium phosphate (pH 6.0) containing 0.5 M NaC1,O.Ol M MgC12, and 0.001 M CaCl2 AU solutes were prepared in this mobile phase. The competing eluent mobile phase consisted of the appropriate weight of the purified MDM added to the above mobile phase. The flow rate used for the separation of proteins was 0.1 mL/min, while the flow rate used for measurement of p-nitrophenylmannose (pNp-mannose) retention time was 1.0 mL/min. Anion Exchange Chromatography. Anion exchange chromatography was carried out under conditions similar to those of Vanecek and Regnier (32). Mobile phase A was 20 mM Tris, pH 7.0, while mobile phase B consisted of 20 mM Tris with 0.5 M sodium acetate added. The pH of this mobile phase was adjusted to pH 7.0 after the addition of sodium acetate to avoid any pH shift due to the acetate. The column used in these experiments was a 4.6 mm i.d. x 15.0 cm Synchropak AX-300 column which was packed according to the manufacturer's instructions using methanol as the packing and slurry solvent at a pressure of 9000 psi. Before the ovalbumin fractions taken from the Con A affinity chromatography could be separated on the anion exchange column, the 0.5 M sodium chloride in the affinity column eluent was removed using Centricon-30 ultrafiltration concentrators from Amicon. The ovalbumin fractions from the Con A affinity column were collected in 5.0-mL Falcon tubes. The collected fractions were then placed in the Centricon filters and centrifuged at 4000 rpm for 20 min in a Beckman 52-21 centrifuge equipped with a JA-20 rotor. Two milliliters of 20 mM Tris, pH 7.0, was added to the concentrate and each fraction was then spun again at 4000 rpm for 20 min. This buffer exchange was repeated once more before the samples were collected by centrifuging a t 2000 rpm for 15 min. After collection each sample was contained in approximately 50 pL of Tris buffer. This volume was brought to 100 pL with mobile phase A so that four 20-pL injections of each sample could be made on the anion exchange column. Isoelectric Focusing. The above desalted ovalbumin fractions were also subjected to isoelectric focusing. One third of a 1-mm Ampholine PAG plate (PI 3.5-9.5) was used in this experiment. Fifteen microliters of each sample was applied to a cellulose applicator strip located midway between the anode and cathode. The middle four lanes were used for samples while the outer two lanes contained a wide PIrange standard from Serva Biochemicals. The plate loaded with the samples was prefocused at a constant voltage of 200 V for 15 min. Power was then turned off and the applicator strips were removed. Focusing was resumed a t 3 W constant power for 71 min, and then at a constant voltage of 1800 V for 30 min. The plates were removed from the cell and then
1119
fixed and stained simultaneously by using the quick stain method of Reisner (33). The gel was stained for 1 h at room temperature and then destained for 30 min by soaking in water followed by soaking for 30 min in the preservative solution consisting of 10% (v/v) glycerol in water. The top of the gel was then protected by applying a cover of Gel-bond. Enzymatic Assay for Horseradish Peroxidase. The method for assaying HRP activity was adapted from Tijssen (34). The amounts of the reagents in the assay were adjusted to give better sensitivity for the dilute peroxidase solutions studied here and consisted of the following: 2.2 mL of 100 mM potassium phosphate, pH 7.0, 0.5 mL of 0.245 mg/mL guaiacol, and 0.4 mL of 9 mM hydrogen peroxide. Peroxide solution was freshly prepared from 30% H202and its concentration was checked via its absorbance (35). The assay was started by rapidly pipetting 20 pL of the HRP solution into the cuvette containing the assay mixture and quickly stirring with a small cuvette paddle. The change in absorbance at 436 nm was recorded for 2 min. Least-squares analysis of the A436 versus time data was then used to calculate the units of activity by the following equation:
U = 4 X dA/dt = 24.4dA/dt
X
(3.12 cm3/25.6 cm-' M-l)
where 3.12 is the final volume of the assay mixture and time (t) is in minutes. U is defined as the number of micromoles of substrate converted per minute. In these experiments, less than 10% of the substrate was converted in the 2 min of data collection. The absorbance versus time curves exhibited some curvature, which was independent of the enzyme concentration. This curvature may be due to the order of addition of reagents. Tijssen recommends that peroxide be the last reagent added to the assay mixture; however, the rapid addition and thorough mixing of 20 pL of enzyme were much easier to accomplish than the addition of 0.4 mL of peroxide. Since the "open tube" runs (see below) and thermal elution fractions of HRP exhibited identical curvature patterns, this observation was not investigated further. Recovery experiments were conducted as follows. A filled loop injection of the HRP solution (10 pL) was made with the Con A column in the column bypass position and the effluent from the detector was collected a t a flow rate of 0.1 mL/min for 40 min from the time of injection. This procedure was then repeated with the column in-line and HRP eluted via a thermal gradient. At the end of 40 min, collection of a second fraction was initiated and the column was then rapidly cooled to 30 "C, as described above. When the column reached 30 "C, it was "pulsed" 5 times with 10 pL of 0.1 M MDM. After 40 min, collection of the second fraction was stopped. The "open tube" data were used as a measure of the total amount of activity of HRP applied to the Con A column. The activity obtained in the first 40-min fraction, when the column was in-line, represents the fraction of HRP recovered during the thermal gradient, while the second 40-min fraction was the amount not removed from the column at elevated temperature, but was still active.
