Anal. Chem. 1996, 68, 629-638
Particle Beam LC/FT-IR Spectrometry Studies of Biopolymer Conformations in Reversed-Phase HPLC Separations: Native Globular Proteins Vincent E. Turula and James A. de Haseth*
Department of Chemistry, University of Georgia, Athens, Georgia 30605-2556
A particle beam LC/FT-IR interface has been employed in the investigation of the effect of chromatographic conditions (e.g., mobile-phase composition, elution process, and stationary phase) on protein secondary structure in reversed-phase HPLC. The ability of the particle beam to obtain IR spectra which are highly representative of protein elution conformation from an HPLC column is demonstrated. Qualitative and semiquantitative measurements of major IR amide bands made from the band intensity and position enabled assessment of the secondary structure content of the eluting protein. Spectra obtained from gradient elutions with n-alkyl silica columns show a degree of randomization during separation. When deposits were redissolved into water and evaporated, however, they reverted to a structure close to the native structure. An n-octadecyl column preserved β-sheet structure, while an n-butyl retained more r-helix structure. IR spectra of lysozyme collected from isocratic elutions with varying concentrations of acetonitrile and 1:1 2-propanol/acetonitrile showed that, when pure acetonitrile was used as the modifier, a partial structural alteration from r to β at high organic concentrations occurred but reverted to r upon reevaporation. No change in conformation was observed with the 2-propanol/acetonitrile mobile phase. With both elution processes, secondary structure changes caused by the chromatography were reversible. Complex biological mixtures that contain proteins and polypeptides possessing a range of molecular weights and diverse amino acid compositions can be separated effectively and rapidly with reversed-phase HPLC (RPC).1-3 The retention of proteins in RPC is complex because the interaction with the stationary phase and the protein is governed by protein amino acid constitution, distribution of hydrophobic residues with the protein, and the three-dimensional structure or conformation of the biomolecules. Highly acidic mobile-phase conditions and nonviscous organic modifiers in the mobile phase are essential for effective separation.1 These solvents alter the tertiary and quaternary levels of structure. Permeation of organic modifier into the compact protein causes exposure of hydrophobic chains previously buried in the core of the protein. A mobile phase with a substantial amount of organic composition induces denaturation and stabilizes the solution state of a denatured unfolded protein, with its (1) Regnier, F. E. Science 1987, 238, 319-323. (2) Geng, X.; Regnier, F. E. J. Chromatgr. 1984, 296, 15-30. (3) Karger, B. L.; Lin, S. J. Chromatogr. 1990, 499, 89-102. 0003-2700/96/0368-0629$12.00/0
© 1996 American Chemical Society
hydrophobic regions now in direct contact with the solvent. Optimal interaction occurs when the protein is denatured, because there is maximum surface contact between the stationary phase ligands and a majority of the peptide residues.1 Geng and Regnier have outlined a general retention model for proteins in RPC and have found a relationship between the amount and type of modifier required to desorb a protein from the stationary phase and the protein molecular weight.2 Karger and Lin found that this model does not hold with partially denatured proteins.3 The conformational state of the protein, therefore, has direct bearing on its retention behavior. Adsorption can cause conformational changes to proteins in addition to conformational changes facilitated by the solvent composition.4-8 These adsorption-induced changes are influenced by adhesion rates, protein concentrations, and protein surface orientation.4 Both the nature of the stationary phase surface and the contact time a protein spends on the surface affect conformation.5 Separation efficiency and sample resolution, which can be manipulated by gradient parameters, reflect how the separation affects conformational order. In some instances, the overall conversion time from native to unfolded forms is comparable to the time of separation. The elution of either form may be obtained with a steep gradient, where the separation time is comparable to the time needed to attain that form.8,9 Proteins with similiar structures (e.g., variants) can be separated from each other if one form can be changed conformationally.10 This would result in a hydrophobic difference between the forms that would enable separation. In addition to the secondary equilibria, conformational interconversion, protein-protein aggregation, metastable adsorption, and multimonomer dissociation degradation may also occur in RPC. 4 In these situations it becomes difficult to differentiate between sample compounds and artifacts.11 Conformation and RPC. Native protein structure can be preserved in RPC; however, the retention process is responsible (4) Hlady, V.; Andrade, J. D. Adv. Polym. Sci. 1986, 79, 1-63. (5) Soderquist, M. E.; Walton, A. G. J. Colliod Interface Sci. 1980, 75, 386392. (6) O’Hare, M. J.; Capp, M. W.; Nice E. C.; Cooke, N. H. C.; Archer, B. G. Anal. Biochem. 