Anal. Chem. 1996, 68, 4006-4014
Conformational Effects on Reversed-Phase Chromatography of Proteins with Particle Beam LC/FT-IR Spectrometry and Free Solution Capillary Electrophoresis Randall T. Bishop, Vincent E. Turula, and James A. de Haseth*
Department of Chemistry, University of Georgia, Athens, Georgia 30605-2556
Examination of Protein Structure by Reversed-Phase HPLC (RPC). Separation of the components of complex biological mixtures, which commonly contain species with widely varying molecular weights and chemical compositions, can be accomplished effectively and rapidly with the use of RPC.1-3 RPC is a powerful analytical tool that enables separation of peptides and proteins that have only minor differences in their amino acid composition.3 Application of RPC has extended into the areas of protein folding/unfolding, purification, and structure/function
correlation studies.4 UV absorption is the primary technique employed for detection in these biomolecular applications. The presence of certain residues (e.g., tryosine or tryptophan) can be useful in the detection of conformational change because of their environmentally sensitive UV absorption behavior. When the absorbance change at these specific wavelengths is monitored, a relationship between absorbance and conformation can be described. Fluorescence detection has also been utilized as a direct probe for tertiary structure change.4,5 A multitude of chromatographic affects (e.g., stationary-phase/solute interactions, adsorption kinetics, elution procedure, and mobile-phase compositions) can cause conformation alterations and, without careful consideration of separation parameters, obfuscate structural assignments by these unidimensional detection techniques. Under the influence of RP chromatographic conditions, multipeak chromatograms are often produced during RPC analysis of homogeneous proteins.6,7 Multipeak chromatograms represent the elution of a single protein in different conformational states, the dissociation of multidomain proteins, or both.2 Ribonuclease A (RNase A) has been observed to produce a multipeak chromatogram with two major peaks: an early-eluting species ascribed to a slightly denatured conformation and a late-eluting peak ascribed to a totally denatured form.7 Due to the inability of UV detection to provide specific conformational information, these peaks are usually assigned to either N or D for “native” and “denatured”, respectively. If a conformational transition occurs on a time scale comparable to that of the separation, the multipeak phenomenon is more pronounced.8 The time dependence of the development of the denatured peak on the chromatographic surface was demonstrated by Cohen.6 Lin and Karger observed that the conformational state of an injected protein is directly related to protein retention and is also correlated to the “Znumber”, a parameter that describes the number of modifier molecules required for desorption.3 Proteins that were disulfidereduced and completely unfolded required a stronger solvent strength for elution and, thereby, a higher Z-number. The mechanisms behind the production of multiple protein conformations during RPC analysis are uncertain. It has been hypothesized that an adsorption/desorption/denaturation equi-
* To whom correspondence should be addressed. FAX: (706) 542-9454. E-mail:
[email protected]. (1) Geng X., Regnier, F. E. J. Chromatgr. 1984, 296, 15-30. (2) Regnier, F. E. Science 1987, 238, 319-323. (3) Lin, S.; Karger, B. L. J. Chromatogr. 1990, 499, 89-102. (4) Oroszlan, P.; Blanco, R.; Lu, X.-M; Yarmush, D.; Karger, B. L. J. Chromatogr. 1990, 500, 481-502.
(5) Wicar, S.; Mulkerrin, M. G.; Bathory, G.; Khundkar, L. H.; Karger, B. L. Anal. Chem. 1994, 66, 3908-3915. (6) Cohen, S. A.; Benedek, K.; Dong, S.; Yitzhak, T.; Karger, B. L. Anal. Chem. 1984, 56, 217-221. (7) Cohen, S. A.; Benedek, K.; Tapuhi, Y.; Ford, J. C.; Karger, B. L. Anal. Biochem. 1985, 144, 275-284. (8) Hearn, M. T. W.; Hodder, A. N.; Aguilar, M.-I. J. Chromatogr. 1985, 327, 47-66.
