Anal. Chem. 1998, 70, 5010-5018
RP-HPLC Binding Domains of Proteins Marie-Isabel Aguilar,* Daniel J. Clayton,† Phillip Holt, Veronica Kronina,‡ Reinhard I. Boysen, Anthony W. Purcell,§ and Milton T. W. Hearn
Department of Biochemistry & Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3168, Australia
Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface. Reversed-phase high-performance liquid chromatography (RPHPLC) is now a central technique for the analysis and purification of biological molecules as a result of the high level of reproducibility, selectivity, and sensitivity that can be achieved.1,2 Due to its ability to monitor subtle changes in molecular conformation, RP-HPLC is also now emerging as a powerful technique for studying the role of lipid-like surfaces in several biorecognition phenomena, such as the action of antimicrobial peptides3 and the role of hydrophobicity in protein folding.4 However, further * To whom correspondence should be addressed. E-mail: mibel.aguilar@ med.monash.edu.au. Fax: +61-3-9905-5882. † Current address: Department of Biochemistry, University of Queensland, St. Lucia, Queensland 4072, Australia. ‡ Current address: Biological Production Facility, Ludwig/Austin Oncology Unit, Austin & Repatriation Medical Centre, Studley Road, Heidelberg, Victoria 3084, Australia. § Current address: Department of Microbiology & Immunology, University of Melbourne, Parkville, Victoria 3052, Australia. (1) Aguilar, M. I.; Hearn, M. T. W. Methods Enzymol. 1996, 270, 3-26. (2) Mant, C. T.; Hodges, R. S. Methods Enzymol. 1996, 271, 3-50. (3) Blondelle, S. E.; Houghten, R. A. Biochemistry 1992, 31, 12688-12694. (4) Hodges, R. S.; Zhu, B. Y.; Zhou, N. E.; Mant, C. T. J. Chromatogr. 1994, 676, 3-15.
5010 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
significant progress in the development of RP-HPLC is impeded by the lack of theoretical models which accurately describe the molecular details of peptide and protein interactions in RP-HPLC. The slow development of detailed physicochemical models is largely due to the complex structural equilibria that peptides, and particularly proteins, can undergo in RP-HPLC systems.3-15 A full understanding of the chromatographic process requires detailed knowledge of the chemical and physical nature of both the mobile phase and the stationary phase and also information on the types of interactions which occur between the solute and the ligand or the solvent. While little is known about the detailed molecular structure of proteins at the chromatographic surface, experimental data with species variants of proteins, as well as recombinant mutants, indicate that proteins interact with the chromatographic surface in an orientation-specific manner.16-18 The retention behavior of proteins, which can be described in terms of the affinity and kinetics of the interaction, is therefore determined by the molecular composition of a specific contact region. Although the contact region for small peptides may involve contributions from the total or a large proportion of the molecular surface of the solute,19,20 for larger polypeptides or proteins, retention data suggest that the contact region represents a relatively small portion of the total solute surface.16-18 The retention properties of larger polypeptides and proteins are (5) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. Anal. Chem. 1993, 65, 30383047. (6) Oroszlan, P.; Wicar S.; Teshima, G.; Wu, S.-L.; Hancock, W. S.; Karger, B. L. Anal. Chem. 1992, 64, 1623-1631. (7) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1995, 711, 71-79. (8) Richards, K. L.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1994, 676, 33-41. (9) Lin, S.; Karger, B. L. J. Chromatogr. 1990, 499, 89-102. (10) Lazoura, E.; Maidonis, J.; Bayer, E.; Hearn M. T. W.; Aguilar, M. I. Biophys. J. 1997, 72, 238-246. (11) Lee, T.-Z.; Thompson, P. T.; Hearn M. T. W.; Aguilar, M. I. J. Pept. Res. 1997, 49, 394-403. (12) Purcell, A. W.; Aguilar, M. I.; Wettenhall, R. E. H.; Hearn, M. T. W. Pept. Res. 1995, 8, 160-170. (13) Blondelle, S. E.; Perez-Paya, E.; Allicotti, G.; Forood, B.; Houghten, R. A. Biophys. J. 1995, 69, 604-611. (14) Blondelle, S. E.; Buttner, K.; Houghten, R. A. J. Chromatogr. 1992, 625, 199-206. (15) Zhou, N. E.; Mant, C. T.; Hodges, R. S. Pept. Res. 1990, 3, 8-20. (16) Regnier, F. E. Science 1987, 238, 319-323. (17) Richards, K. L.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1994, 676, 17-31. (18) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J. Chromatogr. 1995, 711, 61-70. (19) Aguilar, M. I.; Richards, K. L.; Round A. J.; Hearn, M. T. W. Pept. Res. 1994, 7, 207-217. (20) Guo, D.; Mant, C. T.; Taneja A. K.; Hodges, R. S. J. Chromatogr. 1986, 359, 519-532. 10.1021/ac980473c CCC: $15.00