1982, 126, 17-28. (7) Hanson, M.; Unger, K. K.; Schmid, J.; Albert, K.; Bayer, E. Anal. Chem 1993, 65, 2249-2253. (8) Hearn, M. T. W.; Hodder, A. N.; Aguilar, M.-I. J. Chromatogr. 1985, 327, 47-66. (9) Synder, L. R. In HPLC of biological macromolecules: Methods and applications; Gooding, K. M., Regneir, F. E., Eds.; Marcel Dekker: New York, 1990; pp 231-257. (10) Wicar, S.; Mulkerrin, M. G.; Bathory, G.; Khundkar, L. H.; Karger, B. L. Anal. Chem. 1994, 66, 3908-3915. (11) Sadana, A. Bioseparations 1992, 3, 145-165.
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for production of secondary conformational forms, which are observed as either asymmetric or multiple peaks that depend upon the chromatographic conditions. A majority of the literature that concerns conformational effects on RPC is directed to the dynamic state of protein tertiary and quaternary structure, because these structures drastically influence separations. This effect was illustrated in separate studies of the RPC behavior of pure papain12 and ribonuclease A.13 In both cases, two peaks for each protein were observed. The leading peak corresponded to the biologically active native structure, as it had less retention and, hence, poor efficiency. The later eluting peak was ascribed to the denatured form. Lin and Karger correlated the degree of unfolding (that is, denaturation) to the “Z-number”, the retention parameter that accounts for the number of molecules or modifier required to desorb.3 Proteins that had their disulfide linkages reduced were completely unfolded and eluted only with strong solvent strength (higher Z-number). In these cases, denaturation was assessed in terms of tertiary structure and dissociation of quaternary structure. Sadler et al. monitored proteins in mildly acidic 1-propanol (1-PrOH) solutions with fluorescence and UV-CD spectral changes.14 Changes in fluorescence spectra corresponded to exposure of core (native state) tryptophan, and UV-CD changes corresponded to the accumulation of the secondary structure to the alcoholic environment. This change is manifested as the protein coils into an R-helix.15 Chromatography was developed at moderate pH (both pH 4 and pH 7). Similarly, Wicar et al. found that human growth hormone (rhGH, 55% R-helical) retained some of its native state secondary structure in a weak, alcoholic mobile phase, while the tertiary structure was lost. Because the eluted protein was not tightly packed, there was an increased mobility in the side chains that caused changes in the fluorescence signal.10 Overall, however, workers have neglected to examine secondary structure carefully within the aforementioned RPC processes, as there are few good structure probes. Fourier Transform Infrared Spectrometry for the Analysis of Globular Proteins. Protein secondary structure describes the three-dimensional polypeptide backbone orientation (e.g., R-helix, β-sheet, random coil). Infrared spectrometry has been used extensively to examine protein solution structure. The position and intensity of infrared (IR) amide absorption bands are sensitive to globular protein secondary structure content.16 Spectra are complex, and the conformationally sensitive amide absorption bands that are intrinsically overlapped must be extracted from each other. There have been several spectroscopic studies of protein/ surface adsorption, but few have been germane to the investigation of protein adsorption on RPC packings. Morrisey and Stromberg used IR spectroscopy and found that the conformation of proteins adsorbed on silica did not change as surface population increased.17 Jakobsen and Cornell found that additional layers of albumin adsorbed onto germanium surfaces became more disor(12) Cohen, S. A.; Benedek, K.; Dong, S.; Yitzhak, T.; Karger, B. L. Anal. Chem. 1984, 56, 217-221. (13) Cohen, S. A.; Benedek, K.; Tapuhi, Y.; Ford, J. C.; Karger, B. L. Anal. Biochem. 1985, 144, 275-284. (14) Sadler, A. J.; Micanovic, R.; Katzenstein, G. E.; Lewis, R. V.; Middaugh, C. R. J. Chromatogr. 1984, 317, 93-101. (15) Singer, J. S. In Advances in Protein Chemistry; Anfinsen, C. B., Anson, M. L., Baile, K., Edsall, J. T., Eds.; Academic Press: New York, 1962; p 1. (16) Susi, H. In Structure and Stability of Biological Molecules; Timasheff, S. N., Fasman, G. D., Eds.; Marcel Dekker: New York, 1969; pp 575-633. (17) Morrissey, B. W.; Stromberg, R. R. J. Colloid Interface Sci. 1974, 46, 152163.