RESULTS AND DISCUSSION Isolation of Ovalbumin Fractions. Figure 1illustrates the resolution that can be achieved by using temperature gradient elution. In this example, 0.21 mg of ovalbumin (OVA) was resolved into three major fractions by using a linear temperature gradient from 30 to 70 "C during the course of 27 min. The first peak (I) is unretained and accounts for 24% of the total area. Peaks I1 and I11 account for 32% and 44% of the total peak area, respectively. The sample protein was completely eluted from the column by the temperature program, as indicated by the observation that repeated injections of MDM did not show elution of additional protein. Previous studies have shown that this test is a sensitive indicator of complete removal of OVA from the column (12). In addition t o the above three major fractions, there are two minor fractions. The first minor component is evident on the trailing edge of the unretained peak. As the amount of active Con A on the column gradually decreased due t o repeated heat cycles, this fraction merged into the unretained
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
Table I. Effect of Initial Dwell Time on the Retention of Ovalbumin Fractions I1 and IIIn
I
t,,
min
dwell time, min
peak I1
peak I11
4.0 6.0
16.51 17.19
30.76 32.38
Atr
0.68
1.62
"The flow for both hold times was 0.1 mL/min and the solute was 10 p L of ovalbumin (2.0 mg/mL). The temperature program consisted of an initial temperature of 30 "C with an initial hold time as indicated above. The heating rate was 1.5 "C/min to 70 "C with a hold time at the final temperature of 20 min.
I
,
l
j
l
l
,
l
l
0
10
20
30
40
50
60
10
80
l
90 min.
Figure 1. Thermal elution chromatogram and rechromatography of ovalbumin on immobilized concanavalin A. The column is 2.1 mm (i.d.) by 5.0 cm packed with Nucleosil-500 (10 ym) containing 87 mg/mL of concanavalin A. Flow rate is 0.1 mL/min and the detector is set to 0.128 absorbance unit full scale. The temperature program is as follows: 6 min isothermal at 30 "C then increased at 1.5 "C/min to 70 "C. The eluent is as described in the text for Con A chromatography. There is no MDM in the mobile phase. Key: (a) 100 yL of 2.1 mg/mL stock ovalbumin; (b) 100 yL of peak I from chromatogram a; (c) 100 y L of peak I 1 from chromatogram a; (d) 100 y L of peak I 1 1 from chromatogram a; (e) blank gradient base line.
peak. The second minor fraction is not detectable in the chromatogram shown in Figure 1. I t is detected only when a t least 1 mg of OVA is injected onto the column. The strong retention of this fraction (retention time 60 min) combined with its low relative abundance are such that it is only possible to observe this peak on a column that has a low total binding activity. Our results with OVA agree well with those of Iwase et al. (36, 37) who were able to resolve OVA into three majors fractions on Con A-sepharose by means of a gradient of MDM (0 to 0.1 M). In their investigation, they found an unretained component that comprised 16% of the total protein and two retained peaks that amounted to 37% and 41% of the total protein, respectively. I t may be argued that the two retained peaks are merely artifacts due to thermally induced changes in the sample protein. We do not believe that this is the case. First, there is good relative agreement on the amount of the three major fractions with Iwase who used very mild elution
conditions. Second, when new species are generated in a chromatographic column by chemical transformation, the peak shapes are generally very asymmetric and it is impossible to base-line resolve the peaks (38, 39). The peaks shown in Figure 1 are acceptably symmetric and can be base-line resolved by adjusting the heating rate. Third, in control experiments, OVA was incubated at 60 "C for 30 min. These samples were then injected onto the Con A column and eluted as in Figure 1. The only difference between the chromatogram of the preheated samples and that shown in Figure 1 was the presence of a small peak a t 5 min, which accounted for less than about 6% of the total area. That the peaks shown in Figure 1 are inherent in the sample and not generated by the thermal program is proven by the fact that one can isolate and rechromatograph the fractions. As shown in Figure 1 (curves b-d), rechromatography of peak I1 yields only peak I1 and some unretained material. An analogous result is obtained for peaks I and 111. The small unretained peaks seen in curves c and d of Figure 1 were initially thought to be due to the split peak phenomena (40). At the suggestion of a reviewer, we discount this possibility since the association kinetics, residence time, and amount of Con A in the column relative to the amount of sample injected are such as to mandate a very small split peak. There remain several possibilities. First, the small unretained peaks in curves c and d could be due to denaturation of ovalbumin which takes place during the first thermal elution chromatogram. Second, the unretained peak in curve c could be due to some of fraction I co-collected with fraction 11. Third, the unretained peak in curve d could be some desorbed Con A co-collected with fraction I11 (see below). We do not have a n y strong evidence for or against any of these hypotheses. In any case, the unretained peak is small ( < l o % of the total area) and we have not considered it in greater detail. Further studies will be needed to ascertain the source of the unretained material. If the retained peaks were artifacts that derived from a common starting material, the only on-column reaction that could give homogeneous fractions upon rechromatography would be one that went very rapidly to completion during the separation. The results in Table I indicate this cannot be the case since the retention times of peaks I1 and I11 are affected to different extents by the initial dwell time a t 30 "C. Note that at this low temperature, it is most unlikely that OVA will be altered in 2 min. Because the retention of peak I1 increases by only 0.68 min upon a 2-min increase in hold time, the form of OVA represented by fraction I1 must be present in the original sample. Furthermore, a t a temperature of 30 "C, fraction I1 is migrating along the column a t a faster rate than is fraction I11 because its elution time is more strongly influenced by the dwell time at 30 "C than is that of fraction 111. Finally, on columns that have very low Con A activity, isothermal chromatograms (40 "C) of OVA exhibit the same
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
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C
&
1.5 % / r n i n .