In this study, reversed-phase HPLC (RPC), particle beam FT-IR spectrometry, and capillary electrophoresis (CE) were employed to ascertain the dynamic conformational structure empirically of various forms of bovine ribonuclease A (RNase A) to investigate the interdependence of protein conformation and RPC separation. RNase A was analyzed in four conformational states: native, partially denatured, completely unfolded, and completely unfolded and disulfide-reduced (denatured). Each form was analyzed individually and produced similar multizoned RPC profiles composed of several low-response peaks before the primary peak. Particle beam FT-IR spectrometry results from the RPC fractions of the partially denatured and unfolded forms were identical to one another and exhibited a loss of ordered structure. The infrared spectra of the RNase A that was introduced into the HPLC in the denatured and reduced form showed an excess of β-sheet content, particularly the primary peak. We believe this to be a non-native form of RNase A. CE migration times of RNase A samples that were reduced in 2-mercaptoethanol and unfolded in increasing concentrations of urea increased with the degree of unfolding. Mobilities of RNase A RPC fractions were compared to those of the urea unfolded samples. The migration time of the primary RPC fraction of the denatured form showed an intermediate migration rate between that of the native, spherical geometry and denatured, open geometry forms. It is unclear whether this β-structure formed during column propagation or upon elution.
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librium is established between the protein and the nonpolar stationary phase such that multiple conformational states become populated.8,9 As the adsorbed state is highly favored by the partition equilibrium at any point below the critical concentration of organic modifier, however, effectively all protein is in direct contact with the stationary-phase surface.10 This would imply that all relevant conformational transitions occur while the protein is adsorbed to the stationary-phase surface.11 The development of a denatured conformational state on the chromatographic surface is thought to be due to the conformational rearrangement of adsorbed protein such that a more direct contact can be obtained between hydrophobic regions of the protein and the stationaryphase surface, so that a lower free energy state is attained.9 The degree of induced denaturation is related to protein structural integrity and to environmental conditions such as the nature of organic modifier, the presence and nature of an ion-pairing reagent, pH, temperature, and column contact time. As a result of RPC conditions, the conformation of the eluting protein may either remain in the native state or completely or partially denature. Conformational adjustments during RPC analysis result in a significant loss of native quaternary and tertiary structure, which may or may not be reversible, while secondary structure seems to be either preserved or only slightly modified.1 The presence of high concentrations of organic solvent, a detriment to quaternary and tertiary structures, stabilizes the hydrogen-bonding network of secondary structure elements (i.e., helices and β-sheets). This is true because quaternary and tertiary structures are formed primarily to minimize hydrophobic residue/solvent interactions, while the major contributor to secondary structures is hydrogen bonding. The RPC behavior of model peptides with no formal tertiary structure has been shown to correlate with secondary structure, which in the cited studies was the R-helix.12,13 Closely related peptide analogs adopt different helical conformations in solution, which translates into differences in surface hydrophobicity and thereby differences in RPC behavior. Krause et al. observed differences in RPC behavior among D-amino acid replacement sets of an amphipathic R-helical model peptide.14 Extrapolation of peptide secondary structure effects to globular protein RPC behavior is ambivalent, however, as tertiary structure surely mediates secondary structure/stationary-phase interactions. The role and significance of secondary structure in the RPC of globular proteins remains questionable. Particle Beam RPC/FT-IR Spectrometry. The particle beam can trap a sample and preserve the solution secondary structure of a protein. Solution conformation is “frozen” during particle beam desolvation and collection and then analyzed for secondary structure detail in a static solid state by IR microscopy. The particle beam operates at a subambient temperature, so proteins never attain the activation energy necessary for global unfolding during collection. Preliminary experiments have shown (9) Hlady, V.; Andrade, J. D. Adv. Polym. Sci. 1986, 79, 1-63. (10) Koyama, J.; Nomura, J.; Shiojima, Y.; Ohtsu, Y.; Horii, I. J. Chromatogr. 1992, 625, 217-222. (11) Sadler, A. J.; Micanovic, R.; Katzenstein, G. E.; Lewis, R. V.; Middaugh, C. R. J. Chromatogr. 1984, 317, 93-101. (12) Bu ¨ ttner, K.; Blondell, S. E.; Ostresh, J. M.; Houghten, R. A. Biopolymers 1992, 32, 575-583. (13) Blondell, S. E.; Bu ¨ ttner, K.; Houghten, R. A. J. Chromatogr. 1992, 625, 199206. (14) Krause, E.; Beyermann, M.; Dathe, M.; Rothemund, S.; Bienert, M. Anal. Chem. 1995, 67, 252-258.
