High-performance liquid chromatography of amino acids, peptides

jd-endorphin on both C18 and C4 n-alkylsilicas were monitored by examining changes in theexperi- mental bandwidths of these peptides as a function...
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Anal. Chem. 1003, 65,3038-3047

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High-Performance Liquid Chromatography of Amino Acids, Peptides, and Proteins. 123. Dynamics of Peptides in Reversed-Phase High-Performance Liquid Chromatography Anthony W. Purcell, Marie-Isabel Aguilar, and Milton T. W. Hearn' Department of Biochemistry and the Centre for Bioprocess Technology, Monash University, Wellington Road, Clayton 3168, Victoria, Australia

The dynamics of several peptides in reversedphase high-performance liquid chromatography ( R P - H P L C ) have been investigated on both n-octadecyl (C18) silica and n-butyl (C4) silica sorbents. In particular, the conformational interconversions and the relative rates of chromatographic relaxation of bombesin, glucagon, and @-endorphinon both C18 and C4 n-alkylsilicaswere monitored by examining changes in the experimental bandwidths of these peptides as a function of temperature and column residence time under linear gradient elution RP-HPLC conditions. The observed band-broadeningtrends were correlated with previously derived retention parameters and thermodynamic descriptors of the association process determined for bombesin, @-endorphin, glucagon, and a control peptide, penta-L-phenylalanine. This study confirms that bandwidth measurements can be used as an integral experimental component to study the effect of the secondary structure of peptidic solutes on their RP-HPLC retention behavior. Further, the data demonstrate the utility of RP-HPLC as a tool to examine peptide conformational dynamics at hydrophobic surfaces. The relevance of these results to the general phenomenon of peptide-lipid interactions is discussed in terms of the associated evidence for lipid-induced changes in the conformation of these three bioactive peptides. INTRODUCTION Recent experimental investigations on solute retention behavior have provided significant insight into the nature of the interaction between proteins or peptides and chromatographic ligands.'-9 The development of physicochemical models which comprehensively describe polypeptide chromatographic behavior in terms of the thermodynamic and (1) Purcell, A. W.; Aguilar, M. I.; Hearn, M. T. W. J.Chromatogr. 1992, 593, 103-117. (2) Hearn, M. T. W.; Aguilar, M. I. In Modern Physical Methods in Biochemistry, Part B; Neuberger, A., van Deenen, L. L. M., Eds.; Elsevier: Amsterdam, 1988; pp 107-142. (3) Cohen, S. A.; Benedek, K.; Tapuhi, Y.; Ford, J. C.; Karger, B. L. Anal. Biochem. 1985, 144, 275-284. (4) Cohen, S. A.; Benedek, K.; Dong, S.; Tapuhi, Y.; Karger, B. L. Anal. Biochem. 1984, 56, 217-221. (5) Lu, X. M.; Benedek, K.; Karger, R. L. J. Chromatogr. 1986,359, 19-29. (6) Regnier, F. E. Science 1987, 238, 319-323. (7) 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. (8) Oroszlan, P.; Wicar, S.; Teshima, G.; Wu, S.-L.; Hancock, W. S.; Karger, B. L. Anal. Chem. 1992, 64, 1623-1631. (9) Zhou, N. E.; Mant, C. T.; Hodges, R. S. Pept. Res. 1990,3 (1),8-20.

kinetic components of the adsorptionand desorption processes still, however, remains a challenge in the chromatographic sciences. Correlation of predictions derived from such models with the three-dimensional structure and surface topography of the solute at the ligand interface would allow an additional potential of reversed-phase high-performance liquid chromatography (RP-HPLC) in protein and peptide chemistry to be realized. The study of the dependence of chromatographic retention parameters upon column environmental conditions provides experimental insight into factors associated with this interactive process. For example, in a recent paper,lthe reversedphase chromatographic retention behavior of three bioactive peptides,bombesin, 8-endorphin,and glucagon, was examined under a wide range of temperatures with n-octadecyl- (C18) and n-butyl- (C4) based sorbents. Conformational changes associated with the thermal disruption of the solute's interactive structure resulted in changes in the S and log k , values of these polypeptides as the column temperature was elevated. Since the parameter S is related to the magnitude of contact surface area between the solute and the stationary-phase ligands and the log k , value represents the affinity of the solute for the stationary-phase surface,2variations in these two parameters can be used to follow conformational changes of these peptides during the chromatographic process. In addition, the conformational changes evident in the reversedphase retention behavior of these biosolutes was also examined by van't Hoff analysis,' which provided information on the relative flexibility of each peptide in both the bound and free states as revealed from the entropic and enthalpic contributions to the interactive process. Despite the large number of studies on the effect of peptide structure on their RP-HPLC retention behavior, only a few studies have addressed the characterization of the associated bandwidth behavior. In the present investigation, bandwidth data associated with the reversed-phase chromatographic separation of bombesin, glucagon, @-endorphin,and pentaL-phenylalanine were determined and the results correlated with the corresponding chromatographic retention behavior of these peptides. The results are discussed in relation to the kinetics of solute conformational interconversions that occur with polypeptides during chromatographic migration on the C18 and C4 sorbents.