L 0
20
a0
SO
0
20
40
60
SO
0
10
10
60
rnl""I..
Flgure 2. Elution behavior of ovalbumin from immobilized Con A under isothermal conditions. Ten microliters of ovalbumin at 2 mg/mL was injected. The temperature was increased at 6 "C/min to 70 "C and held for 15 min before return to the initial condition. Note the initial activity of Con A had decayed by 33% at the time of this study. No inhibitor was in the mobile phase. Key: (a) initial condition, dwell time 30 min at 30 "C; (b) initial condition, dwell time 60 min at 40 "C; (c) initial condition, dwell time 30 min at 60 "C.
pattern, as shown in Figure 2. Thus, we believe that the fractions show in Figure 1 are real and are not due to thermal stresses on the sample protein. Retention Behavior of Ovalbumin as a Function of Mobile Phase and Gradient Conditions. In contrast to previous attempts by ourselves (12, 14) to resolve OVA by competition elution gradients in HPAC, resolution in temperature gradient affinity chromatography can, as shown in Figure 3, be easily adjusted by changing the rate of temperature change. As expected, the lowest rate gave the best resolution. Increasing the rate resulted in much narrower peaks (in time units) and increased the detectability but caused a decrease in resolution. Also as anticipated, the resolution changes very little upon doubling the rate of temperature change from 6 to 12 "C/min. In fact, above 12 "C/min the resolution is essentially independent of the programmed rate of change of temperature. Inspection of Figure 3 indicates that although the retention time decreases as the rate is increased, the peak separation remains constant above rates of about 5 "C/min. We believe that this results from two factors. The dominant factor is the slow rate of heat transfer from the heater to the column. A program rate somewhere between 6 and 12 deg/min corresponds to the maximum rate of heat transfer into the column under the conditions employed, as far as the inside of the column is concerned. This is supported by the fact that the retention times approach a finite but nonzero time that is independent of heating rate. The second factor that controls the retention and resolution of the ovalbumin fractions is the intrinsic migration rates of the two zones a t the final temperature. As seen in Table 11, the apparent "elution temperature" (with no MDM in the mobile phase) for peak I11 is above the final temperature of the program. Despite the fact that the calculated elution temperature for peak I11 is an artificial number, it serves as a figure of merit to indicate the relative temperatures needed to cause these proteins to elute in a narrow zone a t this level of column activity. Since the final temperature of the program was only 70 "C, the protein elutes only after the column has reached the final temperature. Because the higher rates resemble a step change in temperature, the protein zones at these high rates migrate through the column at a rate determined by their equilibrium binding constants at a temperature equal to the final temperature of the program. Thus, the peak separation under these conditions is independent
I
1
I
I
I
l
0
10
20
30
40
50
mln.
Figure 3. Effect of heating rate on the resolution of ovalbumin on immobilized concanavalin A. All conditions are as in Figure 1 except the detector was set to 0.016 absorbance unit full scale, 10 pL of 2.1 mg/mL of ovalbumin was injected, and the initial dwell time was set to 4 min at 30 "C.
