Anal. Chem. 1999, 71, 2440-2451
Probing the Binding Behavior and Conformational States of Globular Proteins in Reversed-Phase High-Performance Liquid Chromatography Anthony W. Purcell,† Marie-Isabel Aguilar, and Milton T. W. Hearn*
Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia
Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used technique for the separation of proteins under low pH aquo-organic solvent gradient elution conditions, typically carried out at ambient temperatures. These conditions can however induce conformational effects with proteins as evident from changes in their biological or immunological activities. By monitoring the influence of temperature on the retention and bandbroadening characteristics of proteins, the role of conformational processes in these lipophilic environments can be examined. These processes can then be interpreted in terms of a two-state model involving a native (N) and a fully unfolded species (U) or more complex folding/ unfolding models. In the present study, the gradient elution RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been investigated at temperatures between 5 and 85 °C with n-octadecyl (C18)- and n-butyl (C4)-silica reversed-phase sorbents. The interaction of these proteins with these reversed-phase sorbents has also been examined in terms of the contributions that the heme prosthetic group of SWMYO and the disulfide bonds in HEWL make to the stabilization of the native conformation of these proteins in these hydrophobic environments. The observed interconversions of multiple peak zones of SWMYO and HEWL in the presence of C18 and C4 ligands have been subsequently analyzed in terms of the unfolding processes that these proteins can undergo at low pH and at elevated temperatures. The ability of hydrocarbonaceous ligands to trap ensemblies of partially unfolded conformational intermediates of proteins in these perturbing environments has been examined. Pseudo-first-order rate constants have been derived for these processes from analysis of the dependencies on time of the concentration of the different protein species at specified temperatures. The relationship of these processes to the conformational transitions that these proteins can undergo via molten globule-like intermediates (i.e., compact denatured states with a significant amount of residual secondary structure) in solution has also been examined. This study thus further documents an experimental strategy to assess the folding/unfolding behavior of globular proteins in the presence of hydrophobic surfaces and aquo-organic 2440 Analytical Chemistry, Vol. 71, No. 13, July 1, 1999
solvents, whereby the system parameters can potentially affect the preservation of native conformations, and thus the function, of the protein under these conditions. Reversed-phase high-performance liquid chromatography (RPHPLC) with porous n-alkylsilicas is a highly successful technique for the process-scale purification of synthetic organic compounds and other substances of pharmaceutical interest, including polypeptides with molecular weights up to ∼7000. However, this outcome has generally not been replicated for larger polypeptides (MW > 10 000) or globular proteins, although numerous examples of the isolation of functional proteins at the micro- and ultramicropreparative level by RP-HPLC procedures can be found in the scientific literature.1 The combination of the traditionally used acidic elution conditions and the hydrophobicity of n-alkylsilica sorbents has often discouraged practitioners from using RP-HPLC methods for the large-scale purification of proteins. Loss of biological activity, formation of multiple peak zones with samples of high compositional purity, or reduced mass yields with proteins has been observed. These limitations have been attributed either to n-alkyl ligate- or to solvent-induced unfolding processes arising during the RP-HPLC separation under the perturbing influence of the low-pH conditions. While these features may detract from the use of RP-HPLC as the technique of choice for large-scale preparative purification protocols with proteins, these same characteristics provide a unique opportunity to study the folding/ unfolding processes and the conformational stability of proteins in hydrophobic and aquo-organic solvent environments. Over the past several years, analytical gradient elution RPHPLC and high-performance hydrophobic interaction chromatography (HP-HIC) have been increasingly employed as experimental tools to probe polypeptide conformation. For example, RP-HPLC and HP-HIC methods have been used to study the induced stabilization of coiled-coil,2-4 amphipathic R-helical peptides,5-10 * Corresponding author: (fax) 61 + 3 + 9905-5582; (e-mail) milton.hearn@ med.monash.edu.au. † Present address: Department of Microbiology, University of Melbourne, Parkville, Victoria, Australia, 3001. (1) Aguilar, M. I.; Hearn, M. T. W. Methods Enzymol. 