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dered than the initial monolayer.18 In both studies, no gross disruption of conformation was observed with the layer in direct contact with the surface. Moreover, these studies were aimed at the investigation of physiological behavior of particular proteins and were therefore conducted at ambient pressure. In an effort to determine overall conformational effects from RPC, Katzenstein et al. examined several globular proteins adsorbed onto C8 silica particles in a dilution series of 1-propanol solutions with both fluorescence and FT-IR spectroscopy.19 Fluorescence peak shifts observed with increasing concentration of 1-PrOH were indicative of tryptophan residue movement. This tertiary structure change could not be correlated with changes in the IR spectra for samples prepared from the same mobile phase. No trend in diffuse reflectance IR spectra with increasing 1-PrOH was observed either. Protein IR spectra obtained with diffuse reflectance require sampling conditions that are conducive to protein denaturation. Therefore, the IR spectra in the study by Katzenstein et al. are not representative of the protein secondary structure in HPLC experiments and tell little about protein solution conformation. Particle Beam for Use in Secondary Structure Analysis. The particle beam rapidly eliminates solvent (mobile phase) from an analyte solution by evaporation prior to IR detection. The original particle beam device was developed for mass spectrometry,20-22 and later adapted for IR spectrometry.23 The particle beam LC/FT-IR interface functions with both buffered mobile phases24 and reversed-phase chromatography, for example, with small pharmaceutical molecules.25 In normal operation, the chromatographic effluent is pumped through a small capillary and exits as a liquid jet. This column of liquid is then nebulized with a stream of helium. The resulting droplets are directed into a desolvation chamber, where the solvent molecules are stripped by evaporation and eventually removed. The size of the droplets upon nebulization depends on the diameter of the capillary (usually 16-25 µm i.d.). As the volume of the droplets decreases, the relative surface area increases, and hence the evaporation of the droplet is rapid. The desolvation process is endothermic and occurs in a few milliseconds. As the solvent is removed, the protein solution is cooled and does not have time to change conformation. Internal rotations, restricted by the low temperature and rapid desolvation, do not occur. This is, in essence, a rapid lyophilization. Both solute and solvent then travel into a separator to an analysis chamber which is at lower pressure than the desolvation chamber. The protein molecules subsequently strike an infraredtransparent substrate, which is later removed and analyzed by off-line FT-IR microscopy. Hence, a protein from solution can be spectroscopically analyzed and yield a solid state spectrum highly representative of the solution structure. Preliminary studies with (18) Jakobsen, R. J.; Cornell, D. G. Appl. Spectrosc. 1986, 40, 318-322. (19) Katzenstein, G. E.; Vrona, S. A.; Wechsler, R. J.; Steadman, B. L.; Lewis, R. V.; Middaugh, C. R. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 4268-4272. (20) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626-2631. (21) Browner, R. F.; Winkler, P. C.; Perkins, D. D.; Abbey, L. E. Microchem. J. 1986, 34, 15-24. (22) Winkler, P. C.; Perkins, D. D.; Williams, K. W.; Browner, R. F. Anal. Chem. 1988, 60, 489-493. (23) Robertson, R. M.; de Haseth, J. A.; Kirk, J. D.; Browner, R. F. Appl. Spectrosc. 1988, 42, 1365-1368. (24) Robertson, R. M.; de Haseth, J. A.; Browner, R. A. Appl. Spectrosc. 1990, 44, 8-13. (25) Ferguson, G. K. Investigation of pharmaceutical analyses with Monodisperse Aerosol Generator Interface Combining Liquid Chromatography with Fourier Transform Infrared Spectrometry. Ph.D. Dissertation, University of Georgia, Athens, GA, 1993.