Table 11. Effect of Mobile Phase Inhibitor on the Retention Time of Ovalbumin Fractions" concn of
peak I1
peak I11
a-MDM, pM
t,, min
TE, "C
t,, min
TE,"C
0 77.4 149.6 216.4
20.0 f 0.2 15.6 f 0.3 13.3 f 0.3 11.6 f 0.1
54.0 47.4 44.0 41.4
32.9 f 0.2 29.9 f 0.4 27.8 f 0.2 26.0 f 0.1
73.4 68.8 65.7 63.0
The flow rate for all cases was 0.1 mL/min. Ten microliters of ovalbumin (2.0 mg/mL) was injected for each run. The gradient (4 rnin a t 30 "C, 1.5 "C/min to 70 "C, 20 rnin a t 70 "C) was the same for all cases. The apparent elution temperature (TE) was calculated as follows: TE = Ti + r(t, - ti), where Ti is the initial temperature, r is the temperature ramp rate, ti is the hold time a t the initial temperature, and t , is the peak retention time.
of heating rate and is determined primarily by the ratio of the equilibrium constants at the final temperature. A t this time, a direct estimate of how closely the temperature inside the column follows the program is not available since attempts to measure the effluent temperature were
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
1122
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-:, m -
r _
Flgure 4. Delay time in the return of the base line to steady-state versus the heating rate. Conditions are as in Figure 1. Key: (a) 0 , 217 MDM pM in the mobile phase; (b) 0, no MDM in the mobile phase.
unsuccessful. T h a t the lag in temperature is significant and depends on the programmed rate can be inferred from the results given in Figure 3 and is supported by the observations presented in Figure 4. The data given in Figure 4 are the difference in time between when the program reaches the final temperature and when the base-line disturbance reaches a steady state. When there is no MDM in the mobile phase, the baseline return delay time varied from 3 min at 1.5 "C/min to 5 min at 12 "C/min. As was seen for the data on the effect of heating rate on OVA retention, there is no change in the delay time between 6 and 12 "C/min. The delay times with inhibitor in the mobile phase were substantially longer, suggesting that the base-line disturbance, at least in this instance, had a kinetic component. The actual cause of the base-line disturbance with and without inhibitor present in the mobile phase is unknown. For the situation where MDM is present, part of the absorbance shift is undoubtedly due to the desorption of this reagent from Con A at the elevated temperature. The base-line shift without inhibitor may also be partly chemical. Although the exact lag is not known, these results indicate that for heating rates below 6 "C/min the lag time is less than 4 min. The Systec column heater module was specifically chosen for this application because of its low thermal mass. In an experiment where the column heater was attached directly to the detector, without a column or valve in-line, we observed a base-line disturbance within 15 s after a step change in temperature from 30 to 70 "C. The total time for the thermal base-line disturbance to reach its final value was only 1 min. Clearly, the heater responds rapidly and has the necessary power to heat columns of the dimensions used here. Rough calculations on the amount of power needed to raise the column from 30 to 80 "C at a rate of 12 "C/min indicate that 1 W is required a t a flow rate of 0.1 mL/min. This is well below the manufacturer's specification of 3 W. Efficient heat transfer into the column is a critical parameter in determining the ultimate efficiency of this system. In some studies not shown here, the temperature change in the column was generated by moving the column outside the heating element and the insulation. That is, the column was heated only by the warm mobile phase. The resulting peak widths were twice those observed in Figure 1 under otherwise comparable conditions. This observation is not surprising in light of several publications documenting the effect of radial temperature gradients on peak shape (41,42). This effect may also result from the lower achievable temperatures and rate of temperature change. Some evidence to support this claim is presented below. Early results on the retention behavior of OVA and other glycoproteins on Con A stationary phases suggested that they were so strongly retained that the zone of protein injected on the column remained at the head of the column and did not
5
15
12 n :IC H o d Time
_7 --
m ~ ,-
Figure 5. Effect of initial temperature and dwell time on the retention of ovalbumin. The flow rate was 0.1 mL/min. Except for initial temperature and initial dwell time, all conditions are the same as in Figure 1. Solid symbols denote peak I11 and open symbols denote peak I 1 in Figure 1. Key: (a)(A,A)initial temperature 10 "C; (b) (0,W) initial temperature 20 "C; (c) (0,0 )initial temperature 30 "C.