© 1998 American Chemical Society Published on Web 10/29/1998
therefore determined by the specific contact amino acid residues rather than by the entire amino acid sequence. However, the location and identity of these chromatographic contact regions of proteins cannot be readily established. Without this information, it is not possible to predict the molecular basis of the retention behavior of a protein, and this limitation constrains the further development of RP-HPLC as a technique to study proteinsurface interactions. To address this problem, procedures have been developed in this study to identify the chromatographic contact regions of proteins when adsorbed to reversed-phase sorbents. In particular, proteolytic techniques have been used to probe the surface region of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) while adsorbed to an n-butyl (C-4) and n-octadecylsilica (C-18) reversed-phase sorbent. Following proteolytic digestion and characterization of the derived fragments, the results were correlated with the known three-dimensional structure of these two proteins and provide insight into the location of the possible contact regions as well as the orientation of these two proteins at the surface of reversed-phase sorbents. MATERIALS AND METHODS Chemicals and Reagents. Water was quartz-distilled and deionized in a Milli-Q system (Millipore, Bedford, MA). Acetonitrile (HPLC grade) was obtained from Mallinckrodt (Paris, KY) and from EM Industries Inc. (Darmstadt, Germany). Trifluoroacetic acid (TFA) was obtained from Auspep (Parkville, Australia). Recombinant bovine growth hormone (bGH) was generously provided by The Upjohn Co. (Kalamazoo, MI), and horse heart cytochrome c (Cyt c) and pepsin were obtained from Sigma (St. Louis, MO). Trypsin and chymotrypsin were purchased from Worthington Inc. (Freehold, NY). Ammonium bicarbonate was obtained from Mallinckrodt, and acetone and calcium chloride were from BDH Chemicals (Kilsyth, Australia). Apparatus. Chromatographic analyses of protein digestions were carried out with a Beckman System Gold chromatographic system (Beckman Instruments, Inc., Fullerton, CA), consisting of a dual-pump programmable solvent module 126 and a variable UV detector module 166 and controlled using System Gold software (version 5.0). All chromatographic profiles were monitored at 214 nm. Chromatographic analyses of growth hormone digestions were performed with a Waters Associates (Milford, MA) liquid chromatograph 484 system, consisting of two model 6000A solvent delivery pumps, a U6K universal injector, a WISP model 712B sample processor, and an M660 gradient programmer. The detector used was a Lambda-Max model 484 LC spectrophotometer operating at 215 nm, and the total system was controlled by Maxima 820 operating software. Chromatographic Procedures. Analyses of protein digests were performed on a Bakerbond Widepore n-butylsilica column (J.T. Baker Chemicals, Phillipsburg, NJ) with dimensions of 250 × 4.6 mm i.d., containing sorbents of 5 µm nominal particle size and 30 nm average pore size or a Zorbax 300 SB-C8 reversedphase column with dimensions of 4.6 mm × 15 cm, with particles 5 µm in diameter and 300 Å average pore size (Rockland Technologies, Newport, DE). Bulk solvents were degassed by sparging with helium. Linear gradient elution was performed from 0.1% (v/v) TFA in deionized water (buffer A) to 0.09% (v/v) TFA with 50% (v/v) aqueous acetonitrile (buffer B) over gradient times
of 50 and 150 min for Cyt c and from 0.1% (v/v) TFA in deionized water (buffer A) to 0.09% (v/v) TFA with 80% (v/v) aqueous acetonitrile (buffer B) over gradient times of 30, 60, and 120 min for bGH, both at a flow rate of 1 mL/min. Injection size of peptide solutions varied between 30 and 100 µL in analytical separations and between 0.5 and 1.5 mL in preparative separations. In Situ Tryptic Cleavage of Proteins. Binding of Cyt c to RP-HPLC Sorbents in a Batch Procedure. The sorbents used for all in situ digestion experiments were Vydac C-4 or C-18 silica (The Separations Group, Hesperia, CA). Fifteen milligrams of Vydac C-18 silica (5 µm particle size and 30 nm pore diameter) was washed and vortexed with 1 mL of methanol and then three times with 1 mL of 35% acetonitrile to suspend the sorbent material. After each washing step and centrifugation, the solvent was discarded. The sorbent then was resuspended for 2 h in 1 mL of 0.1% TFA containing 7.5 mg of Cyt c under continuous agitation to allow maximal binding. After centrifugation, the pellet was washed four times with 1 mL of cleavage buffer (50 mM ammonium bicarbonate, 2 mM calcium chloride) to remove all nonbound Cyt c. After each centrifugation, the