EXPERIMENTAL SECTION Chemicals and Reagents. HPLC grade acetonitrile was obtained from J. T. Baker (Phillipsburg, NJ) and 2-propanol from Fisher Chemical (Fairlawn, NJ). Water was deionized to 18 MΩ with a Barnstead NANO ultrapure water system. The HPLC solvents were degassed continuously in their reservoirs with helium. Two buffers were employed to control sample pH, a phosphate buffer which, depending upon buffer strength, varied in both the amounts of potassium phosphate monobasic and sodium phosphate dibasic anhydrous, both supplied by J. T. Baker, and a Tris buffer consisting of TRIZMA hydrochloride (tris(hydroxymethyl)aminomethane hydrochloride) and TRIZMA base (tris(hydroxymethyl)aminomethane) from Sigma Chemical Co. (St. Louis, MO). Trifluoroacetic acid (TFA) was supplied by Aldrich Chemical Co. (St. Louis, MO). The four globular proteins, ribonuclease A (RNase A), R-chymotrypsin, lysozyme, and trypsin, analyzed in this study were of the highest available grade purity, purchased from Sigma Chemical, and were used without further purification. All reagents for the stop rate activity assay of ribonuclease A were purchased from Sigma Chemical. These include type VI ribonucleic acid from torula yeast and sodium acetate trihydrate. Chromatography. Reversed-phase HPLC separations were performed on a Hewlett-Packard 1090L binary gradient HPLC system (San Jose, CA) with a six-port Rheodyne injection valve (Cotati, CA) and a 10 µm frit filter (Upchurch Scientific, Oak Harbor, WA). Separations were performed with n-alkyl silica support columns: a narrow-bore (250 mm × 2.1 mm i.d.) Macrosphere 300 Å n-butyl 7 µm (Alltech Associates, Deerfield, IL), and analytical (250 mm × 4.6 mm i.d.) 300 Å n-butyl and n-octadecyl 5 µm columns with similar surface loads (Vydac, Hesperia, CA). The columns were thermostated at ambient temperature (∼25 °C) with the use of a column heater (FIAtron Systems Inc., Milwaukee, WI). Two organic modifiers were
used: pure acetonitrile and 1:1 2-propanol/acetonitrile. Because of its viscosity, pure 2-propanol caused a tremendous back pressure and could not be used with the particle beam. Detection was carried out with a Kratos Spectroflow 783G variable wavelength detector set for 10 mV output at 0.25 AU. The signal output was captured by a PE Nelson Model 1022 digital integrator (Perkin-Elmer, Norwalk, CT) with a single channel and scanned at 600 points/min for these experiments. Postrun process was performed with both PE Nelson integrator software and GRAMS 386 software (Galactic Industries Corp., Salem, NH). A GRAMS Array Basic program, prepared by the authors, was used to convert the chromatogram abscissa from data points to minutes. Depending upon the experiment, several different size injection loops were used (20, 10, and 5 µL), but for a majority of the separations and particle beam collections, sample concentrations were prepared to 1-0.5% (w/v) with the 10 µL loop, which gave a 100-50 µg on-column injection. In determinations of the capacity factor (k′), a solution of Uracil (Aldrich), monitored at 214 nm, was injected to mark the unretained peak retention time, t 0. Particle Beam Apparatus. The operation of the particle beam in the context of this application was described above. A detailed explanation of the hardware is available elsewhere.27 A significant modification to the aerosol generator was implemented because the previous liquid stream probe22 was prone to leak and moved when HPLC pressure fluctuated. The probe is a brassbored cylinder that houses the connection between the HPLC tubing and the fused silica capillary in the aerosol generator. To improve reproducibility and ease of use, a universal fused silica sleeve and finger-tight nut secure the fused silica capillary into a standard 1/16 in. hex union, as shown in Figure 1. The length