move. Only after inhibitor was introduced did the protein zone begin to migrate and move to the column outlet. The results in Figure 5 show that this is not the case. These experiments were carried out by extending the dwell time at the initial temperature from 4 to 15 min. The same temperature program was then applied to the column. If the initial hypothesis that the zone does not migrate unless inhibitor or a temperature gradient is applied is correct, then the protein will spend the same amount of time migrating under program conditions regardless of the initial hold time (i.e., the difference between the retention time and the hold time). As the data in Figure 5 indicate, the time under thermal gradient conditions decreases, meaning that the protein has migrated some finite distance along the column during the initial dwell period. In fact, peak I1 elutes isothermally at 30 OC, so the hypothesis cannot be true. Peak I11 is influenced much less by the dwell time and migrates very little a t 30 "C. Due to the imprecision in the area of these broad peaks, we did not observe any trend in the area as a function of dwell time. The extent of zone migration under isothermal conditions is most likely a very strong function of the amount of active Con A bound to the support. A t the time these experiments were carried out, this column had been subjected to more than 50 heat cycles and had much less active Con A on it than was initially present. This explains previous observations from this laboratory (12-14) that OVA did not elute under isothermal and isocratic conditions. In previous work, the binding activity of the columns was very high. Mobile phase inhibitors can have a very strong effect on the retention of glycoproteins during thermal gradient elution. Table I1 shows that the temperatures needed to achieve elution can be decreased by adding a low level of inhibitor to the mobile phase. As little as 217 pM MDM is sufficient to lower the elution temperature of peak I1 by 13 "C and to cause peak I11 to start eluting before the final temperature of 70 OC is reached. Interestingly, the added inhibitor improves the resolution of the bound ovalbumin fractions since the retention of peak I11 is not decreased as much as is that of peak 11. The reason for this observation is not clear but may have to do with the extent of nonspecific binding for peak 111. If the retention of peak I11 is controlled to a significant extent by a temperature-sensitive but nonbiospecific interaction, then one would expect a smaller effect of the inhibitor on its retention time. Another interesting observation is that a low concentration of inhibitor has a rather large effect. Isothermal experiments at 25 "C or lower required 0.1 M or more MDM to elute OVA (12). In addition, inhibitor gradients were not able to resolve the two retained forms of ovalbumin seen here. The higher
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
\
4 t ' t
2;
94
C
1\
\ C
B
A
1123
~
1C
20
30
40
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60
70
80
H e a t Cycle k!
Flgure 6. Retention of p-nitrophenylmannose versus the number of thermal cycles. Retention times for pNp-mannose were measured as descrlbed in the Experimental Section. Temperaure gradients for heat
a.
treatment are the same as described in Flgure 1, except the final temperature was 80 O C . Key: (a) 0 , Con A immobilized by the glutaraldehyde method, column heated directly and by mobile phase flow; (b) 0, Con A immobilized by glutaraldehyde, column heated by mobile phase flow only; (c) A, Con A immobilized by carbonyldiimidazole, column heated directly and by flow of the mobile phase; (d) A,as in c above but after 50-70 I.LM MDM was added to the mobile phase. temperatures used here have increased the desorption rate to a point where the inhibitor can now kinetically compete more effectively with glycoproteins for Con A binding sites. Despite these observations, the most important result of the inhibitor studies should not be overlooked. That is, inhibitors lower the temperature needed for elution. This is especially important when the temperature stability of the proteinaceous solutes or of the stationary phase is a problem. Stability of the Affinity Phase. In order to ascertain the thermal stability of the affinity phase toward repeated heating and cooling cycles, the retention of p-nitrophenylmannoside was checked periodically. Since p-nitrophenylmannoside binds only to active Con A, a change in its retention is a fairly good indicator of a change in the relative amount of active Con A on the column. The results of these experiments are given in Figure 6. There is a clear-cut decrease in retention with thermal cycle number. We believe that this decrease corresponds monotonically to the decrease in the amount of active Con A remaining on the column. Because the stability of an affinity phase could well depend on the chemistry used to immobilize the ligand, we examined stationary phases prepared by immobilizing Con A to an aminopropylsilica via the glutaraldehyde method as well as by attachment to a diol phase via the carbonyldiimidazole method. Comparison of parts a and c of Figure 6 shows that the glutaraldehyde immobilized Con A is far less stable than is carbonyldiimidazole immobilized Con A. The amount of activity lost per cycle is roughly 7 times greater for the glutaraldehyde immobilized material, as measured by the ratio of the slopes in Figure 6a,c. The reason behind this major difference in stabilities is not known, although the mechanism of loss appears to be due to leaching of Con A, since the initial base-line disturbance blank thermal chromatogram of a new glutaraldehyde column is about 5 times higher than that for a new carbonyldiimidazole column. Both phases should have stable covalent linkages between the support and ligand. Glutaraldehyde chemistry, after reduction of the Schiff base with sodium borohydride, results in a stable secondary amine linkage (43). Diolcarbonyldiimidazole chemistry results in a carbamate link between the ligand and support (31). The greater instability of the glutaraldehyde-Con A phase may indicate that the borohydride reduction is largely incomplete, and consequently, the majority of the linkages between Con A and the support are still in the Schiff base form. This explanation might also
b.
C.
