Biotechnol. Frog. 1992, 8, 540-545
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Cation-Exchange Displacement Chromatography of Proteins with Protamine Displacers: Effect of Induced Salt Gradients Joseph A. Gerstner and Steven M. Cramer* Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180-3590
Protamine was investigated for its utility as a protein displacer in cation-exchange systems. Although the protamine solution contained several variants of the molecule, the high affinity of all of the components in this heterogeneous biopolymer enabled it to act as an efficient protein displacer. To facilitate parameter estimation of the protamine, a preliminary purification was carried out by preparative elution chromatography. Chromatographic parameters of both the feed proteins and protamine displacer were obtained for use in a multicomponent steric mass action ion-exchange displacement model. Model simulations were compared to displacement results under both moderate and intense induced salt gradient conditions. In both cases, excellent agreement was obtained between the displacement experiments and theoretical predictions. In addition, these studies serve to dramatize the importance of induced salt gradients in ion-exchange displacement systems.
Introduction Displacement chromatography is rapidly emerging as a powerful preparative bioseparation technique due to the high throughput and product purity associated with the process (Horvath, 1985;Frenz and Horvath, 1988; Cramer and Subramanian, 1990; Cramer, 1991). This technique offers several advantages in preparative chromatography as compared to the traditional elution mode. The displacement process takes advantage of the nonlinearity of the isotherms such that a larger feed can be separated on a given column with the purified components recovered at significantly higher concentrations. Furthermore, the tailing observed in preparative elution is greatly reduced in displacement chromatography due to the self-sharpening boundaries developed during the process. In contrast to preparative elution where the feed components are diluted during the separation, the feed components can be significantlyconcentrated during the displacement procedure. These advantages combine to make displacement chromatographyan extremely attractive preparative technique for the isolation of biomoleculesfrom the dilute solutions often encountered in biotechnology processes. Anion-exchange displacement chromatography of proteins has been studied by several investigators. Peterson and co-workers (Peterson, 1978;Peterson and Torres, 1983; Torres et al., 1984; Torres et al., 1985;Torres et al., 1987; Dunn et al., 1988)have used carboxymethyldextrans (CMDs) as displacers for various protein mixtures. Chondroitin sulfate has been employed to displace @-galactosidase(Liao and Horvath, 1990)and j3-lactoglobulins (Liao et al., 1987; Lee et al., 1988). Jen and Pinto have performed protein displacements using relatively low molecular weight dextran sulfate (1991) and poly(viny1sulfonic acid) (1990) as displacers. Ghose and Mattiasson (1991) have examined the purification of lactate dehydrogenase using a carboxymethyl starch displacer. Cramer and co-workers (Subramanian et al., 1988;Subramanian and Cramer, 1989; Subramanian et al., 1989) have examined a variety of cation-exchange displacement systems. Recent work in our laboratory (Jayaraman et al., 1992) has examined the
* Author to whom correspondence should be addressed. 8756-7938/92/300&0540$03,00/0
effects of molecular weight on the efficacy of both DEAEdextran and dextran sulfate displacers. In these studies, we have investigated the utility of protamine as a potential displacer for cation-exchange systems. Protamines are a group of basic proteins found in sperm cellswhich act by replacing histones in chromatin during spermatogenesis (Subirana, 1975). Typically less than 35 amino acids in length (McKay et al., 19861, protamines are highly positively charged due to high lysine and arginine contents (45-80% ) (Subirana, 1982). The amino acid sequences for several species of fish (including salmon) have been reported (Andoet al., 1973;Croft, 1980). Structurally, protamine is composed of four a-helical sections connected via partially flexible joints (Warrant and Kim, 1978), which should facilitate interactions of the charged site of the molecule with the stationary-phase material. These properties produce a relatively flexible, low molecular weight (4-5 kDa) protein with approximately 20 positively charged sites per molecule. It is this unique combination of properties that led us to examine the utility of protamine as a high-affinity displacer for cationexchange systems. A potential drawback associated with protamine is the natural heterogeneity of the protein. Within a single species of fish, it has been reported that the composition of protamine varies between individuals of the same species and is a function of fish age and location of fish harvest (Ando et al., 1973). McKay et al. (1986) report that protamine isolated from a single trout testis possessed six variations of protamine. The effect of displacer heterogeneity has been examined for polymeric displacers by Torres et al. (1987). Jen and Pinto (1991) have indicated that heterogeneous displacers can be employed for protein purification provided that the componentsof the displacer have a higher affinity for the stationary-phase material than the feed proteins. In this article, we present results on the purification of proteins using both purified and heterogeneous protamine displacer solutions. In addition, a comparison between theoretical predictions and displacement experimentswill be presented under both moderate and intense induced salt gradient conditions.