of the probe allows its use in both cross-flow and coaxial aerosol generators. In addition, the position of the fused silica is fixed relative to the dispersion gas, which is critical to alignment. A 5/ in. taper allows easy alignment with an open-end wrench. The 8 overall experimental setup is pictured in Figure 2. To minimize dead volume and therefore reduce band diffusion, 0.005 in. i.d. hypodermic tubing soldered to 1/16 in. fittings was plumbed throughout the HPLC system. The in-line UV-visible detection, alerting the presence of the eluting protein, enabled much more accurate and thorough analyte collection. Because the particle beam IR interface requires no pressure differential for collection (as does the MS interface), and because greater transport efficiency has been observed with a single-stage momentum separator, an open stainless-steel spacer replaced the small orifice skimmer that is present in the MS interface. Lastly, a thermocouple tube and gauge (Kurt J. Lesker Co., Clairton, PA) were used to monitor interface vacuum integrity. Infrared Spectrometry. Several reference spectra are presented here. The following describes the preparation and acquisition of both reference particle beam and solution spectra. The reference particle beam spectra were collected from pure water mobile phase with no column. Solution spectra were collected by transmission and attenuated total reflectance (ATR) spectrometry. The concentration of the protein solutions used varied and depended upon the sensitivity of the experiment: for ATR, 1% w/v (10 mg/mL); solution transmission, 5% w/v (50 mg/mL); particle beam, 0) for each run. The separation that maintained the highest degree of activity was the n-octadecyl/acetonitrile run. Interestingly, the n-octadecyl activity exceeded that found for both the evaporated and acidic mobile-phase particle beam collection. Presumably, the amount of structurally intact RNase A at the point of nebulization was greater than that from the acidic mobile phase. The extremely rapid particle beam evaporation functioned to retain any native RNase A; this explains why there was more activity with the n-octadecyl column than with the sample that was simply evaporated. Moreover, in the separations, the enzyme was in contact with an organic modifier. This medium enabled the enzyme-bound water to remain associated, more so than acidic water, and therefore preserved activity. The 2-propanol cut modifier had substantial improvement over pure acetonitrile on the n-butyl separations. Given the identical RPC conditions, we concluded that the n-butyl stationary phase caused more surface unfolding than the n-octadecyl surface. It can be deduced from these analyses, which are consistent with findings from the IR spectra, that the n-octadecyl separation maintained both secondary and tertiary conformation slightly more than n-butyl separations with these modest-sized proteins. Full mid-IR spectra of R-chymotrypsin are illustrated in Figure 6. The liquid phase spectrum B was obtained with ATR spectrometry and shows much structural detail, including the amide III; the amide III pattern observed is consistent with the high β-sheet content of the sample. Previous IR studies produced estimations of secondary structure consistent with our results: for D2O solution, 12% R-helix, 51% β-sheet;32 for particle beam H2O, 23% R-helix, 63% β-sheet.34 The resultant IR spectra of R-chymotrypsin show RPC trends identical to those observed for RNase A. The n-octadecyl spectrum (C) shows the randomization of the R-helix also. Here, however, the split peak maximum favors the β-sheet region (1636 cm-1), as expected. Compared to the n-butyl spectrum (D), this showed a vast reduction in ordered structure. (34) Turula, V. E.; de Haseth, J. A. Proc. Soc. PhotoOpt. Instrum. Eng. 1993, 2089, 516-517.