MATERIALS AND METHODS Apparatus. All chromatographic measurements were per-

formed on a Perkin-Elmer (PE) Series 4 chromatograph (Perkin Elmer, Norwalk, CT)utilizing a PE ISS-100 autosampler, a PE LC-95 ultraviolet-visible spectrophotometer, and a PE 7500 professional computer with the CHROM 3 software package installed. All peak profiles were routinely monitored at 215 nm, stored on the Winchester disc of the PE 7500 computer, and processed simultaneouslyby a PE LCI-100computingintegrator. Further peak analysis was performed using software routines included in the CHROM 3 program framework. Temperature

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ANALYTICAL CHEMISTRY, VOL. 05, NO. 21, NOVEMBER 1, 1993

was controlled either by immersing the column in a thermostated column water jacket coupled to a recirculating cooler (FTS Systems, New York) or by an 1.C.I TC 1900 column oven (I.C.I. Instruments, Dingley, Victoria, Australia). Chromatography was performed on Bakerbond Widepore n-butylsilica and n-octadecylsilica columns (J. T. Baker Chemicals, Phillipsburg, NJ) with dimensions of 250 X 4.6 i.d. mm and containing sorbents on 5-pm nominal particle size and 30-nm average pore size. All pH measurementswere made with an Orion Model SA520 pH meter (Orion, Cambridge, MA). Chemicals and Reagents. Acetonitrile (HPLC grade) was obtained from Mallinckrodt (Paris, KY) and HPLC-grade trifluoroacetic acid (TFA)acquired from Pierce (Rockford, IL). Water was distilled and deionized in a Milli-Q system (Millipore, Bedford, MA). Penta-L-phenylalanine, bombesin, and bovine or porcine pancreaticglucagon were all obtained from Sigma (St. Louis, MO) and were of >95% purity as determined by RPHPLC and amino acid compositionanalysis. Human @-endorphin ofsimilarpurity(>95%)wasobtainedfromeitherOrganon (OSS, the Netherlands) or Sigma; both batches of &endorphin gave identical HPLC profiles and amino acid composition during routine solute analysis. Chromatographic and Computational Procedures. Bulk solvents were filtered and degassed by sparging with nitrogen, and solvent reservoirs were maintained under 75-kPa pressure in a nitrogen atmosphere. Linear gradientelution was performed using 0.1% TFA in water (buffer A) and 0.09% TFA in 65% aqueous acetonitrile (buffer B)over gradient times of 15,30,45, 60,75,90,120,150, and 180 min with a flow rate of 1mL/min. Column temperature was maintained at temperatures of 5, 15, 25,37,45,55,65,75, and 85 "C and varied typically less than 1 "C. To ensure rapid temperature equilibration of the mobile phase, the mobile phase was passed through a 50 cm X 0.13 mm i.d. piece of microbore tubing, inserted into the equilibrated column jacket or oven before the column inlet. Peptide solutions were made by dissolving the solute at a concentrationof 0.1 mg/mL in 0.1% TFA (buffer A), while the injection size varied between 1 and 5 pg of peptide. Samples injected at these concentrations(bombesin 61 pM, 8-endorphin 29 pM and glucagon 28 pM) will most likely be monomeric, since these peptides are known to exist in the monomeric form at concentrationsfar exceeding those employed in this study.1w12 All data pointa were derived from at least duplicate measurements with retention times and bandwidths between replicates typically varying less than 1% and less than 5 % ,respectively. The column dead volumewas taken asthe retention time of the noninteractive solute, sodium nitrate. The various chromatographic retention and thermodynamicparameters S, log k,, AH", and AS", were calculated as previously reported.' The bandwidth data were plotted as 3D-mesh surfaces representing an XY plane of temperature w gradient length extended in the Z axis with the experimentally observed bandwidth. The XZ plane for all 3D plots represents the dependence of bandwidth on temperaturefor a particular gradient time while the YZ plane representsthe dependence of bandwidth on gradient time for any given temperature. The 3D mesh was initially constructed from a grid of 81 duplicated data points derived from experimentally measured bandwidth for the nine gradienttimes and nine different temperatures employed in this study. A mesh surface was then interpolated with the data to form a grid of 30 X 30 points using the softwarepackage Sigmaplot 5.0 (Jandel Scientific, San Rafael, CA) and inverse distance calculations with a statistical data weighting of 3.0.

RESULTS AND DISCUSSION Analysis of Experimental Bandwidths for Interconverting Systems. The kinetic processes that dictate the (10) Cavatorta, P.; Spisni, A.; Szabo, A. G.; Farruggia, G.; Franzoni, L.; Masotti, L. Biopolymers 1989,28 (l),441-463. (11) Taylor, J. W.;O~termm,D. G.; Miller, R. J.; Kaiser, E. T. J. Am. Chem. Soc. 1981,103,6965-6966. (12) Kom, A,; Ottensmeyer, F. J . Theor. Biol. 1983, 105, 403-425.