0 1992 American Chemical Society and Amerlcan Institute of Chemical Englneers
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Materials and Methods Materials. Protein-Pak SP-8HR (100 X 5 mm i.d. and 50 X 5 mm i.d.) columns and a Protein-Pak SP-15HR (100 x 20 mm i.d.) column were gifts from Waters Chromatography, Division of Millipore (Milford, MA). Protamine sulfate was purchased from ICN Biomedicals (Costa Mesa, CA). Sodium phosphate dibasic, sodium chloride, ammonium sulfate, a-chymotrypsinogen A, cytochrome c, and lysozyme were purchased from Sigma (St.Louis, MO). Apparatus. Preparative Chromatograph. A Waters Delta Prep 3000 (Waters Chromatography, Division of Millipore, Milford, MA) was used which consisted of a 600E system controller, a pump fitted with 400-pL heads, a Lambda-Max Model481LC UV-vis detector, and a 745B data module. Sample was introduced onto the column using a Valco Model EClOW 10 port electrically actuated injector (Valco Instruments, Houston, TX). Analytical Chromatograph. The system employed for analysis consisted of an LKB 2150 HPLC pump (Pharmacia-LKB Biotechnology, Uppsala, Sweden), connected to the column through a Rheodyne 7125 injector fitted with a 20-mL sample loop (Rheodyne, Cotati, CA). The column effluent was monitored using a Spectroflow 757 UV-vis detector (Applied Biosystems, Ramsey, NJ), with data acquisition and chromatographic analyses performed using a Maxima 820 chromatography workstation (Waters Chromatography, Division of Millipore, Milford, MA). For displacements, the Rheodyne injector was replaced with a Valco Model EClOW 10 port electrically actuated injector (Valco Instruments), and a LKB Model 2212 Helirac (Pharmacia-LKBBiotechnology) was used to collect fractions of the column effluent. Ancillary Equipment. Ultrafiltration was performed using a Minitan acrylic system (Millipore, Bedford, MA) fitted with lo00 NMWL regenerated cellulose membranes. A Labconco freeze-dry system Model Lyph Lock 4.5 was employed for lyophilization (Labconco, Kansas City, MO). Sodium analysis was performed on an atomic adsorption spectrophotometer (Model 3030, Perkin-Elmer, Norwalk, CT). Methods. Preparative Purification of Protamine. A Protein-Pak SP-15HR (100 X 20 mm i.d.1 cationexchange column was equilibrated with a carrier of 1.05 M sodium chloride in 25 mM sodium phosphate dibasic buffer, pH 7.3. A total of 19 mL of a 10 mg/mL protamine sulfate solution was introduced onto the column. A product cut of the column effluent was collected from 10 to 25 min, after which a regenerant solution of 1 M ammonium sulfate in 0.1 N sodium hydroxide was passed through the column for 10 min. The column was then reequilibrated with the carrier for 5 min. The flow rate was kept constant at 16 mL/min, and the column effluent was monitored at 237 nm. Operation of Displacement Chromatography. In all displacement experiments, aProtein-Pak SP-8HR (100 X 5 mm i.d.) column was sequentially perfused with carrier, feed, displacer, and regenerant solutions. Fractions of the column effluent were collected throughout the displacement runs and were assayed by analytical chromatography as described below. The regenerant was 0.85 M ammonium sulfate in 0.1 N sodium hydroxide. In all displacement experiments, the feed was loaded at a flow rate of 0.2 mL/ min, and the displacement was performed a t 0.1 mL/min. Displacement Chromatography with the Purified Displacer. The displacement experiment was carried out using a feed mixture containing 10.8 mg of a-chymotrypsinogen A, 7.9 mg of cytochrome c, and 17.3 mg of lysozyme in 1.7mL of a 12.5 mM sodium phosphate, dibasic
0
2
4
6
8
10
12
nlltwTon (h)
Figure 1. Analytical chromatogram of crude protamine sample: column, 50 x 5 mm i.d. Protein-Pak SP-8HR gradient conditions, solvent A, 25 mM sodium phosphate buffer, pH 7.5, solvent B, A + 1M ammonium sulfate; gradient, 60 to 100% B in 6 mL, feed, 20 r L of 10.0 mg/mL protamine sulfate; flow rate, 0.75 mL/min.