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Figure 7. Effect of concentration of acetonitrile modifier on isocratic lysozyme retention. Log capacity factor k′ vs % acetonitrile in isocratic elution mode. The chromatography was performed on a Vydac n-butyl 250 mm × 4.6 mm column with a flow rate of 0.35 mL/min; mobilephase composition, solvent A, 0.1% TFA in H2O, solvent B, 0.1% TFA/39-44% acetonitrile/60.9-55.9% H2O. Particle beam collections were made at each data point. Wavenumber positions (in cm-1) per data point indicate amide I (open text) and amide II (solid text). Figure 6. R-Chymotrypsin IR spectra. (A) Reference, particle beam deposit from pure H2O (no column). (B) Reference, ATR solution phase 2% (w/v) in H2O. (C) Particle beam deposit from C18 gradient elution (acetonitrile modifer). (D) Particle beam deposit from C4 gradient elution (acetonitrile modifier). (E) Evaporated film deposit D dissolved with pure H2O and reevaporated on CaF2 substrate. (F) Particle beam deposit from C4 gradient elution (1:1 2-propanol/ acetonitrile modifier). (G) Evaporated film deposit F dissolved with pure H2O and reevaporated on CaF2 substrate.
Recasting from reconstituted RPC n-butyl deposits (with both modifiers, spectra E-G) showed again a conversion back to a structure very close to that of the native, with an intensity indicative of the high β-sheet content. Lastly, it was clear that less β-sheet structure transversed the n-butyl columns intact than on the n-octadecyl. R-Chymotrypsin is folded into two antiparallel β-barrels, with a cylindrical arrangement of six β-strands each.29 The rigid n-butyl ligand network forces disorganization of these surface β-sheet frames during adsorption. Isocratic Elution of Lysozyme. Because the elution process employed in RPC governs the manner in which a protein propagates through an RPC column, it was considered important to investigate how isocratic elution affects secondary structure compared to gradient elution. Koyama et al. have already described the differences in the rate of propagation through RPC columns of varying length between gradient and isocratic elution.35 In gradient elution, protein velocity is dynamic, changing with the mobile phase, whereas in isocratic elution, it is constant. In isocratic elution, the mobile-phase strength determines the rate at which a protein moves through; it is the equilibrium or partition between mobile/stationary phases that changes. The capacity factor (k′) is the retention parameter that provides insight regarding rate. Small changes in the percentage organic modifier (35) Koyama, J.; Nomura, J.; Shiojima, Y.; Ohtsu, Y.; Horii, I. J. Chromatogr. 1992, 625, 217-222.
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(acetonitrile, acetonitrile/2-propanol) have drastic effects upon k′.9,35 At very low values of k′, protein bands move through the column at an appreciable rate, and there is little stationary phase interaction. Lysozyme was included in the above gradient studies. The spectra were consistent in being highly symmetric, with the amide I peak maximum centered at 1660 cm-1. Figure 7 illustrates how k′ changed on the analytical n-butyl column with increasing organic modifier (pure acetonitrile) and, with particle beam IR detection, how lysozyme structure was affected as well. When the mobile phase strength included 43% acetonitrile, retention was poor, as the dropoff of k′ indicated. At this concentration, the IR spectra show splitting of the amide I, along with a concomitant shift in amide II. At 44%, there is virtually no retention, and an IR spectrum identical to 43% is again obtained. Because little partitioning occurred with this mobile-phase concentration, surfaceinduced alteration could not have been responsible for the observed IR behavior. Severe peak spreading became pronounced at these low modifier concentrations. To determine if the mobilephase composition was solely responsible for this structure change, the solution spectrum of lysozyme prepared in the mobile phase (44% acetonitrile-56% water) was acquired (Figure 8, spectrum A). A modest shoulder at 1622 cm-1 indicated that this solvent system might have altered the hydrogen bonding or hydrophobic environment such that some β-sheets formed in a protein that has little proclivity to do so. Spectrum B shows the particle beam collection of the 44% acetonitrile elution. The definitiveness of the 1635 cm-1 shoulder clearly indicates the presence of the β-sheet. Although the bands at 1622 and 1635 cm-1 differ by 13 cm-1, they are representative of β structure in the liquid and solid states, respectively. When the particle beam deposit was redissolved in pure water, evaporated, and scanned, it produced a spectrum similar to that of the native lysozyme. The 1450 cm-1 band is not from a protein but a surface artifact, as it
Figure 8. Lysozyme IR spectra from isocratic acetonitrile elutions. (A) Reference solution spectrum, ATR CIRcle cell 2% (w/v). Solution composition, 44% acetonitrile/56% H2O/TFA. (B) Particle beam deposit (from 44% acetonitrile 56% H2O elution with the n-butyl column). (C) Evaporated film deposit B (44% acetonitrile) dissolved with pure H2O and reevaporated on CaF2 substrate. (D) Evaporated film from 44% acetonitile/56% H2O.