1996, 270, 1-25. (2) Lau, S. Y. M.; Taneja, A. K.; Hodges, R. S. J. Chromatogr. 1984, 317, 129140. (3) Hodges, R. S.; Semchuk, P. D.; Taneja, A. K.; Kay, C.M.; Parker, J. M. R.; Mant, C. T. Pept. Res. 1988, 1, 19-30. (4) Zhou, N. E.; Mant, C. T.; Hodges, R. S. Pept. Res. 1990, 3, 8-20. 10.1021/ac9808369 CCC: $18.00
© 1999 American Chemical Society Published on Web 05/22/1999
or insulin-related polypeptides11-13 by nonpolar ligands. Compared to these studies with peptides, much less work has been reported on the conformational behavior of globular proteins in similar hydrophobic environments. As a consequence, the pathways of the solvent- and/or nonpolar ligate-induced denaturation noted with, e.g., growth hormone,14,15 cytochrome c,16,17 lysozyme,18 ribonuclease A,19 R- and β-lactalbumin,20-22 trypsin,23 or soybean trypsin inhibitor,24 have not yet been fully enunciated. This incomplete knowledge has arisen, in part, because these earlier studies have typically examined the interactive behavior of proteins in these hydrophobic environments at only a single temperature. The combined influences of temperature and residency time25 on the thermodynamic and extrathermodynamic parameters, such the affinity of association or the contact area terms of a protein under isocratic and gradient elution RP-HPLC conditions with different n-alkylsilicas, have not been extensively studied. Similar considerations also apply to the effect of temperature and residency time changes on the kinetics of interconversion between different conformational species or folded states of proteins when adsorbed to nonpolar surfaces. In the present investigation, the RP-HPLC behavior of sperm whale myoglobin (SWMYO) and hen egg white lysozyme (HEWL) has been studied over a wide range of temperature conditions with both C18 and C4 n-alkylsilica sorbents. The choice of these two proteins in these investigations was influenced by the knowledge that the structural and conformational properties of SWMYO and HEWL in bulk solution environments have already been the subject of over 4450 and 520 publications, respectively. (5) Aguilar, M. I.; Hodder, A. N.; Hearn. M. T. W. J. Chromatogr. 1985, 327, 115-138. (6) Aguilar, M. I.; Hodder, A. N.; Hearn. M. T. W. J. Chromatogr. 1986, 352, 52-66. (7) Blondelle, S. E.; Buttner, K.; Houghten, R. A. J. Chromatogr. 1992, 625, 199-206. (8) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. Anal. Chem. 1993, 65, 30383047. (9) Krause, E.; Beyermann, M.; Dathe, M.; Rothemund, S.; Biernet, M. Anal. Chem. 1995, 67, 252-258. (10) Purcell, A. W.; Aguilar, M. I.; Wettenhall, R. E. H.; Hearn, M. T. W. Pept. Res. 1995, 8, 160-170. (11) Hearn, M. T. W. In HPLC: Advances and Perspectives; Horvath, Cs., Ed.; Academic Press: New York, 1983, Vol. 3, pp 87-155. (12) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1995, 711, 61-70. (13) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1995, 711, 71-79. (14) Hearn, M. T. W.; Aguilar, M. I.; Nguyen, T.; Fridman, M. J. Chromatogr. 1988, 435, 271-284. (15) Oroszlan, P.; Wicar, S.; Wu, S.-L.; Hancock, W. S.; Karger, B. L. Anal. Chem. 1992, 64, 1623-1631. (16) Richards, K. L.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1994, 676, 17-31. (17) Richards, K. L.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1994, 676, 33-41. (18) Lu, X. M.; K. Benedek, K.; Karger, B. L. J. Chromatogr. 1986, 359, 19-29. (19) Cohen, S. A., Benedek, K.; Tapuhi, Y.; Ford, J. C.; Karger, B. L. Anal. Biochem. 1985, 144, 275-284. (20) Benedek, K. J. Chromatogr. 1988, 458, 93-104. (21) Grinberg, N.; Blanco, R.; Yarmush, D. M.; Karger, B. L. Anal. Chem. 1989, 61, 514-520. (22) Oroszlan, P.; Blanco, R.; Lu, X.-M.; Yarmush, D. M.; Karger, B. L. J. Chromatogr. 1990, 500, 481-502. (23) Hearn, M. T. W.; Hodder, A. N.; Aguilar, M. I. J. Chromatogr. 1985, 327, 47-66. (24) Cohen, K. A.; Schellenberg, K.; Karger, B. L.; Grego, B.; Hearn, M. T. W. Anal. Biochem. 1984, 140, 223-235. (25) Hearn, M. T. W. In Protein Purification; Janson, J. C., Ryden, L., Eds.; VCH Publ.: New York, 1998; pp 239-282.