carrier, pH 6.0. The displacer was 15 mg/mL protamine in the carrier. Displacement Chromatography cum Elution. Displacement chromatography with an induced salt gradient elution of a feed component was performed using a feed mixture containing 7.2 mg of a-chymotrypsinogen A and 14.2 mg of lysozyme in 1.7 mL of a 50 mM sodium phosphate, dibasic carrier, pH 6.0. The displacer was 13.1 mg/mL purified protamine in the carrier. Displacement Chromatography with the Crude Displacer. The displacement experiment was carried out using a feed mixture containing 7.6 mg of cytochrome c and 14.7 mg of lysozyme in 1.5 mL of a 12.5 sodium phosphate, dibasic carrier, pH 6.0. The displacer was 15 mg/mL crude protamine in the carrier. Analytical Chromatography. Fractions collected during displacement experiments were analyzed by HPLC on a Protein-Pak SP-8HR (50 X 5 mm i.d.) column as follows. Column effluent was monitored at 280 nm for proteins and 230 nm for protamine. The flow rate employed in these analyses was 0.75 mL/min. Purified protamine displacer was analyzed using isocratic elution with 0.85 M ammonium sulfate in 25 mM sodium phosphate dibasic, pH 7.3, as the eluent. Heterogeneous (crude) protamine displacer was analyzed using a linear gradient separation. An 8-min (6 mL) linear gradient of 0.6 to 1.0 M ammonium sulfate in 25 mM sodium phosphate buffer, pH 7.5, was employed. Protein analysis was performed using isocratic elution with 0.1 M ammonium sulfate in 25 mM sodium phosphate dibasic, pH 6.0, as the eluent.
Results and Discussion Purification of Protamine. The salmon protamine employed in this study consisted of a heterogeneous mixture composed of several proteins with the basic protamine structure but with minor mutations in the amino acids sequence. A chromatogram of the commercially obtained protamine illustrates this heterogeneity (see Figure 1). In order to carry out a rigorous comparison of protamine displacement experiments and theoretical predictions obtained with the steric mass action (SMA) model (Brooks and Cramer, 19921, it was first necessary to obtain pure material. Preliminary experiments indicated that linear gradient purification of the protamine was impractical due to the low separation factors of the major components. For preparative elution purification, the f i s t major component was designated the desired compound to facilitate devel-
Biotechnol. Frog., 1992, Vol. 8,No. 6
542
I 0
5
10
15
m
25
0
2
ndal Thn (h)
Figure 2. Chromatogram of preparative elution purification of protamine: column 100 X 20 mm i.d. Protein-Pak SP-15HR eluent, 1.05 M sodium chloride in 25 mM sodium phosphate, dibasic; feed, 19 mL of 10 mg/mL crude protamine sulfate; flow rate, 16 mL/min; product collection from 10 to 25 min. opment of the purification protocol. A chromatogram of the preparative elution experiment is shown in Figure 2. The desired component was recovered in essentially pure form in an effluent cut taken from 10 to 25 min (240mL total volume) as indicated in the figure. Several runs were carried out to obtain the desired quantity of the purified material (500mg). Appropriate fractions from the various preparative elution separationswere subsequently pooled, desalted using ultrafiltration, and lyophilized to yield the purified protamine shown in Figure 3. Induced Salt Gradient. In ion-exchange systems, a salt counterion is associated (bound) with each exchange site on the stationary phase to maintain electroneutrality constraints. When a biopolymer binds to an adsorption site, it displaces the salt counterion. In displacement chromatography, where the displacer is fed at conditions such that it binds to a significant number of sites on the stationary phase, the amount of displaced counterions (salt)becomes considerable. Peterson (1978)firstreported induced salt gradients during the purification of serum proteins by the displacer carboxymethyldextran. Subramanian et al. (1989)examined the effect of the induced salt gradients by comparing displacement and step gradient chromatographyperformed under similar initial feed and carrier conditions. The induced salt gradient can drastically alter the salt microenvironment that each