does not change in intensity from B to C. An evaporated film (spectrum D) prepared from this mobile phase showed a symmetric amide I band centered at 1660 cm-1. Most likely, heterogeneous and slow evaporation gave the lysozyme time to acclimate to the aqueous solvent and reconstitute back to its native structure. This demonstrated that the particle beam can catch dynamic protein conformation, whereas evaporation cannot. This alteration of lysozyme to β from predominantly R with isocratic elutions was not observed in any of the gradient elutions performed previously, irrespective of column packing. The experiment was repeated with a modifier system of 1:1 2-propanol-acetonitrile (Figure 9). Most striking is the improved retention; not until the modifier concentration was at 50% did the k′ reduce to near nonabsorption, while it was 43% for the acetonitrile modifier experiment. As indicated by the amide I and II wavenumber positions, no β-sheet formation was observed, either. IR spectra of a particle beam collection at 45% modifier were found to be identical to the reference spectrum of lysozyme in pure water solution and a particle beam collection, also from pure water. Each spectrum has amide I and II bands in the R-helix positions. From these studies, we concluded that secondary structure alteration can occur with strong mobile phases in isocratic elution, and 2-propanol can preserve three-dimensional structure better than acetonitrile. This may not be the case for proteins with high β-sheet content because alcohols are known to stabilize R-helices.15,36,37 It was deduced that surface degrada(36) Nelson, J. W.; Kallenbach, N. R. Biochemistry 1989, 28, 5256-5261. (37) Fink, A. L.; Painter, B. Biochemistry 1987, 26, 1665-1671.
Figure 9. Effect of concentration of 1:1 2-propanol/acetonitrile modifier on isocratic lysozyme retention. Log capacity factor k′ vs % acetonitrile in isocratic elution mode. Chromatography identical to that used in Figure 7, with the exception of the mobile-phase composition which was solvent A, 0.1% TFA in H2O, solvent B, 0.1% TFA/3850% acetonitrile/61.9-49.9% H2O. Particle beam collections were made at each data point. Wavenumber positions (in cm-1) per data point indicate amide I (open text) and amide II (solid text). Table 3. Trypsin Concentrations versus Capacity Factor and Detector Response on C4 Columns injection (µg) 500 250 100 50 25 5 50 25 2.5
retention time (min)
k′
detector response (mV)
26.0 25.8 25.9 25.6 25.9 25.5
1.84 1.82 1.83 1.80 1.83 1.79
39.8 17.8 13.6 7.6 2.9 1.4
IR amide bands I II 1660 1659 1660 1660 1660 1659
1538 1536 1537 1536 1537 1537
Alltech Narrow-Bore C4 250 mm × 2.1 mm 25.8 4.39 5.1 1659 26.5 4.53 4.5 1658 26.5 4.53 0.3 1660
1540 1541 1535
tion was not a factor in these situations, and it is speculated that a combination of organic modifier, pressure, and velocity is responsible for changes. These changes would most likely be different for different proteins and sets of RPC conditions. Injection Quantity with Trypsin. The load of the analytical scale RPC column can vary in quantity from 200 to 1 µg injected. Because stationary-phase coverage may not be complete with low concentrations, and overload can result in protein aggregation at high concentrations, an experiment was run to determine if the injected quantity affects secondary structure. Trypsin, an R/β protein similar to R-chymotrypsin in conformation, was injected in concentration ranges from 0.5 mg to 2.5 µg on both the analytical and narrow-bore n-butyl columns. Particle beam collections were made on each run. The IR spectra collected in each case showed no amide I or II peak shift or intensity change for any run (see Table 3). The elution secondary structure was comparable to that of R-chymotrypsin from the gradient elution experiments and showed some loss of ordered structure. It appears that, at low pH (approximately pH 2), protein secondary structure will disorder upon RPC elution regardless of the quantity Analytical Chemistry, Vol. 68, No. 4, February 15, 1996
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Figure 10. IR spectrum of particle beam deposit of 100 ng injection of R-chymotrypsin, gradient elution on an Alltech n-butyl 250 mm × 2.1 mm column. The spectrum was acquired with 2000 scans at 8 cm-1 resolution. The shaded portions of the spectrum correspond to mobilephase material that carried through with R-chymotrypsin.