supernatant was analyzed by RP-HPLC as described above to show the degree of Cyt c removal. Calculation of Binding Efficiency. To calculate the amount of bound Cyt c, the adsorbed protein was eluted from the C-18 sorbent with 75% acetonitrile/0.1% TFA (2 × 0.25 mL). An aliquot of the eluate was then analyzed by RP-HPLC and the amount of bound Cyt c measured by comparison with a Cyt c standard curve. Cleavage of Accessible Sites of Cyt c following Binding. The sorbent with bound Cyt c was incubated with TPCK-treated trypsin in cleavage buffer under continuous agitation to cleave accessible cleavage sites. Based on the calculation of the binding efficiency, a protein:trypsin ratio of 10:1 was chosen. The cleavage process was stopped after 18 h with the addition of two drops of 2 M HCl. Preparation of Cyt c Peptides. After centrifugation of the sorbent, an aliquot of the collected supernatant containing the tryptic peptides which did not bind to the C-4 or the C-18 sorbent (the nonbound fragments) in the presence of the cleavage buffer was analyzed by RP-HPLC under the same chromatographic conditions as a tryptic digest of Cyt c generated in the absence of sorbent material, with the same protein:protease ratio and the same cleavage conditions (the free solution digest). The sorbent was washed four times with 1 mL of cleavage buffer to remove peptides which had not bound to the sorbent. An aliquot of the last supernatant was analyzed by RP-HPLC to confirm the absence of residual amounts of free peptides. The remaining tryptic peptides which were bound to the sorbent (the bound fragments) were eluted with 75% acetonitrile/ 0.1% TFA (2 × 0.25 mL). After centrifugation, an aliquot of the combined supernatants was analyzed by RP-HPLC. An in situ experiment was also performed using cleavage buffer without trypsin. In addition, trypsin was incubated in cleavage buffer in the absence of both Cyt c and sorbent, under the same conditions as for the analysis of the free solution digest of Cyt c to distinguish Cyt c fragments from autocatalytic trypsin fragments. Identical procedures were used for the in situ tryptic digestion of bGH. Cleavage of Proteins with Chymotrypsin and Pepsin. Proteolytic in situ and solution cleavages of Cyt c and bGH with Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Figure 1. Schematic representation of the in situ proteolytic digestion procedure for proteins adsorbed to reversed-phase sorbents.
chymotrpysin and pepsin were carried out under conditions similar to those described for cleavage with trypsin but with the following cleavage buffers. For chymotryptic cleavage, 50 mM sodium bicarbonate, pH 7.9, was used, and the reaction was stopped by acidification with 2 M HCl. Pepsin cleavage was performed in 0.1% TFA, and the reaction was stopped by neutralization with 2 M sodium hydroxide. Analysis of Tryptic Fragments. Amino acid composition analysis of tryptic peptides was performed using a Waters (Milford, MA) Picotag amino acid analyzer. N-Terminal sequencing was performed on an Applied Biosystems (ABI) 470A gas-phase protein sequencer modified to use 475A chemistry with on-line HPLC analysis and an ABI120A analyzer coupled to a 900A ABI data analysis module (Foster City, CA). Electrospray mass spectrometry analysis was performed by Chiron Mimotopes (Clayton, Australia) using a Perkin-Elmer Sciex API III mass spectrometer (Perkin-Elmer-Sciex, Thornhill, ON, Canada). Capillary electrophoresis was performed using a Beckman P/ACE System 5000 instrument with a variable P/ACE UV absorbance detector and System Gold (version 8.1) software. A 57-cm (50 cm to detector) fused silica capillary tubing of 50 µm internal diameter was used for all analyses. Computer analysis of peptide fragments was performed with the program Rasmol (version 2.6). The solvent-accessible surface areas for each protein were calculated using the Connolly algorithm21 using a Silicon Graphics Indigo2 workstation. The operating software program was Insight II from Molecular Simulations (San Diego, CA). RESULTS AND DISCUSSION Optimization of the in Situ Cleavage Conditions. The procedure developed for the in situ proteolytic cleavage of proteins bound to reversed-phase sorbents involved adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage of the adsorbed protein. The procedure is illustrated schematically in Figure 1. The concept implicit in this approach is that the region of the protein surface in contact with the sorbent remains intact and bound during the digestion step, while the region(s) of the protein that is (are) exposed to the solvent are accessible to proteolysis. The cleaved fragments (21) Connolly, M. L. J. Appl. Crystallogr. 1983, 16, 548-558.