protein experiences during a displacement separation. Since protein adsorption is a strong function of salt concentration, large fluctuations in the local salt concentration can cause significant changes in the displacement profile. The effect of the induced salt gradient is to depress the adsorption isotherms of the feed proteins. In the extreme, when a protein adsorption isotherm is depressed such that it no longer intersects with the displacement operating line, that protein will elute from the column ahead of the displacement train. The traditional approach has been to predict the final displacement profile utilizing adsorption isotherms of the feed components which were measured under the initial carrier conditions. For ion-exchange systems, the induced salt gradient must be predicted and explicitly accounted for in order to accurately predict the final displacement profile. Parameter Estimation for the SMA Model. The steric mass action ion-exchange model (SMA) developed by Brooks and Cramer (1992)explicitly accounts for steric effects in multicomponent protein equilibria and is able to predict complex behavior in ion-exchange displacement systems. Furthermore, this formalism can be used to predict displacement profiles under induced salt gradient
4
6
8
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Figure 3. Analytical chromatogramof purified protamine: feed, 20 pL of 10 mg/mL purified protamine. All other conditions were the s m e as given in Figure 1. Table I. Steric Mass Action Parameters species
characteristic steric Qequilibrium charge (Y) factor ( a ) (mM) constant ( K ) ~~
a-chymotrypsinogen Aa cytochrome co lysozymea protamine
4.83 6.02 5.32 17.5
49.15 53.64 38.01 11.73
10.396 9.407 12.951 19.5
0.0092 0.0107 0.0886 1 X lo9
From Jayaraman et al. (1992). Table XI. Amino Acid Sequence for Salmon Protamine A l a Pro-Arg-Arg-Arg-Arg-Ser-Ser-Ser-Arg-Pro-Val-AreArg-Are Arg-Arg-Pro-Arg-Val-Ser-Arg-Arg-Arg-Arg-Arg-Arg-GlyGly-AreArg-AreArg Reproduced with permission from Croft (1980). Copyright 1980 John Wiley and Sons.
conditions. The required model parameters for each compound are characteristic charge, v, steric factor, u, maximum stationary-phase concentration, Qmax,and the equilibrium constant, K. The SMA parameters for proteins are readily determined from linear elution experiments and nonlinear frontal experiments. Since the protamine binds very strongly, it is impossible to perform linear elution experiments for parameter estimation. Accordingly, SMA parameters for the purified protamine were determined using the methodology presented by Gadam et al. (1992). Model parameters employed for the simulations in this manuscript are presented in Table I. The characteristic charge of the purified protamine was determined to be 17.5. A representative amino acid sequence for a protamine isolated from salmon (salmine A l ) is given in Table 11. Interestingly, the number of charged amino acids (arginine residues) in this protamine variant is 20,which closely corresponds to the characteristic charge determined in our experiments. This result indicates that many of the charged moieties in this relatively small biopolymeric displacer can interact with the stationary-phase material. Total Displacement. The efficacy of the purified protamine as a displacer is illustrated in Figure 4A. In this displacement, a three-component feed mixture composed of a-chymotrypsinogen A, cytochrome c, and lysozyme is readily purified with minimal cross contamination of the proteins. The SMA formalism of Brooks and Cramer (1992)can be employed to calculate the isotachic displacement profile under induced salt gradient conditions. The slope of the displacement operating line can be given by
Under isotachic conditions, the induced salt gradient
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Biotechnol. Rog., 1992, Vol. 8, No. 6 3.5 T
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Figure 5. (A) SMA adsorption isotherms under initial conditions (25mM Na+). (B) SMA adsorption isotherms under the following induced salt gradient conditions: protamine, 25 mM Na+; lysozyme, 70 mM Na+; cytochrome c, 74 mM Na+; a-chymotrypsinogen A, 78 mM Na+.