As a consequence, the interactive behavior of SWMYO and HEWL in RP-HPLC environments can be readily compared and contrasted with experimental data obtained in these previously used experimental systems in terms of similarities or differences in the conformational properties and folding/unfolding behavior of these proteins,26,27 To this end, the interactive behavior of these two proteins was characterized by examining changes in the gradient elution retention parameters and peak shape properties in response to alterations in the operating temperature or residency time of the RP-HPLC system. The presence of the heme prosthetic group in SWMYO and the intramolecular disulfide bonds in HEWL provide stabilizing influences on the overall tertiary structure of these molecules. Hence, the contribution of these moieties to the maintenance of “nativelike” interactive structures of these proteins in hydrophobic environments was examined by comparison with the corresponding interactive behavior of apoSWMYO and carboxymethylated HEWL (cm-HEWL). The results provide insight into the unfolding processes of SWMYO and HEWL, in terms of the binding characteristics of conformational intermediates that are induced in the presence of hydrophobic surfaces and low-pH aquo-organic solvent conditions. MATERIALS AND METHODS Chemicals and Reagents. Chromatographic-grade acetonitrile and methanol was obtained from Mallinckrodt (Paris, KY). Trifluoroacetic acid (TFA) was obtained from Pierce Chemical Co. (Rockford, IL). Water was deionized in a Milli-Q system (Millipore, Bedford, MA) and distilled. Sperm whale skeletal muscle myoglobin (type II), bovine hemin, and hen egg white lysozyme were obtained from Sigma Chemical Co. (St. Louis, MO), purified to >95% purity and characterized as described elsewhere26-29 by differential scanning UV spectroscopy, tricinePAGE analysis,30 amino acid compositional analysis, analytical RPHPLC, and electrospray mass spectroscopy (ES-MS) using a Sciex API-1 (Thornhill, ON, Canada), Micromass Platform II (Altrincham, Cheshire, UK), or Finnigan LCQ ion trap mass spectrometer (San Jose, CA). Eluted peaks were characterized in an analogous manner to confirm the compositional integrity and authenticity of the recovered components. Acetone, HCl, NaOH, orthophosphoric acid, ammonium sulfate, ammonium hydrogen carbonate, ammonium acetate, and dimethyl sulfoxide were all obtained from Merck Australia (Kilsyth, Victoria, Australia). Preparation of Sperm Whale Apomyoglobin. Apo-SWMYO was prepared by a modification of the method developed31 for hemoglobin. Briefly, myoglobin (1 mg) was dissolved in 1 µL of 0.1 M HCl, incubated for 10 min, and added dropwise into 20 volumes of ice cold acetone/0.1 M HCl. The apoprotein formed as a precipitate and was collected in pellet form following centrifugation for 10 min at 4000 rpm and 4 °C on a Sorvall RT6000 benchtop centrifuge (Dupont, Wilmington, DE). The pellet was subjected to a second acetone/0.1 M HCl extraction and (26) Pfeil, W.; Privalov, P. L. Biophys. Chem. 1976, 4, 23-32. (27) Ragone, R.; Colonna, G.; Bismuto, E.; Irace, G. Biochemistry 1987, 26, 21302134. (28) Hearn, M. T. W. In Theory and Practice of Biochromatography; Vijayalakshmi, M. A., Ed.; Harwood Academic Publ.: Newark, NJ, in press. (29) Hearn, M. T. W.; Quirino, J. P.; Terabe, S. Anal. Biochem., submitted. (30) Scha¨gger, H.; von Jagow, G. Anal. Biochem. 1987, 166, 368-379. (31) Rossi-Fanelli, A.; Antonini, E.; Capato, A. Biochim. Biophys. Acta. 1958, 30, 608-615.