injected. The particle beam LC/FT-IR detection limits are substantially lower than those used for this experiment (nanogram levels). Given column capacity, however, it was possible to cover the analytical column load limits within the range studied here. A separation of the three enzymes on the narrow-bore n-butyl column at injection quantities of 100 ng for each component has been made previously.38 A 100 ng R-chymotrypsin sample was collected with the particle beam, and good IR spectra were obtained. As shown in Figure 10, there is structural detail, with a definitive β-sheet shoulder closer to the native structure than in the spectra in Figure 6. A considerable carry-through of a material of unknown origin is present (Figure 10, shaded bands). From these results, it can be postulated that proteins directly adsorbed on n-alkyl silica particles are not drastically altered. This is consistent with other IR surface studies.17,18 Because the loads used in the previous experiment were much greater than the 100 ng quantities used here, excess protein ensured more than complete coverage. In this case, the majority was aggregated atop of the layer in direct contact with the stationary phase. It is difficult to determine the cause of denaturation frequently observed in most of this work. One possibility was the ablation of the excess layers by pressure gradients and eddy currents. Nevertheless, the denatured species dominated spectra and gave the appearance of being significantly altered. CONCLUSIONS For the enzymes examined, column efficiency was better with an n-octadecyl stationary phase but was more flow rate-dependent than with the n-butyl column. Also, slightly more native secondary structure was retained on the C18 column with each protein examined in this study. With the proteins that possess a mixture of structure, more R-helicity was lost when run through the n-octadecyl column. On the n-butyl columns, both RNase A and R-chymotrypsin lost β-structure: the 2-propanol/acetonitrile modi(38) Turula, V. E.; de Haseth, J. A. Dynamic Solution Structure of Globular Proteins by Reversed Phase HPLC Particle Beam LC/FT-IR Spectrometry. Presented at the 46th Pittsburgh Conference and Exposition on Analytical Chemistry, New Orleans, LA, March 6-10, 1995.
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fier seemed to preserve native R-helix well but did little to preserve the β-sheet structure. On the n-butyl columns, proteins randomized upon elution but reverted when redissolved and evaporated from water. Although a good portion of the β-sheet reorganized, the percent β content had dropped relative to the reference spectra. The enzymes ribonuclease A, lysozyme, and R-chymotrypsin have been reversibly unfolded with reversed-phase chromatography. Very distinct IR amide band contours showed a predominance of random structure on the secondary level upon elution from butyl and octadecyl silica columns. Both isocratic and gradient elution spectra of the particle beam deposit, redissolved, evaporated, and rescanned, showed a secondary structure similar to that from the reference spectra. Thus, the column interaction, solvents, pressure, and, to some extent, surface alter protein secondary structure, but not irreversibly. The activity assays of ribonuclease A illustrated how the particle beam rapidly desolvated proteins such that the actual solution conformation was maintained for collection and analysis. Further charactization of the secondary strucuture constitution of all RPC peaks, including both native and denatured, with particle beam, diode array detection, and freesolution capillary electrophoresis is ongoing. More accurate understanding of stationary phase ligand affects is still being sought, in particular with an n-octyl stationary phase. ACKNOWLEDGMENT This paper was presented in part at the 46th Pittsburgh Conference and Exposition on Analytical Chemistry, New Orleans, LA, March 5-10, 1995. The authors thank the Perkin-Elmer Corp. for generous support of this work.
Received for review August 24, 1995. Accepted November 27, 1995.X AC950874H X
Abstract published in Advance ACS Abstracts, January 15, 1996.