results in the followingelevated salt concentrations in each purified protein i zone:
The isotachic concentration of the displaced feed component i can than be calculated directlyfrom the expression A l/ui A - (C')*"[i7;;] c*i= (vi + u ~ ) A i = 2,3,..., n + 1 (3)
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The displacement profile predicted by the SMA model is shown in Figure 4B. As seen in the figure, there is good agreement between the model and experiments. In this experiment, the induced gradient produced by the displacer front increases the salt microenvironment for each protein by approximately 50 mM. The adsorption isotherms of the feed components and protamine displacer are shown in Figure 5 for both the initial carrier and induced salt conditions in the isotachic displacement train. A solute movement analysis based on the intersection of the operating line with the isotherms under the initial carrier conditions(Figure 5A) would result in an incorrect prediction of the displacement profile. A solute movement analysis based on the intersection of the operating line with the isotherms under the induced
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Mobile Phase Concentration (mM) Figure 6. (A) SMA adsorption isotherms under initial conditions (100 mM Na+). (B) SMA adsorption isotherms under the following induced salt gradient conditions: protamine, 100 mM Na+; lysozyme, 131 mM Na+; a-chymotrypsinogen A, 158 mM Na+.
salt gradient conditions produces the correct elution order a t concentrations of 0.7, 0.9, and 1.7 mM for a-chymotrypsinogen A, cytochrome c , and lysozyme, respectively. This is in good agreement with the concentrationsobserved in the displacement experiment, namely, 0.65,0.90, and 1.95 mM for a-chymotrypsinogen A, cytochrome c, and
Biotechnol. Prog., 1992, Vol. 8, No. 6
544 3r
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column ahead of the displacement train as predicted using the SMA formalism (Figure 7B). Clearly, the phenomena of induced salt gradients can greatly affect the final displacement pattern. Heterogeneous Protamine Displacer. The utility of heterogeneous protamine as a displacer was examined as shown in Figure 8. As seen in the figure, the heterogeneous protamine is also an efficient displacer for cation-exchange systems. These results are comparable to those obtained with the purified protamine displacer. This experiment confirms that heterogeneous biopolymers can be employed as efficient displacers, providing that all of the displacer components have a greater affinity for the stationary-phase material than the feed proteins.
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In this article, we have demonstrated that both purified and heterogeneous protamine are efficient protein displacers for cation-exchange systems. The effects of induced salt gradients were explored under both displacement and "induced elution" conditions. The SMA formalism of Brooks and Cramer (1992) was able to successfully predict induced salt gradient and complex displacement behavior in these systems.
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Figure 7. (A) Displacement cum elution: column, 100 X 10mm i.d. Protein-Pak SPdHR; eluent, 100 mM sodium phosphate, dibasic; feed, 7.22 mg of a-chymotrysinogen A and 14.20mg of lysozyme in 1.7 mL of eluent; displacer, 13.1 mg/mL protamine; fraction size, 200 pL, 0-5 mL, and 100 pL, 5-7.5 mL. (B)SMA
simulation of separation.