5012 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
which are not bound to the sorbent and hence are not part of the contact region can then be recovered from the supernatant by centrifugation. The remaining portion(s) of the protein which is (are) still adsorbed to the sorbent can then be eluted with 75% (v/v) aqueous acetonitrile-0.1% TFA. This procedure was developed and validated using two well-characterized model proteins, Cyt C and bGH. In situ proteolytic cleavage of sorbent-bound protein was carried out in a batch mode and required the optimization of two factors: (1) the protein:sorbent ratio and (2) the time over which the protein adsorption was carried out. The aim of these optimization steps was to select the appropriate experimental conditions for the adsorption of protein onto the reversed-phase sorbent. In the first instance, the optimum protein:sorbent ratio which maximized the coverage of the surface with protein was investigated by adding increasing amounts (between 0.6 and 3.0 mg) of Cyt c to 3 mg of C-4 sorbent and shaking for 6 h. Similar procedures were used for the adsorption of protein to a C-18 sorbent. Nonadsorbed Cyt c was removed by centrifugation of the slurry mixture and removal of the supernatant. After the sorbent was washed with 0.1%TFA, the bound Cyt c was desorbed and the amount determined by RP-HPLC. Figure 2a shows the plot of the relative peak area of Cyt c recovered versus the protein: sorbent ratio. This figure shows that the optimum ratio after which no additional Cyt c is adsorbed corresponds to approximately 0.5 mg of protein:1 mg of sorbent. This ratio thus reflects the maximal coverage of the reversed-phase sorbent by the Cyt c, thereby ensuring that readsorption of any nonbound fragments onto the sorbent after cleavage was minimized. The optimum time for adsorption of Cyt c was then investigated over a 24-h time period using a Cyt c:sorbent ratio of 0.5:1. Figure 2b shows a plot of relative peak area of Cyt c (recovered from the sorbent material) versus the adsorption time up to the first 2 h. The data show a rapid adsorption of protein over the first 20 min, which then remained constant over the ensuing 22 h. An adsorption time of 2 h was thus chosen for subsequent experiments. These optimized conditions were then used for a preliminary tryptic digestion experiment. C-4 sorbent containing the bound protein at protein:sorbent ratios of 1:2 (optimized value), 1:10, and 1:20 was resuspended in 50 mM ammonium bicarbonate and 2
Figure 3. RP-HPLC separation of the bound fragments in situ tryptic digestion of Cyt c bound to a C-4 sorbent. Cyt c was adsorbed onto the sorbent at a ratio of (a) 1:20, (b) 1:10, and (c) 1:2 mg of protein: milligram of sorbent, subjected to in situ tryptic digestion, and analyzed as described in the Materials and Methods section. The retention time (min) for intact Cyt c is indicated.
Figure 2. Optimization of the conditions for adsorption of Cyt c onto the C-4 sorbent. (a) Plot of relative peak area of recovered Cyt c versus protein:sorbent ratio. (b) Plot of relative peak area of recovered Cyt c versus adsorption time. See Materials and Methods section for other details.
mM CaCl2. Trypsin was then added to the sorbent slurry at a trypsin:protein ratio of 1:20 on the basis of the amount of Cyt c bound to the sorbent. Digestion proceeded with agitation for 24 h and was stopped by acidification with 2 mM HCl. The mixture was then centrifuged and the supernatant, which contained the nonbound fragments, collected. The sorbent was then washed twice with 0.1% TFA, and the washings were combined. The sorbent pellet was then washed with 75% (v/v) acetonitrile-water/ 0.1% TFA and recentrifuged. The supernatant, which contains the bound fragments, was collected and the sorbent pellet subjected to two additional washings. Both the bound and nonbound fragments were analyzed by RP-HPLC. The chromatograms obtained for the bound fragments at each of the three protein: sorbent ratios are shown in Figure 3. It can be seen that, for protein:sorbent ratios of 1:20 and 1:10, there is only one main peak which corresponds to Cyt c. These results demonstrate that, under these conditions, there is incomplete coverage of the sorbent material, which resulted in adsorption of the trypsin, which in turn prevented digestion from occurring. In contrast, tryptic digestion proceeded to completion at a ratio of 1:2 (Figure 3c),
as evidenced by the large number of peaks recovered in the bound fraction. These results confirm that a protein:sorbent ratio of 1:2 represents an optimum set of conditions for subsequent in situ digestion experiments. Furthermore, these results demonstrate that proteins are able to undergo tryptic digestion while adsorbed to a chromatographic surface and that proteases can selectively digest adsorbed proteins. Analysis of the tryptic fragments was then undertaken to determine the location of the hydrophobic binding domain. Analysis of Tryptic Digests. The in situ digestion procedure involved the following five steps, as depicted in Figure 1: 1. Adsorption of protein onto the reversed-phase material. 2. Digestion of adsorbed protein with trypsin. 3. Removal of nonbound fragments. 4. Elution of bound fragments. 5. Analysis of nonbound and bound peptide fragments. Cytochrome c. In situ cleavage of Cyt c bound to a C-18 silica sorbent was performed using a trypsin:Cyt c ratio of 1:10, and digestion was allowed to proceed for 24 h. The amount of Cyt c bound to the C-18 material was 20% of the quantity of Cyt c in the solution exposed to the sorbent, and this value was used to calculate the amount of trypsin required. Cyt c was adsorbed to C-18 silica, trypsin was added, and the samples were shaken overnight and then acidified to stop the digestion. The nonbound and bound fractions were separated by RP-HPLC and compared to the tryptic map obtained in the absence of the C-18 silica, i.e., Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
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Table 1. Free Solution Tryptic Fragments of Cytochrome c peptide
position
sequence
molecular mass (Da)
observed in HPLC
T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T20 T21 T22
1-5 6-7 8 9-13 14-22 23-25 26-27 28-38 39 40-53 54-55 56-60 61-72 73 74-79 80-86 87 88 89-91 92-99 100 101-105
GDVEK GK K IFVQK CaAQCaHTVEK GGK HK TGPNLHGLFGR K TGQAPGFTYTDANK NK GITWK EETLMEYLENPK K YIPGTK MIFAGIK K K TER EDLIAYLK K ATNE
571.6 203.2 146.2 633.8 1016.2 260.3 283.3 1168.4 146.2 1470.6 260.3 603.7 1495.9 146.2 677.8 779.0 146.2 146.2 404.5 964.2 146.2 433.4
x x x x x x x x x
x
a The heme prosthetic group is covalently attached to cysteine-14 and cysteine-17.