that no significant structural alteration of the protein occurs during particle beam operation.15 It was also demonstrated that dried particle beam protein deposits retained the solution conformation that the protein possessed prior to nebulization. Secondary structure estimates from IR spectra of 10 globular proteins, obtained by several transmission and reflectance techniques, correlated very well with particle beam spectra.16 Recently, the particle beam was employed in the investigation of the effect of chromatographic conditions on protein secondary structure in RPC.17 Particle beam spectra of a mixture of three enzymes, RNase A, lysozyme, and R-chymotrypsin, injected into an RPC system in their native forms, all showed a high degree of structural randomization upon elution. More R-helicity was preserved on an n-butyl column, and conversely, more β-sheet remained intact when analyzed with an n-octadecyl column. Very distinct IR amide band contours showed that for each enzyme a substantial amount of both the R-helices and β-sheet domains, randomized by RPC, reorganized when deposits were dissolved and re-evaporated. Particle beam spectra of RNase A and R-chymotrypsin injected and collected individually from RPC again showed a high degree of structural randomization related to both RPC conditions and interaction with RPC media.18 Capillary Electrophoresis (CE) and Protein Conformation. CE has been used to study protein conformations19-21 and in the elucidation of folding/refolding structures.22-24 CE is a nonretentive separation technique in which protein conformation can be preserved. Parameters such as capillary coating, temperature and length, and buffer strength and pH can be varied to achieve either maintenance or perturbation of conformation. Sample migration in free solution CE is governed by molecular constitution, shape, and size.25 For these reasons it is possible to resolve protein conformers as in the investigation of folding mechanisms. Strege and Lagu demonstrated that with CE the native form of bovine trypsinogen was resolved from intermediate refolded forms.24 To maintain the unfolded-state of human transferrin, Kilar and Hjerten et al. included high concentrations of urea in the run buffer and were able to resolve the several folding intermediates from the native species.23 Hilser was able to thermally unfold lysozyme when the CE was operated at elevated temperatures.22 The two distinct forms in the slow thermal transition of lysozyme, folded a unfolded, were resolved from each other. The protein was unfolded at 62 °C but migrated with a higher mobility, as did the peptide standard that was included. The increased rate was due to a drastic reduction in the buffer viscosity that in turn offered less resistance to the protein. Larmann et al. built an on-line RPC/ CE multidimensional chromatographic system for the resolution (15) Turula,V. E.; de Haseth, J. A. Appl. Spectrosc. 1994, 48, 1255-1264. (16) Turula, V. E.; de Haseth, J. A. Proc. Soc. Photo-Opt. Instrument. Eng. 1993, 2089, 516-517. (17) Turula, V. E.; de Haseth, J. A. Anal. Chem. 1996, 68, 629-638. (18) Bishop, R. T.; de Haseth, J. A. Mikrochim. Acta, in press. (19) Grossman, P. D.; Colburn, J. C.; Lauer, H. H.; Nielsen, R. G.; Riggin, R. M.; Sittampalam, G. S.; Rickard, E. C. Anal. Chem. 1989, 61, 1186-1194. (20) Zhao, Z.; Malik, A.; Lee, M. L. Anal. Chem. 1993, 65, 2747-2752. (21) Malik, A.; Zhao, Z.; Lee, M. L. J. Microcolumn. Sep. 1993, 5, 119-125. (22) Hilser, V. J.; Worosila, G. D.; Freire, E. Anal. Biochem. 1993, 208, 125131. (23) Kila´r, F.; Hjerte´n, S. J. Chromatogr. 1993, 638, 269-276. (24) Strege, M. A.; Lagu, A. L. J. Chromatogr. 1993, 652, 179-188. (25) Palmieri, R.; Nolan, J. A. Protein capillary electrophoresis: Theoretical and experimental considerations for methods development. In CRC Handbook of Capillary Electrophoresis: A practical approach Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1994; pp 325-368.