1
1
1
1
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l
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0
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80
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mln. Flgure 7. Effect of the loss of Con A binding activity on the peak shape of horseradish peroxidase. Column as in Figure 1 but with 68 mg/mL of Con A, detector set at 0.008 absorbance unit full scale, 10 pL of HRP (1.2 mg/mL) injected. Key: (a) after 28 heating cycles, temperature program is initial temperature 30 "C for 4 min then heat at 2 OC/min to 80 O C and hold for 30 min, 514 I.LM MDM present in the mobile phase; (b) after 27 heating cycles, temperature program is initiil temperature 40 O C for 4 min then heat at 2 "C/min to 70 O C and hold for 20 min, 514 pM MDM in the mobile phase; (c) after 11 heating
cycles, temperature program is initial temperature 30 O C then heat at 1.5 'C/min to 70 O C and hold for 20 min, 275 I.LM MDM in the mobile phase. account for some instability in the glutaraldehyde-linked Con A column observed a t temperatures of only 30 "C. There is still some controversy over what reactive species is involved in glutaraldehyde activated supports and the nature of the linkage with ligands (44). In any case, it is evident that thermal cycling experiments provide a rather straightforward approach to investigating the relative stability of different methods of immobilizing an affinity ligand. Part of the activity loss associated with long-term thermal cycling may be due to the rapid cooling of the column to its initial temperature. The large peaks between 60 and 70 min (see Figures 7 and 8) occur in blank thermal gradients and are not related to the injection of any sample. Assay of the collected fractions from such blank runs indicates that the material is a protein. This observation can be readily explained by the fact that lower temperatures favor the existence of the dimer form of Con A over the tetramer (45). Rapid cooling probably results in conversion of some immobilized
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
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0
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60
70
80
I
90 min.
Flgure 8. Thermal elution chromatogram of ribonuclease B on a concanavalin A column. The column and mobile phase condffions are as in Figure 1, except the column was prepared with 68 mg/g of Con A. The detector was set at 0.008 absorbance unit full scale. Ten microllters of 2.5 mg/mL of ribonuclease B was injected. Temperature program is initial temperature 30 "C for 4 min then heat at 1.5 "C/min to 70 "C. MDM (275 1M) present in the mobile phase.
tetramer to dimer, with one dimer unit remaining covalently bound to the support while the other subunit, which was not covalently attached to the surface, is released into the mobile phase. This is supported by the observation that the rate of cooling has a pronounced effect on the size of the peak observed. At higher cooling air flow rates, this peak is fairly large, but it is not observable a t very low cooling rates. In addition, such peaks are not observed at all after the column has lost about 50% of its initial activity. This supports the above hypothesis that this peak is due to noncovalently bound Con A dimers. A detailed study of the rate of activity loss with and without enhanced cooling has not been done to determine if column life could be extended. From the results shown above, it is quite likely that less activity would be lost if the column were cooled very slowly. Direct heating of the column, in contrast, to heating by the mobile phase, results in much higher temperatures being generated inside the column. As is seen from the slopes of Figure 6a,b, a directly heated column (Figure 6a) loses activity much more quickly than does an indirectly heated column (Figure 6b). Since both of these columns were made from the same material, and since the programmed final temperature (80 "C) was the same for both, the increased stability must be due to a lower final temperature being realized for column B. For the glutaraldehyde-Con A columns heated only via
the mobile phase, the actual final temperature is low enough to result in a stability comparable to the carbonyldiimidazole-Con A material (Figure 6c), as shown by the near equality in slopes. The use of an inhibitor in the mobile phase results in a marked increase in stability of the affinity phase (Figure 6d). This seems to indicate that the binding of a ligand by Con A stabilizes a particular conformation which is more resistant to thermal denaturation and dissociation. This effect has been observed by Doyle et al. for Con A in solution (28). Another advantage of adding inhibitor to the mobile phase is the greater ease of eluting glycoproteins from the column. Because elution is facilitated under these conditions, the final temperatures can be lowered with a concomitant increase in stability. The loss of Con A activity with repeated heat cycles is not entirely detrimental. Figure 2 illustrates the elution of ovalbumin at several fixed temperatures. The column used in these experiments had experienced more than 80 heat cycles and roughly 33% (as calculated by the decrease in retention of pNp-mannoside) of its initial activity remained. A t the initial level of activity, no elution of protein other than the unretained fraction was observed under isothermal conditions. After the loss of some activity, peak I1 of ovalbumin eluted at 30 "C with a k'of 8.5 while elevated temperatures were still needed to elute peak 111. Higher