Table 2. Nonbound Tryptic Fragments of Cytochrome c
Figure 4. RP-HPLC separation of (a) solution tryptic fragments, (b) nonbound fragments, and (c) bound fragments of Cyt c. The fragments in (b) and (c) were generated after tryptic digestion of Cyt c adsorbed to a C-18 sorbent. See Materials and Methods section for other details.
a solution control digestion. These chromatograms, corresponding to the control proteolytic tryptic map obtained in solution, are shown in Figure 4a, while the tryptic maps obtained for the nonbound and bound fragments when Cyt c was adsorbed to the reversed-phase material are shown in Figure 4b and c, respectively. Comparison of these chromatograms reveals significant differences in the digestion patterns. In particular, the peptides obtained in the nonbound tryptic map (Figure 4b) generally eluted earlier than those peptides obtained in the bound tryptic map (Figure 4c). This result indicates that more hydrophobic and/or longer peptides were present in the bound fraction, which suggests that Cyt c interacts with the C-18 sorbent through specific hydrophobic contact regions. To identify the spatial location of the fragments generated in each tryptic map within the three-dimensional structure of Cyt c, all fragments were collected and subjected to amino acid analysis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Cyt c contains 104 amino acid residues, with a total of 19 lysine residues and 2 arginine residues. The sequences and molecular masses of the expected tryptic fragments for Cyt c are listed in Table 1, while the sequences and molecular masses of the tryptic fragments found in the nonbound and bound fractions are listed in Tables 2 and 3. Almost all the expected fragments were recovered in the free solution digest and are identified in 5014 Analytical Chemistry, Vol. 70, No. 23, December 1, 1998
peptide
position
sequence
molecular mass (Da)
T1 T9-10 T10 T8+ T8
1-5 39-53 40-53 28-38 28-38
GDVEK KTGQAPGFTYTDANK TGQAPGFTYTDANK TGPNLHGLFGR TGPNLHGLFGR
571.6 1598.7 1470.6 >1168.4 1168.4
Figure 4a according to their code in Table 1. Analysis of the peptides in the nonbound fraction revealed that Cyt c was cleaved by trypsin at positions 5, 27, 38, 39, and 53 (Table 2) with the release of three major fragments, T1, T8, and T10. Assuming there was a specific orientation of the Cyt c bound to the C-18 ligands, some of the peptides present in the nonbound fraction would also be expected to occur in the free solution tryptic digest and result in coincident peaks in their respective chromatograms. Comparison of Figure 4a and b reveals the presence of T1, T8, and T10 in both the free solution digest and the nonbound fragments, indicating that these sequence regions do not form part of the chromatographic binding domain. Analysis of the eight major bound fragments revealed additional cleavage points at positions 7, 8, 13, 25, 55, and 86. The heme prosthetic group is covalently attached to Cyt c through cysteine-14 and cysteine-17 and will, therefore, be attached to any peptides containing the T5 sequence. In summary, the analysis shows that the N-terminal lysine-22, as well as the C-terminal lysine residues 60, 72, 73, 79, 87, 99, and 100 and arginine-91, was inaccessible to tryptic digestion. In addition, the major peak in the bound fraction, corresponding to T12-22, exhibited chromatographic retention times very similar to those of intact Cyt c. These results indicate that the sequence region encompassing residues glycine-56glutamate-104 is involved in the hydrophobic binding domain.
Table 3. Bound Tryptic Fragments of Cytochrome c peptide
position
sequencea
molecular mass (Da)
T7-8 T8 T2-6 T3-4 T4 T5-7 T12-16 T12-22
26-38 28-38 6-25 8-13 9-13 14-27 56-86 56-105
HKTGPNLHGLFGR TGPNLHGLFGR GKKIFVQKCaAQCaHTVEKGGK KIFVQK IFVQK CaAQCaHTVEKGGKHK GITWKEETLMEYLENPKKYIPGTKMIFAGIK GITWKEETLMEYLENPKKYIPGTKMIFAGIKKKTEREDLIAYLKKATNE
1433.6 1168.4 2187.6 762.0 633.8 1523.8 3630.5 5763.1
a The lysine and arginine residues which were inaccessible or partially accessible to digestion are shown in italic type. b The heme prosthetic group is covalently attached to cysteine-14 and cysteine-17.
Figure 6. Plot of solvent-accessible surface area of amino acid residues in the three-dimensional structure of Cyt c. The sequence positions of phenylalanine, leucine, and tryptophan residues are highlighted by the filled circles, while the sequence positions of arginine and lysine residues are indicated by open circles. Figure 5. Three-dimensional structure of Cyt c, showing the bound regions in black and dark gray and the nonbound regions in light gray. The black regions contain arginine and lysine residues within the sequence region 56-104 (excluding lysine-86, which is dark gray) that were totally inaccessible to tryptic digestion, while the dark gray region contains the arginine and lysine residues (except lysine-22, which is black) within the sequence region 6-25 that were partially accessible to digestion. The protein backbone is shown in a ribbon representation, and the heme moiety is shown in ball-and-stick mode.