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of peptides in tryptic maps.26 The complement of CE to RPC provides additional information for the molecular conformation and geometry of the separated species. In this study, we determined how the initial, injected threedimensional structure affects chromatography and how that structure is changed during propagation through the column. The utility of the particle beam to trap transient species that occur upon column elution is demonstrated. Additionally, precise secondary structure detail of satellite conformer peaks within the multizoned chromatograms was examined. RNase A was an ideal model protein for this study because it was readily prepared and stabilized in several conformational states.27 RNase A is a singlechain pancreatic nuclease with a molecular weight of ∼13 690, a secondary structure that has a majority of β-sheets with some R-helix and random regions28 and a folding/refolding behavior that has been studied extensively.29-32 By examination of the different forms of RNase A upon RPC elution with particle beam FT-IR spectrometry and CE, a complete conformational analysis was possible; infrared spectrometry conveyed secondary structure information, and CE the topology. EXPERIMENTAL SECTION Chemicals and Reagents. Bovine ribonuclease A, a basic protein with a pI ) 8.88,20 was of the highest available grade purity purchased from Sigma Chemical Co. (St. Louis, MO) and was used as received. Urea and HPLC grade acetonitrile were obtained from J. T. Baker (Phillipsburg, NJ). Water was deionized to 18 MΩ with a Barnstead NANOultrapure water system. Trifluoroacetic acid (TFA) and triethylamine (TEA) were supplied by Aldrich Chemical Co. (St. Louis, MO). 2-Mercaptoethanol was obtained from Sigma. Two buffers were employed to control sample pH; they were a phosphate buffer, with sodium phosphate monobasic and phosphoric acid all supplied by J. T. Baker, and an acetate buffer, sodium acetate trihydrate (Sigma) and acetic acid (J. T. Baker). Preparation of Denatured Ribonuclease A. The native protein was prepared by dissolution of the appropriate amount of ribonuclease A in either water or buffer, which was experiment dependent. Partially denatured RNase A was dissolved in 0.1% TFA. To prepare the unfolded and disulfide-reduced protein, RNase A was incubated for 24 h in a nitrogen-sealed solution of various amounts of urea up to 8 M (complete unfolding) and 0.3 M 2-mercaptoethanol. To ensure dissociation of the four disulfide linkages, the pH was adjusted within the range of 7-8.5 with TEA.27 The unfolded protein was prepared in a manner identical to the unfolded/reduced species, with the exclusion of 2-mercaptoethanol. Chromatography and Particle Beam FT-IR Spectrometry. The HPLC system consisted of a Perkin-Elmer 200 Series quaternary pumping system and a Perkin-Elmer 235C diode array detector. The detector output was captured with the use of a PE (26) Larmann, J. P.; Lemmo, A. V.; Moore, A. W.; Jorgenson, J. W. Electrophoresis 1993, 14, 439-447. (27) White, F. H. Methods Enzymol. 1967, 54, 481-484. (28) Fersht, A. Enzyme Structure and Mechanism, 2nd ed.; W. H. Freeman and Co.: New York, 1985, p 30. (29) Garel, J.-R.; Nall, B. T.; Baldwin, L. Proc. Natl. Acad. Sci. U.S.A. 1975, 73, 1853-1857. (30) Creighton, T. E. J. Mol. Biol. 1980, 137, 61-80. (31) Kim, P. S. Methods Enzymol. 1986, 131, 136-156. (32) Udgaonkar, J. B.; Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 8197-8201.