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resuspended in 100 mM NH4HCO3 (pH 7.0); the product lyophilized. Reduction and Alkylation of Hen Egg White Lysozyme. Hen egg white lysozyme was reduced and alkylated according to the method described for bovine insulin.13 Briefly, HEWL (1 mg) was dissolved in 250 µL of guanidine hydrochloride buffer (i.e., 6 M guanidine hydrochloride, 200 mM Tris, 2 mM EDTA, pH 8.0). The mixture was incubated at 37 °C for 1 h, and after cooling, dithiothreitol (1 mg, Boehringer Mannheim GmbH) in 30 µL of guanidine hydrochloride buffer was added to the solution. This mixture was immediately flushed with nitrogen and incubated at 37 °C for 3 h. The solution was cooled and iodoacetic acid (1.9 mg, Fluka) in 70 µL of 1 M Tris-HCl (BDH) (pH 8.0) added. This mixture was incubated at room temperature in the dark for 15 min. Excess β-mercaptoethanol (Sigma) was then added to terminate the reaction, and the cm-HEWL precipitated overnight in 30 volumes of chilled (-20 °C) methanol (Mallinckrodt). The precipitate was collected following centrifugation for 10 min at 4000 rpm with a Sorvall RT-6000 centrifuge (Dupont) and resuspended in chilled methanol (1 µL). A stream of high-purity nitrogen was used to dry the cm-HEWL to a fluffy white product. Chromatographic Procedures. Procedures for the acquisition of the experimental data were based on a previously described8,32 instrumental approach using a Perkin-Elmer (Norwalk, CT) series 4 HPLC system. In brief, SWMYO, apo-SWMYO, HEWL, or cm-HEWL were chromatographed on Bakerbond C18 and C4 sorbents (J. T. Baker, Philipsburg, NJ), nominally of 5 µm particle size and 30-nm average pore diameter, packed into columns of dimensions of 250 × 4.6 µm i.d., using a linear gradient of 0.1% TFA in water (buffer A) and 0.09% TFA in 65% aqueous acetonitrile (buffer B) with gradient times of 30-90 min and column temperatures between 5 and 85 °C. On-line photodiodearray measurements were made using an LKB 21440 rapid spectral detector (Pharmacia Biotech AB, Uppsala, Sweden) and the derived spectra analyzed using the Wavescan software. Protein solutions were prepared by dissolving the solute at a concentration of 0.1 mg/µL in 0.1% aqueous TFA (buffer A). Injection sizes varied between 0.5 and 2 µg of protein, depending on the absorbency and peak heights of the individual species. All injections were made with SGE syringes (Melbourne, Australia), and pH measurements were recorded with an Orion model SA520 pH meter (Cambridge, MA). All data points were derived from at least duplicate measurements with retention times varying less than (0.5%. Prolonged use of n-alkylsilica columns at high temperatures under some elution conditions can result in decreased resolution or column capacity of polypeptides.33 To ascertain whether the effects of temperature on solute retention observed in this study were solely due to secondary conformational equilibria of the proteins or whether degradation of the column contributed to the observed phenomena, control solutes (N-acetyltryptophanamide, N-acetylphenylalanine ethyl ester, penta-L-phenylalanine) were run concurrently with the protein samples. Retention and peak shape characteristics8,32 of these low-molecular-weight solutes were monitored, and when significant changes in these parameters of (32) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1992, 593, 103-117. (33) Guo, D.; Mant, C. T.; Taneja, A. K.; Hodges, R. S. J. Chromatogr. 1986, 359, 519-532.
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these control substances were observed, the columns were replaced. Columns of the same batch were used in order to minimize variations due to the manufacturing process with these sorbents. The relevant parameters, i.e., S and log ko, where ko is the median capacity factor as the median volume fraction of the organic modifier φ j f 0, were derived using experimentally determined gradient retention data that were processed with the Pek-n-Eze software program. Variations in these derived parameters from replicate experiments with different columns of the same manufacturer’s batch number were