The location of these sequence regions within the threedimensional structure of Cyt c are highlighted in Figure 5. Residues 56-104 are shown in black, residues 6-25 are shown in dark gray, and the remainder of the protein sequence is shown in light gray. It is evident from this figure that a relatively large region on the surface of Cyt c is protected by the reversed-phase sorbent from tryptic digestion. There are two sequence regions identified as peptides in the nonbound fractions, corresponding to residues 1-5 and 28-53, that are located on opposite sides of the Cyt c structure. The observation that the N-terminal residues 1-5, which are spatially located within the large putative hydrophobic binding domain, are susceptible to tryptic cleavage suggests that there may be conformational changes associated with the adsorption of Cyt c to the C-18 sorbent. As these N-terminal residues will be highly flexible in solution, it is possible that Cyt c binds with a conformation in which residue lysine-5 remains accessible to tryptic cleavage. In addition, while the main bound fragment consisted of residues 56-104, a small degree of cleavage was also observed at position 86, which is close to the N-terminal residues 1-5 in the three-dimensional structure of Cyt c. This result may indicate that the changes in conformation which expose
residues 1-5 to tryptic attack may also partially expose residue 86 to proteolytic cleavage. Moreover, the region glycine-6-lysine25 of the bound Cyt c showed partial (incomplete) cleavage, generating limited amounts of the tryptic fragments T4-5. As noted in Table 1, T2, T3, and T6 were not recovered from the RP-HPLC separation of the tryptic digest of free or bound Cyt c. The digestion of Cyt c was also carried out with the proteolytic enzymes chymotrypsin and pepsin. However, no in situ digestion was observed with these enzymes, even though complete digestion was observed in the absence of the reversed-phase material. These results can be explained in terms of the nature of the amino acid residues at which chymotrypsin and pepsin cleave. These two enzymes cleave specifically at hydrophobic and/or aromatic amino acid residues, which are located largely within the interior of the three-dimensional structure of Cyt c. This characteristic of these nonpolar residues is illustrated in Figure 6, where a plot of the relative solvent accessibility of phenylalanine, leucine, and tryptophan determined using the procedure of Connolly21 is shown. Specifically, the accessible surface area of each residue in the X-ray crystal structure of Cyt c can be defined as the area accessible to a water molecule (1.4 Å).22 As is evident from Figure 6, the sequence positions of these hydrophobic residues which are targeted by chymotrypsin and pepsin correspond to minima in this plot; i.e., they are buried within the hydrophobic core of the folded protein. As observed in the present study, cleavage of Cyt c by these enzymes was observed under bulk solution conditions, (22) Richards, F. M. Annu. Rev. Biophys. Bioeng. 1977, 6, 151-176.
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suggesting that the conformational flexibility of Cyt c results in a certain degree of solvent exposure of one or more of these internalized hydrophobic residues. Partial proteolytic cleavage then leads to further unfolding of the protein and subsequent cleavages until the complete digestion has occurred. The inability of chymotrypsin and pepsin to cleave bound Cyt c demonstrates that the adsorbed Cyt c is conformationally constrained, remaining resistant to proteolytic attack. By comparison, Figure 6 also indicates that the sequence position of arginine and lysine residues corresponds to maxima in the accessibility plot; i.e., these residues are largely located on the surface of Cyt c and are, therefore, readily accessible to trypsin in solution or when orientated away from the reversed-phase sorbent with bound Cyt c. The in situ tryptic digestion of Cyt c was also carried out with the protein adsorbed to a C-4 sorbent material. Digestion patterns observed for the bound and nonbound experiments were almost identical to those obtained with the C-18 sorbent (results not shown). This result demonstrates that the orientation of Cyt c at a hydrophobic surface is not strongly influenced by the structure of the n-alkyl ligands that are immobilized to the reversed-phase material. In a previous study on the retention properties of Cyt c in RP-HPLC, it was found that significantly different retention behavior was observed for Cyt c when it was chromatographed on a C-18 and a C-4 sorbent.18 The results of the present study indicate that these differences in the retention behavior are not due to Cyt c adopting a significantly different orientation at the sorbent surface, but rather arise from differences in the relative affinity of the protein for each ligand, together with the differences in dynamic flexibility of the short n-butyl ligand compared to the longer n-octadecyl ligand.23 Growth Hormone. The general applicability of the in situ digestion procedures developed with Cyt c to other proteins was tested with the digestion of bovine growth hormone (bGH), which was subjected to in situ tryptic digestion in the presence of a C-18 sorbent under conditions similar to those used for Cyt c. The tryptic map obtained for bGH in free solution is shown in Figure 7a, while the RP-HPLC chromatographic profiles of the nonbound and bound fractions are shown in Figure 7b and c, respectively. Comparison of these chromatograms reveals significant differences in the digestion patterns obtained for the control solution tryptic map and the peptide fragments generated in situ. In particular, as was observed for Cyt c, the peptides present in the nonbound fraction all eluted much earlier than the peptides obtained in the bound fraction, demonstrating the more hydrophobic nature of the bound fragments. The tryptic fragments from each experiment were collected and analyzed by amino acid analysis, N-terminal sequencing, and electrospray mass spectrometry. bGH contains 191 amino acid residues, with a total of 11 lysine residues and 13 arginine residues. The sequences and molecular masses of the expected tryptic peptides in solution are listed in Table 4. Tables 5 and 6 list the peptide fragments identified from the chromatographic analysis of the nonbound and bound fractions in Figure 7b and c. Almost all of the expected fragments were recovered in the control solution digestion. It can be noted that the fragments T5-18 and T23-25 were obtained, which contain the disulfide bonds between (23) Yarovsky, I.; Aguilar, M. I.; Hearn, M. T. W. Anal. Chem. 1995, 67, 21452153.