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Notation mobile-phase concentration of solute i, mM mobile-phase concentration of displacer, mM feed concentration of solute i, mM isotachic mobile-phase concentration of solute i, mM carrier salt concentration, mM equilibrium constant, dimensionless stationary-phase concentration of solute i, mM stationary-phase concentration of displacer, mM isotachic stationary-phaseconcentrationof solute i, mM isotachic stationary-phase concentration of displacer, mM stationary-phase saturation capacity of solute i, mM stationary-phasesaturation capacity of displacer, mM volume (width) of the isotachic zone containing feed component i, mL feed volume
pro7 6
7
8
9
Elution Volume (ml)
Figure 8. Displacement chromatography using heterogeneous protamine. Conditions were the same as given Figure 4,except the following: feed, 7.58 mg of cytochrome c and 14.74 mg of lysozyme in 1.5mL of eluent; displacer, 15mg/mL heterogeneous
protamine.
lysozyme, respectively. Thus, the SMA formalism is able to predict both the induced salt gradient and isotachic displacement profile in this protamine displacement system. Displacement cum Elution. While it is important to account for induced salt gradients in order to predict the correct displacement profile, under sufficiently large induced salt gradients the system can be transformed from a displacement to an elution regime. The adsorption isotherms under the initial and induced salt gradient conditions are presented in Figure 6 for a displacer concentration of 13.1 mg/mL in a 50 mM sodium phosphate, dibasic carrier. While a solute movement analysis under the initial carrier conditions predicts displacement of the two feed proteins, under the induced salt gradient conditions the a-chymotrypsinogen A isotherm lies below the operating line. The displacement experiment and SMA simulation are presented in Figure 7. Under these conditions, the a-chymotrypsinogen A eluted from the
Greek Symbols A slope of the displacer operatingline, dimensionless A stationary-phase capacity (monovalent salt counterion), mM Y characteristic charge, dimensionless U steric factor, dimensionless
Acknowledgment The authors thank Professor Joyce Diwan for her assistance with the atomic adsorption analysis. This research was supported by Millipore Corporation and a Presidential Young Investigator Award to S.M.C. from the National Science Foundation.
Literature Cited Ando, T.; Yamasaki, M.; Suzuke, K. Protamines Isolation, Characterization, Structure and Function, Molecular Biology and Biochemistry and Biophysics 12; Springer-Verlag: New York, 1973.
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Brooks, C. A.; Cramer, S. M. Calculation of Isotachic Displacement Profiles and Induced Salt Gradients Using A Multicomponent Steric Mass-Action Ion-Exchange Equilibrium. AIChE J., in press. Cramer, S. M. Displacement Chromatography. Nature 1991, 351,251-252.
Cramer, S. M.; Subramanian, G. Recent Advances in the Theory and Practice of Displacement Chromatography. Sep. Purif. Methods 1990.19 (11, 31-91. Croft, L. R. Handbook of Protein Sequence Analysis; John Wiley and Sons: Chichester, England, 1980, p 470. Dunn, B. E.; Edberg, S. C.; Torres, A. R. Purification of Escherichia coli Alkaline Phosphatase on an Ion-Exchange High-Performance Liquid Chromatographic Column using Carboxymethyl Dextrans. Anal. Biochem. 1988,168,25-30. Frenz, J.; Horvath, Cs. High Performance Liquid Chromatography, Advances and Perspectives; Horvath, Cs., Ed.; Academic Press: Orlando, FL, 1988; Vol. 8, p 211. Gadam, S.; Jayaraman, G.; Cramer, S. M. Characterization of Non-Linear Adsorption Properties of Dextran-Based Polyelectrolytes in Ion-Exchange Systems. J. Chromatogr., in press. Ghose, S.; Mattiasson, B. J. Evaluation of Displacement Chromatography for the Recovery of Lactate Dehydrogenase from Beef Heart under Scale-up Conditions. J. Chromatogr. 1991, 547, 145-153.