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Nelson Model 1022 digital integrator (Perkin-Elmer, Norwalk, CT). Postrun processing was performed with both PE Nelson integrator software and GRAMS/386 software (Galactic Industries Corp., Salem, NH). RPC was performed on an analytical (250 × 4.6 mm i.d.) n-butyl silica column with a surface load of ∼4 µmol/m2, 5 µm particle size, and 300 Å pore size (Vydac, Hesperia, CA). The column was thermostated at ambient temperature (∼25 °C) with the use of a column heater (FIAtron Systems Inc., Milwaukee, WI). HPLC mobile phase was (A) 0.1% TFA in 18 MΩ H2O, (B) 0.1% TFA in acetonitrile. Gradient program: 5-95% acetonitrile over 40 min at a flow rate of 0.35 mL/min. Detection for the illustrative chromatograms in Figure 1 was at 220 nm. A complete description of the operation of the particle beam, in the context of the biopolymer conformational analysis, is available elsewhere.15,33 Protein deposits were collected at the indicated elution times onto a calcium fluoride window (25 mm dianeter × 2 mm) and interrogated off-line with the use of a Spectra Tech IR-Plan infrared microscope (Shelton, CT) interfaced to a Perkin-Elmer 1725X FT-IR spectrometer. All spectra were obtained from 2000 co-added scans at 8 cm-1 resolution. All spectral manipulations including second-derivative and deconvolution were performed with GRAMS/386 software (Galactic Industries Corp.). Capillary Electrophoresis. A Bio-Rad Biofocus 2000 capillary electrophoresis system was used in this study. A 50 cm × 50 µm i.d. fused-silica capillary column (Polymicro Technologies, Inc., Phoenix AZ) was coated with UCON H-75-90,000 (Alltech Associates, Deerfield, IL) poly(ethylene-propylene glycol), a weakly hydrophobic coating as previously described.21 The reagents were mixed in a proportion that produced a polymer film thickness of 100 nm. The conditions for the CE experiments were as follows: 15 kV run voltage (300 V/cm), 50 and 75 mM phosphate, and 50 mM acetate run buffers, 220 nm detection wavelength, 20 °C capillary temperature, cathodic destination reservoir, and the hydrodynamic injection technique, 4 psi s. Current values ranged from 15 to 25 µA for 50 mM phosphate buffer systems with this capillary. Buffers were filtered with a 0.2 µm pore Nylon syringe filter (Gelman Sciences, Ann Arbor, MI). The protein was prepared in the lowest buffer strength that would stabilize the conformation and maintain a narrow pH range and sample zone. Sample buffers varied in concentration from 10 to 50 mM phosphate. In the determination of migration times of unfolded RNase A, urea was added only to the sample buffer and not included in the run buffer. The polymer capillary coating reduced the electroosmotic flow to such a low level that measurement of the migration time of a neutral marker was difficult. To allow migration rate comparisons between the various conformational forms of RNase A, a migration index (MI) was recorded. The MI was the ratio of the migration rate of the RNase A from either a urea unfolding or RPC fraction to that of the native form of RNase A. Chromatography and CE Hyphenation. Portions of the RNase A RPC elution profile collected for CE analysis were identical to those collected during the particle beam analysis. To preserve the conformations of the eluted proteins, the chromatographic fractions were collected in precooled vessels which were subsequently submerged in a dry ice/methanol bath. Samples (33) Turula, V. E. Dynamic Solution Conformation of Biopolymers by Particle Beam/FT-IR Spectrometry. Ph.D. Dissertation, University of Georgia, Athens, GA, 1995.
Figure 1. Chromatographic profiles of RNase A analyzed in different conformational states. The shaded areas are fractions of eluent collected with the use of the particle beam interface. Chromatographic conditions: Vydac 250 × 4.6 mm n-butyl column; 0.35 mL/min flow rate; gradient from 5 to 95% acetonitrile over 40 min; UV detection at 220 nm. Solvent A, 0.1% TFA in 18 MΩ deionized water; solvent B, 0.1% TFA in acetonitrile.
were quickly thawed and analyzed individually to minimize the dead time between thaw and injection, which was approximately 3-4 min. Fractions 1 and 2 were collected as one and diluted with 200 mM phosphate stock solution due to their low concentrations in the RPC effluent, while fraction 3 was diluted with 100 mM phosphate solution. A combined sample of all three fractions was diluted with 100 mM phosphate solution as well. With each experimental run acquired, an electropherogram of a native RNase A was also collected for comparative purposes. RESULTS AND DISCUSSION Chromatography and Particle Beam FT-IR Spectrometry. RNase A was chromatographed in four different conformational states and analyzed postcolumn with UV spectrometry and particle beam LC/FT-IR spectrometry. These conformational states were prepared as follows: native in H2O, partially denatured in 0.1% TFA, unfolded in 8 M urea, and denatured, that is unfolded and disulfide reduced, in 8 M urea and 0.3 M 2-mercaptoethanol. Portions of these multipeak chromatograms, shown in Figure 1, were isolated with the particle beam and later interrogated by FT-IR spectrometry. The chromatographic behavior (e.g., retention times and peak profiles) of RNase A injected in the native (prepared in H2O) and partially denatured states (prepared in 0.1% TFA) were identical, so only the partially denatured form was analyzed with the particle beam technique. As was observed by Karger and Lin,3 chromatographic retention increased as the injected form of the protein became more unfolded (Figure 1). Resulting infrared spectra of the chromatographic peaks in Figure
Figure 2. Infrared spectra of native RNase A in H2O and RPC fractions of RNase A prepared in 0.1% TFA prior to analysis. Amide I and II regions for the RPC fractions are illustrated along with the corresponding second-derivative spectra.