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Figure 7. RP-HPLC separation of (a) solution tryptic fragments, (b) nonbound fragments, and (c) bound fragments of bGH. The fragments in (b) and (c) were generated after tryptic digestion of bGH adsorbed to a C-18 sorbent. See Materials and Methods section for other details.
cysteines 53-164 and 181-189. In contrast, the N-terminal residues arginine-17 and lysine-30 and the C-terminal residues arginine-77 and -95 were inaccessible to tryptic attack when bGH was adsorbed to the C-18 sorbent, while incomplete digestion was noted at arginine-34 and -106. The sequence regions corresponding to alanine-1-arginine-42 and serine-71-lysine-112 appear to constitute the major portion of the chromatographic binding domain of bGH. This result demonstrates that bGH also interacts with the reversed-phase material in an orientation-specific manner through specific sequence regions. The locations of the nonbound and bound fragments within the three-dimensional structure of bGH are shown in Figure 8. This figure demonstrates that the bound fragments are predominantly localized along helix 1, helix 2, the intervening extended coil, and the N-terminal section of helix 3. It can also be seen in Figure 8 that the disulfide bond between cysteine-53 and cysteine164 restricts the accessibility of trypsin to the potentially cleavable sites at arginine-42. If the conformation of bGH, when adsorbed onto the C-18 sorbent, was identical to the known X-ray structure, it would be expected that amino acid residues within helix 3 and/ or helix 4 would also be involved in the hydrophobic binding domain. However, the results presented here suggest that there is some destabilization of the conformation of bGH during
Table 4. Free Solution Tryptic Fragments of Bovine Growth Hormone peptide
position
sequence
molecular mass (Da)
observed in HPLC
T1 T2 T3 T4 T5+18 T6 T7 T8 T9 T10 T11 T12 T13 T14 T15-16 T17 T19 T20 T21 T22 T23-25
1-17 18-30 31-34 35-42 43-64 ∼ 158-166 65-70 71-77 78-95 96-108 109-112 113-114 115-125 126-133 134-139 140-150 151-157 167 168-171 172-177 178-180 181-191
AFPAMSLSGLFANAVLR AQHLHQLAADTFK EFER TYIPEGQR YSIQNTQVAFCFSETIPAPTGKNYGLLSCFR NEAQQK SDLELLR ISLLLIQSWLGPLQFLSR VFTNSLVFGTSDR VYEK LK DLEEGILALMR ELEDGTPR AGQILK QTYDKFDTNMR SDDALLK K DLHK TETYLR VMK CRRFGEASCAF
1765.5 1480.2 579.6 963.3 3474.6 806.9 845.1 2085.1 1443.1 537.7 259.4 1259.7 916.2 629.0 1418.9 761.0 146.2 507.7 782.1 376.6 1246.7
x x x x x
Table 5. Nonbound Tryptic Fragments of Bovine Growth Hormone peptide
position
sequence
molecular mass (Da)
T14 T21 T4 T17 T20-21 T15-16 T24-25
134-139 172-177 35-42 151-157 168-177 140-150 183-191
AGQILK TETYLR TYIPEGQR SDDALLK DLHKTETYLR QTYDKFDTNMR RFGEASCAF
629.0 782.1 963.3 761.0 1281.8 1418.9 987.3
adsorption to the C-18 surface. Changes in the three-dimensional structure of human growth hormone have been previously observed upon adsorption to C-18 sorbents6 and a series of alkylated silicas and self-assembled monolayers.24 The in situ tryptic digestion of bGH was also carried out with a C-4 RP-HPLC material, and the digestion patterns observed in the bound and nonbound fractions were very similar to those obtained with the C-18 material (results not shown). In addition, bound bGH was also resistant to proteolysis by chymotrypsin and pepsin (results not shown), suggesting that any changes in conformation upon adsorption to either the C-4 or C-18 sorbent are not associated with significant unfolding of the protein structure. Orientation Effects of Cyt c and bGH. The gradient elution separation of peptides and proteins in RP-HPLC involves the initial adsorption of the solute onto the sorbent surface in aqueous buffer, generally containing 0.1% TFA, followed by the elution of the solute by an increase in organic solvent concentration. The experimental conditions used in the present study to initially adsorb Cyt c and bGH onto the C-18 and C-4 sorbents, therefore, correspond to the conditions experienced during the initial stage of a gradient elution experiment. The subsequent exposure of the adsorbed protein to pH 7 during the tryptic digestion may result in some changes in conformation of the exposed regions of the proteins. However, these conformational changes are unlikely to signifi(24) Buijs, J.; Britt D. W.; Hlady, V. Langmiur 1998, 14, 335-341.