isothermal temperatures result in narrower zones but can also give distorted peaks such as those observed at 60 "C. Peak distortion a t higher temperatures is most likely due to denaturation of OVA after the rapid change in temperature from 30 to 60 "C. Although no hard experimental evidence is available, the multizoning of OVA at 60 "C is probably due to increased nonspecific interactions between ovalbumin and Con A as a consequence of unfolding of the OVA. Interestingly, this multizoning effect is not seen when linear temperature gradients are employed. The primary interaction (biospecific) between Con A and glycoproteins may help stabilize the protein to such an extent that the protein can withstand elevated temperatures, provided that the glycoprotein and Con A are in contact when the temperature is increased. If the glycoprotein is not associated with Con A when the temperature increases, it then unfolds rapidly. This latter scenario is equivalent to what happens in the isothermal experiments at high temperature (Figure 2c). The ease of eluting glycoproteins after substantial activity loss has some important implications for the design of highly efficient affinity phases. Obviously, the initial Con A loadings used in these studies are too high. The elution of glycoproteins which bind multivalently to Con A, such as HRP, makes elution extremely difficult. As illustrated in Figure 7c, HRP barely starts to elute a t 40-50 min, even though the column has been at 70 "C for 30 min. At these ligand densities, higher initial temperatures (Figure 7b), higher final temperatures (Figure 7a), or lower ligand densities are needed to elute multivalent glycoproteins in reasonably small volumes. Univalent glycoproteins, such as ovalbumin or ribonuclease B, which has a single polysaccharide chain at Asn-34 (46), are much easier to elute (Figure 8). A more detailed study of ligand density effects on the retention of univalent and multivalent proteins will be presented elsewhere. Thermal Denaturation Studies. At a temperature where the van der Waals and other noncovalent forces that maintain the tertiary structure of a protein no longer prevail over long range thermally driven motions of the protein, the native structure is lost and the protein is said to be denatured (47). For most proteins, the temperature range where this occurs is between 45 and 85 "C. Once the protein is cooled below its midpoint temperature for denaturation (Tm),the process
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
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Flgure 9, Anion exchange chromatography of ovalbumin and fractions 1-111 shown in Figure 1. The mobile phase conditions are given in the text. Detection is at 280 nm with 0.032 absorbance unit full scale. Key: (a) stock ovalbumin; (b) fraction I from Con A column; (c) fraction I1 from Con A column; (d) fraction 111 from Con A column.
is usually reversible. Because thermal denaturation can occur in a temperature region where it is desirable to carry out temperature programming, an investigation into the possible modification of the eluted proteins was carried out. Since ovalbumin has no easily measurable bioactivity, potential alterations of this protein were studied via anion exchange chromatography and isoelectric focusing. The so-called “molecular footprint” (48) is sensitive to a particular set of amino acid residues on the surface of the protein and their exact spatial arrangement. Under denaturing conditions, this arrangement is disturbed and retention is altered. This alteration in retention has been used to study the denaturation of proteins under a variety of conditions (49-51). Figure 9 shows the results of running the ovalbumin stock, as well as the first, second, and third thermal fractions from a Con A column, on a high-performance anion exchange column. comparison of the major features of the anion exchange chromatogram of the stock OVA and fraction I, the low-temperature fraction, isolated from the Con A column shows that the two types of chromatography, that is, anion exchange and Con A affinity, are not a t all sensitive to the same types of OVA heterogeneity. The concept of a
Table 111. Peak Areas and Retention Times for the Three Major Peaks in the Anion Exchange Chromatography of the Ovalbumin Fractions from Con A Affinity Chromatographya peak no.
solution
fraction I
fraction I1
fraction I11
Retention Time, min 1 2 3
13.75 f 0.02 13.54 f 0.03 13.71 f 0.11 13.78 f 0.03 19.47 f 0.02 19.31 f 0.02 19.33 f 0.09 19.39 f 0.02 21.78 f 0.01 21.58 f 0.04 21.58 f 0.05 21.70 f 0.01
1 2 3
6.2 ak 0.4 49.8 f 9.1 35.6 f 23
total area % 91.6
Area, % 12.7 f 1.0 35.4 f 7.1 30.4 k 12
6.0 f 0.3 66.5 + 2.0 22.2 f 2.4
5.20 f 0.001 69.70 f 2.9 21.20 f 5.9
98.5
94.7
95.9
Retention time and area data are the results from three repetitions of each solution. All solutions were desalted according to the procedure given in the Exuerimental Section.
“molecular footprint? is clearly applicable. Comparison of the anion exchange results for the stock OVA and all three fractions taken from a Con A column shows that no drastic denaturation has taken place as a result of the use of elevated
1126
ANALYTICAL CHEMISTRY, VOL. 61. NO. IO. MAY 15. 1989 1
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CATHODE
-7.2
Std. S
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111
Std.
ANODE
Flgun 10. IJOelectrlc focusing of Ovalbumin fractions obtained from Con A column: SM. derides two standard lanes. S i s h stock SolUWan of ovalbumin. and 1-111 denote the three fractions w a n in Figure 1 on a Con A column.