1 (Figures 2-4) show the eluite to be a primarily unordered species. There are significant differences in secondary structure, however, between the fractions of the disulfide-reduced species and disulfide-intact species. The third fraction spectra that corresponds to the denatured peak show evidence of the presence of a β-sheet structure at 1630 cm-1 in all samples. This feature becomes more pronounced as the initial conformational state of the protein becomes more unfolded (i.e., partially denatured to denatured, disulfide reduced). This feature is also evident to a lesser extent in the spectra of the first and second fractions as well. The second-derivative spectra of the third fractions (Figures 2-4) show the presence of unordered (1660 cm-1), β-sheet (1629 cm-1), and β-turn bands (1695 cm-1). After initial infrared analysis, the protein deposits were dissolved in a small amount of water (5 M urea was completely unfolded. Migration rates decreased only slightly until the critical urea concentration was reached (>5 M), when a majority of the protein became unfolded and a considerable decrease in migration rate was observed, as shown in the electropherograms in Figure 7 and from the data in Table 2. In 8 M urea the protein required ∼22 min to migrate the effective capillary length (Figure 7). This behavior was ascribed to the increased frictional coefficient and solvent acces4012 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
sibility of the more denatured forms of the protein. This is consistent with the findings of Kila´r and Hjerte´n, who observed that the reduced form of transferrin migrates slower than the native form.23 Detector response was similar for each sample and ranged from 4 to 9 milliabsorbance units. As illustrated in Figure 7, symmetrical peak shape was compromised in the urea series. It is believed that a small portion (not greater than 20%) of the unfolded form refolded once the denaturing stimulus was removed.29-31 Given the effective capillary length, and the refolding kinetics of RNase, the unfolded and regenerated portions would have had insufficient time for resolution in this experiment; thereby an asymmetric peak shape was produced. Complementary to the RPC/FT-IR investigation, the RPC/CE experiments enabled a complete three-dimensional description of the RNase A upon elution. 400 µg of the three conformational forms of RNase A, partially denatured, unfolded, and denatured, were injected separately onto an n-butyl analytical bore column. This was the upper protein load level for this column. Fractions were collected at times identical to those of the particle beam analysis (Figure 1). Fractions 1 and 2 were collected as one due to their low concentration. The collected volume of fractions 1 and 2 combined was approximately 400-450 µL, and for fraction 3 (main peak) 375-400 µL. Despite several collections, fractions 1 and 2 were of poor recovery and CE detection was weak, with migration rates that were not reproducible. The migration rate of the third fraction of RNase A injected in the native form was identical to that of native RNase A with a MI of 1 (Figure 8A,B). Although RNase A was completely unfolded in 8 M urea, without disulfide reduction, the protein, depending upon conditions, rapidly refolded upon removal of the denaturant. We observed a modest, ∼12 s difference in migration rates, MI ) 0.98, between the native standard, and the third fraction from RNase A injected into the RPC from 8 M urea (Figure 8C,D). Table 2 provides the migration rates for RNase A in both the urea series and RPC fractions. When RNase A was unfolded and disulfide reduced, a vast majority of ordered secondary structure was lost. As described earlier, RPC retention was enhanced, and in particle beam spectra, a stable and abundant β-sheet structure was observed. In Figure 8E,F, electropherograms show a difference in migration rates between the RPC-eluted denatured and native forms of over 3 min, with an MI of 0.81. The MI of the denatured RPC component is intermediate between those of RNase A in the 4 and 6 M urea in the unfolded series. This behavior was ascribed to a difference in protein contour from that of a open, random
Figure 8. (A) Electropherogram of the primary RPC fraction (third peak) RNase A injected in native form. (C) Electropherogram of the primary RPC fraction (third peak) of unfolded RNase A injected from 8 M urea. (E) Electropherogram of the primary RPC fraction (third peak) of denatured RNase A injected from 8 M urea and 0.3 M 2-mercaptoethanol. (B, D, and F) Native RNase A standard electropherograms in 50 mM phosphate run immediately before each respective non-native RNase A analysis.