x x x x x x x x x x x x
cantly affect the composition or location of the chromatographic binding domains of these proteins. Both Cyt c and bGH exhibit very high binding affinities for each sorbent at both pHs; i.e., neither protein could be eluted in 0.1% TFA or 50 mM ammonium bicarbonate. Major changes in conformation and/or orientation upon exposure to pH 7 can also be discounted, as both bound proteins were totally resistant to cleavage by either chymotrypsin (pH 7.9) or pepsin (pH 2.5). While direct spectroscopic evidence using, for example, in situ techniques25 is required in order to verify these conclusions, the lack of cleavage by chymotrypsin and pepsin provides evidence that the conformation of the bound Cyt C and bGH remained constrained. During the elution procedures, a protein or peptide may also experience significant changes in secondary and/or tertiary structure upon exposure to organic solvent. In the case of peptides, particularly those that are amphipapthic in nature, adsorption onto the reversed-phase sorbent together with exposure to organic solvent has been shown to induce a significant degree of secondary structure.5,10,12,25,26 While there is less direct evidence for changes in the secondary and tertiary structures of proteins, the often poor yields obtained in terms of mass and biological activity suggest that proteins can undergo significant changes in conformation during gradient elution. While it is difficult to verify spectroscopically, the extent of unfolding is likely to be restricted to changes in tertiary structure. Secondary structural units will tend to be less affected and may be stabilized because of interaction with a hydrophobic surface. When the R-helical and/or β-sheet regions of a protein are stabilized by the internal hydrophobic core of the protein, the exposure of the protein to a reversed-phase surface may cause the protein to turn “inside-out”; i.e., the internal hydrophobic residues can come into contact with the sorbent surface, allowing the regions of secondary structure to remain intact. Further studies are, therefore, required (25) Blondelle, S. E.; Ostresh, R. A.; Houghten, R. A.; Perez-Paya, E.; Biophys. J. 1995, 68, 351-359. (26) Steer, D. L.; Thompson, P. E.; Blondelle, S. E.; Houghten, R. A.; Aguilar, M. I. J. Pept. Res. 1998, 51, 401-412.
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Table 6. Bound Tryptic Fragments of Bovine Growth Hormone peptide
position
T5+18
43-64 158-166 1-17 96-108 1-42 71-95 1-34 71-108 71-112
T1 T9 T1-4 T7-8 T1-3 T7-9 T7-10 a
sequencea
molecular mass (Da)
YSIQNTQVAFCFSETIPAPTGKNYGLLSCFR
3474.6
AFPAMSLSGLFANAVLR VFTNSLVFGTSDR AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFERTYIPEGQR SDLELLRISLLLIQSWLGPLQFLSR AFPAMSLSGLFANAVLRAQHLHQLAADTFKEFER SDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDR SDLELLRISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEK
1765.5 1443.1 4734.6 2912.2 3789.3 4979.3 5499.0
The lysine and arginine residues which were inaccessible or partially accessible to digestion are shown in italic type.
Figure 8. Three-dimensional structure of bGH, showing the bound regions in black and the nonbound regions in light gray. The protein backbone is shown in a ribbon representation.
in order to elucidate the specific effect of organic solvents on the bound conformation of proteins in order to fully characterize the orientation of proteins at reversed-phase sorbent materials. (27) Yarovsky, I.; Hearn, M. T. W.; Aguilar, M. I. J. Phys. Chem. 1997, 101, 10962-10970.
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CONCLUSIONS The procedures developed in this study have permitted the identification of the chromatographic contact regions of two wellcharacterized globular proteins bound to a C-18 and a C-4 RPHPLC sorbent. In particular, it has been shown that distinct areas on the protein surface become inaccessible to proteolytic cleavage when the protein is adsorbed to the sorbent surface. The results of the present study demonstrate that largely continuous regions of Cyt c and bGH were in contact with the C-18 and the C-4 sorbent. As more data are obtained on the precise location of the binding domains, interactive docking experiments27 can be performed between the protein and the reversed-phase surfaces. In the absence of spectroscopic techniques that can accurately determine the orientation of proteins upon adsorption to reversedphase sorbents, the present study provides an experimental approach to enzymatically identify the chromatographic binding domains of proteins and represents a further step in the development of a fully mechanistic model which accurately describes the molecular basis of protein retention in RP-HPLC. ACKNOWLEDGMENT The financial support of the Australian Research Council is gratefully acknowledged. The authors also thank Tzong-Hsien Lee for his assistance in the production of the illustrations. Received for review April 30, 1998. Accepted August 25, 1998. AC980473C