temperature. No new peaks have appeared and retention times for the three major peaks in the anion exchange chromatograms are constant within experimental error (Table III). There are, however, some noticeable differences in the relative m o u n t s of the three major ion exchange peaks between the different ovalbumin fractions. For instance,peak 1in Fraction I has a shoulder on the tail which doubles in area in comparison to the other fractions. In addition, the small triplet of peaks between 15and 19 min differs between fractions. The cause of these differences is not known. The different forms observed could be due to amino acid point mutations in ovalbumin’s primary structure or to differences in phosphorylation of the carbohydrate chain. Since 13 different carbohydrate chains have been observed in ovalbumin (52),this seems to be the most likly explanation a t this point. Structural characterization of the various peaks in Figure 9 is needed to resolve this issue. This observation deserves further investigation since it has not been previously reported. Isoelectric focusing was also used to assess whether the protein was altered by thermal elution. Because this method is sensitive to the total charge on a protein and not just the distribution of a limited number of surface residues and because the pZ of a protein is very sensitive to denaturation (53), this method might pick up subtle changes in tertiary structure not observable by ion exchange. T h e gel uhoto shown in Figure 10 shows no differenceln p l of the ovalbumin stock in lane 2 and the three fractions obtained bv thermal elution from Con A shown in lanes 3, 4, and 5. All four samples indicate that the PI of the major component is 4.1,which agrees with the published PI of ovalbumin (54). Again, there are some differences in the intensity of the bands between fractions, as seen in the ion exchange chromatograms. On the basis of these results, it appears that there is no appreciable modification of the ovalbumin as a result of thermal gradient elution. The possibility still exists that these methods rue too insensitive to the changes in structure that do occur upon thermal elution. Alternatively, the protein is partly denatured as it elutes from the column, but sufficient time has elapsed before the above chromatographic and isoelectric focusing studies could be done, such that the protein refolded and
I
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l 40
l
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l
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mln. Flpure 11. Thermal elution chromatcgam of horseradlsh p%roxk!ase on a Con A column with a reduced activity due to thermal cycling. Detection Is at 0.016 absorbance unk. Ten microllters of 2 mg1mL of HRP was injected. The temperature program is initial temperature 30 O C for 8 min than heat at 4 ‘Clmln to 70 “C and hold for 20 min. No MDM Is present In the Mbile phase.
Table IV. Recovery of Horseradish Peroxidase Activity under Thermal Gradient and Isothermal Conditions
W activity samole open tube run 0-40 min thermal gradient fraction M O min thermal gradient fraction with 513 M a-MDM in the mobile phase pulse elution with a-MDM at 30 OC HRP stock with 1 mM Ca2+incubated 40 min at 70 “C HRP stock without Ca” incubated 40 min at 70 OC
units
* 0.2 * 0.1 3.34 * 0.2
recovered
3.50 3.16
3.45 i 0.1
90 95
6.4
98.5 77
0.8
10
exhibited behavior identical with that of the native protein. As a further test of protein modification in response to thermal elution. the activitv of HRP was determined both before and after’ thermal elition from a Con A column. Because of the extreme sensitivity of HRP elution to the amount of active Con A on the support (Figure 7), this work was carried out on a column whose activity had been reduced to the point where the well-shaped peak shape for HRP shown in Figure 11 was obtained. Assays were done on a fraction collection for 40 min. This fraction included the unretained peak and the major peak a t 20 min. This analysis was compared to an open tube run, as described in the Experimental Section. The results of these experiments are given in Table IV. Ninety percent of the applied protein is recovered in the 40-min fraction. In fact, even more H R P can be recovered if the activity of Con A is further reduced. Addition of in-
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989
hibitor (MDM) to the mobile phase increases the amount of protein recovered for two reasons. First, multivalent interactions between HRP and Con A are reduced, making elution easier, and second, the protein elutes more quickly and spends less time at an elevated temperature. Seven 10-pL pulses of 0.1 M MDM at 30 "C resulted in nearly quantitative recovery of the enzyme activity. Comparison of this value with those obtained with the thermal elution results suggests that 9% of the activity of the H R P was lost as a result of elevated temperature. The actual loss in activity may, in fact, be less than this amount since some H R P may still be active, but remains bound to the Con A. As a final test to see how sensitive H R P is to temperature, the protein was incubated at 70 "C for 40 min and assayed for activity. The enzyme is sensitive to the presence of calcium. This result is foreshadowed by the fact that it is a calcium-requiring enzyme (55). In our study of the recovery of activity, the mobile phase contained 1mM calcium, thereby stabilizing H R P to some extent. It is interesting to note that the recovery is higher for the protein eluted from the column than that which was treated at an equivalent temperature in solution. There are several reasons for this. First, while on the column H R P did not spend the entire 40 min at 70 "C since this was a temperature gradient experiment. Second, the protein incubated a t 70 "C was more concentrated than that run on the column, and aggregation may have contributed to protein loss. Finally, the close contact between Con A and H R P may have actually stabilized the enzyme through protein-protein interactions. Regardless of the reason for the greater stability for HRP during thermal elution, these results indicate that thermal gradient elution has potential in reducing some of the kinetic limitations to resolution in HPAC.
CONCLUSION In contrast to the more common methods employing biospecific elution with a competitive inhibitor, linear temperature gradients resulted in the resolution of glycoproteins bound to Con A-silica. The proteins are eluted in narrow zones (approximately 1 mL) and in a short time (