Horvath, Cs. The Science of Chromatography; Brunner, F., Ed.; Elsevier: Amsterdam, 1985; p 185. Jayaraman, G.; Gadam, S.; Cramer, S. M. Ion-Exchange Displacement Chromatography of Proteins: Dextran-Based Polyelectrolytes as High Affinity Displacers. J. Chromatogr., in press. Jen, S.-C.; Pinto, N. G. Use of the Sodium Salt of Poly(VinylsulfonicAcid) as a Low-Molecular-Weight Displacer for Protein Separations by Ion-Exchange Displacement Chromatography. J. Chromatogr. 1990,519, 87-98. Jen, S. C. D.; Pinto, N. G. Dextran Sulfate as a Displacer for the Displacement Chromatography of Pharmaceutical Proteins. J. Chromatogr. Sci. 1991,29,478-484. Lee, A. L.; Liao, A. W.; Horvath, Cs. Tandem Separation Schemes for Preparative High-Performance Liquid Chromatography of Proteins. J. Chromatogr. 1988,443, 31-43. Liao, A. W.; Horvath, Cs. Purification of &Galactosidase by Combined Frontal and Displacement Chromatography. BioChemical Engineering VI,Annals of the New York Academy of Sciences; Goldstein, W. E., Dibiasia, D., Pedersen, H., Eds.; New York Academy of Sciences: New York, Vol. 589, pp 182191.
Liao, A. W.; El Rassi, Z.; LeMaster, D. M.; Horvath, Cs. High Performance Displacement Chromatography of Proteins: Separation of &Lactoglobulins A and B. Chromatographia 1987,24,881-885.
McKay, D. J.; Renaux, B. S.; Dixon, G. H. Rainbow Trout Protamines Amino Acid Sequences of Six Distinct Proteins from a Single Testis. Eur. J . Biochem. 1986, 158, 361-366. Peterson, E. A. Ion-Exchange Displacement Chromatography of Serum Proteins, Using Carboxymethyldextrans as Displacere. Anal. Biochem. 1978,90,767-784. Peterson, E. A.; Torres, A. R. Ion-Exchange Displacement Chromatography of Proteins, Using Narrow-Range Carboxymethyldexrans and a New Index of Affinity. Anal. Biochem. 1983,130,271-282. Subirana, J. A. In The Biology of the Male Gamete;Duckett, J. G., Racey, P. A., Eds.; Academic Press: London, 1975; p 239. Subirana, J. A. In Proceedings of the Fourth International Symposium on Spermatology; Andre, J., Ed.; Martinus Nijhoff: The Hague, 1982; p 197. Subramanian, G.; Cramer, S. M. Displacement Chromatography of Proteins Under Elevated Flow Rate and Crossing Isotherm Conditions. Biotechnol. Prog. 1989,5 (31, 92. Subramanian, G.; Phillips, M. W.; Cramer, S. M. Displacement Chromatography of Biomolecules. J. Chromatogr. 1988,439, 341-351.
Subramanian, G.; Phillips, M. W.; Jayaraman, G.; Cramer, S. M. Displacement Chromatography of Biomolecules with Large Particle Diameter Systems. J. Chromatogr. 1989,484, 226236.
Torres, A. R.; Dunn, B. E.; Edberg, S. C.; Peterson, E. A. Preparative High-Performance Liquid Chromatography of Proteins on Ion-Exchange Columns with Carboxymethyldextrans as Displacers. J. Chromatogr. 1984, 316, 125-132. Torres, A. R.; Krueger, G. G.; Peterson, E. A. Purification of Gc-2 G l o b u l i n f r o m H u m a n S e r u m by D i s p l a c e m e n t Chromatography: A Modelfor the Isolationof Marker Proteins Identified by Two-Dimension Electrophoresis. Anal. Biochem. 1985,144,469-476.
Torres, A. R.; Edberg, S. C.; Peterson, E. A. Preparative HighPerformance Liquid Chromatography of Proteins on an Anion Exchanger Using Unfractionated Carboxymethyldextran Displacers. J. Chromatogr. 1987, 389, 177-182. Warrant, R. W.; Kim, S.-H. a-Helix-double helix interaction shown in the structure of a protamine-transfer RNA complex and a nucleoprotamine model. Nature 1978, 271, 130-136. Accepted September 28, 1992.