chain in the urea series to that of an oblong, planar structure in the denatured RPC fraction. Thus, the CE results further confirmed the presence of the β-structure observed in RPC/ particle beam spectra. CONCLUSIONS Both CE and infrared spectral data support the hypothesized formation of a β-sheet pattern in RNase A upon elution from RPC analysis. Particle beam infrared spectra showed that the secondary structure of the primary chromatographic peak was primarily β-sheet structure with additional unordered segments. The leading chromatographic peaks are primarily disordered structures, but with considerably less of the β-sheet structure found in the primary peak. In CE analysis of the primary chromatographic peaks, the overall geometry of this β-structure assumed an oblong, planar shape which migrated at a slower rate than did the analogous compact native form, but faster than the disordered and opened denatured form. As evidenced by both IR and CE analysis, however, this intermediate structure was not stable in the disulfide intact species and reorganized upon dissolution to a more nativelike conformation. The β-sheet structure was more stable in the disulfide-reduced species as solution refolding was hindered by the lack of cooperativity provided by intact disulfide linkages. This work also showed that RPC separations are affected by the initial conformation of a protein. Stationary-phase/protein interaction was enhanced with an increasing degree of denaturation. Ordered secondary structure was lost uniformly despite the differences in stationary-phase affinity between the conformational forms. The disulfide intact forms show much structural randomization yet some residual secondary structure remains, while the disulfide-reduced species showed more extensive randomization with a particular loss of R-helicity. We speculate that the degree of unfolding due to both chemical treatment prior to injection and structural randomization during column propagation was so great that refolding occurred
Figure 9. Correlations among empirical data related to the third RPC faction of RNase A chromatographed in three different conformational states: (A) capillary electrophoresis migration indexes and HPLC retention times; (B) HPLC retention times and normalized β-sheet peak areas; (C) capillary electrophoresis migration indexes and normalized β-sheet peak areas. β-sheet peak areas were normalized to the total amide I area.
upon desorption. In view of the postcolumn conformational similarities between the disulfide-intact and disulfide-reduced species, it seems that, with a given chromatographic system, RPC randomizes different conformations in a similar manner: the same IR absorption bands are evident in the eluites with different injected conformations. CE and IR data point to the reversibility of the denaturation in more robust protein structures, e.g., those with intact disulfides. It is the susceptibility to structural change of the injected protein and the interaction time upon the column that has the most bearing on both the final elution conformation and the potential for refolding to the native conformation. Good correlation of R2 ) 0.980 was found between CE migration indexes and RPC retention time of the third peak of all conformational states, as shown in Figure 9. Modest correlations were evident between the normalized β-sheet peak area and the MI (R2 ) 0.874) and the RPC retention times (R2 ) 0.952), respectively. Obviously, the β-sheet content cannot completely Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
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account for the variability in retention data within each data set, which is expected as other factors are also influential in the individual separation techniques. With the use of the particle beam we have observed protein unfolding on RPC.17 The RNase A trapped and described in this study strongly resembled an early β-sheet refolding intermediate previously described.32 We believe that the formation of ordered, secondary structure elements in large globular proteins analyzed through RPC may be due to refolding after a loss of structure within the column. Structural detail of these species can be obtained when the protein is trapped with the particle beam. Presently, the S-carboxymethylated, thiol-blocked, disordered form of RNase A is under examination by both RPC/IR and RPC/CE. Future work involves application of this methodology to the study the RPC behavior of proteins with quaternary structure such as multidomained R-chymotrypsin, and β-lactoglobulin. We are
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continuing efforts to trap and examine folding intermediates with a modified particle beam system. ACKNOWLEDGMENT The authors thank Dr. Abdul Malik of the University of South Florida for advice on capillary coating. We also acknowledge Mr. T. G. Venkateshwaran of the College of Pharmacy, University of Georgia, for assistance with chromatography. This paper was presented in part at the 15th International symposium on Protein, Peptide, and Polynucleotide Analysis, Boston, MA, November 1922, 1995. Financial support from the University of Georgia Research Foundation is gratefully acknowledged. Received for review March 26, 1996. Accepted August 28, 1996.X AC960295S X
Abstract published in Advance ACS Abstracts, October 1, 1996.