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band-broadening behavior of low molecular weight, rigid organic molecules in gradient elution are influenced by axial dispersion in the bulk mobile phase, dispersion due to slow mass transfer across the solvent-particle boundary and in the intraparticulate spaces and dispersion due to slow mass transfer of the solute at the stationary-phase surface.13J4For organic compounds, where the molecular surface area and the respective diffusivity of an individual solute remain essentially constant throughout the Chromatographic analysis, each of these band-broadening contributions can be satisfactorily accounted for by current theoreticaltreatments.2113J4 For a peptidic or protein solute on the other hand, the assumption that the surface area or hydrodynamic shape can be characterized in terms of a constant value throughout the chromatographic separation will only be valid for conformationally rigid molecules. In the absence of monomer-multimer association or solute aggregation, the major cause of band broadening with biomacromolecules in RP-HPLC systems is believedZJ'J6 to be time-dependent changesin the conformationand molecular composition of the hydrophobic contact area established between the solute and the chromatographic ligands. Interconversions between such conformational states lead to changes in hydrodynamic volume, the formation of multiple interactive regions involving the surface of the molecule and the immobilized ligands, and changes in the adsorption kinetics, all of which may be manifested as bandwidth changes in reversed-phase chromatography. However, the change in the interactive region of the solute will have a more significant effect on experimental bandwidth than any corresponding changes in solute diffusivity. The ability to detect and resolve these kinetic intermediates in RP-HPLC will depend on the sensitivity of the on-line spectroscopic technique, the chromatographic relaxation times associated with the different phenomena, the magnitude of the differences in retention times for the different species, and the peak variance of each species. The simplest experimental manifestation of conformational interconversion of a polypeptide or protein is illustrated by the appearance of two peaks which may, for example, correspond to the native or folded (N) and unfolded (U)structure, as depicted in Figure 1. For the folded and unfolded species to be resolved as individual peaks, they must have significantly different surface regions in contact with the chromatographic ligands. Although present theoretical considerations2*5J4J6of solute conformationalinterconversions often involve the two-state dynamic model shown in Figure 1, the mechanism of such interconversions may be more complex.17-lQ For example, the formation of stable intermediates with half-lives commensurate with the chromatographic process can occur, however, such as molten globular structures and other intermediates which are not necessarily on the N to U trajectory, as evident from recent studies with trypsin: soyabean trypsin inhibitor,Nand growth hormone.w1 (13) Snyder, L. R. In High Performance Liquid Chromatography. Aduances and Perspectives; Horvttth, Cs., Ed.; Academic Press: New York, 1980; Vol. 1, pp 207-316. (14) Aguilar, M. I.; Heam, M. T. W. In HPLC of Proteins, Peptides and Polynucleotides: Contemporary Topics and Applicatiom; Hearn, M. T. W.; Ed.; VCH Publishers: New York, 1991; pp 247-275. (15) Hearn, M. T. W.; Hodder, A. N.; Aguilar, M. I. J. Chromatog. 1986,327,4746. (16) Melander, W. R.;Lin, H. J.; Jacobson, J.; HorvBth, Cs. J. Phys. Chem. 1984,88,4527-4236. (17) Creighton, T. E. Protein Structure: A Practical Approach IRL Press: Oxford, UK, 1989; Chapters 1-3. (18) Weiasman, J. S.;Kim, P. S.Science 1991,253, 1386-1393. (19) Brems, D.N.;Brown, P. L.; Becker, G. W. J. Biol. Chem. 1990, 265, 5504-5511. (20) Cohen, S. A.; Dong, S.; Benedek, K.; Karger, B. L. In Affinity Chromatography and Biological Recognition; Chaiken, I. M., Wilchek, M., Parikh, I., Eds.; Academic Press: Orlando, FL, 1983; pp 479-487.

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frog skin28with the amino acid sequence and molecular weight shown in Table I. Bombesin has a wide range of biological activities both in vivo and in vitro which are mediated by the adoption of a C-terminal a-helical structure.10~29JO In the present study the propensity of bombesin to adopt a signifSOLVENT icant amount of secondary structure in the presence of a PARTICLE K6 BOUNDARY chemical hydrophobic surface (i.e., an n-alkylsilica) was further characterized through analysis of bandwidth data. The results also provide insight into the dynamics of these conformational interconversions. Figure 2A showsthe temperature dependence of bandwidth for bombesin chromatographed on the C18sorbent at gradient times of 15-180 min and temperatures ranging from 5 to 85 "C. Figure 2A demonstrates that a t gradient times less than 60 min the bandwidth remained relatively constant over the temperature range studied. For gradient times of 160 min significant increases in bandwidth were observed over a very Flgure 1. Model describing a two-state dynamic interconversionfrom discrete temperature range. The nonplanar nature of the a fully folded (N) molecule to a fully unfolded structure (U)in solution 3D-mesh surface depicted in Figure 2A for bombesin is in during interaction with stationary-phase ligands. The rate constants direct contrast to the behavior of penta-L-phenylalanine, kl-k8 are displayed for each stage of the cycle for the interconversion shown in Figure 3, which demonstrated essentially planar between the free structures (N and U) and the bound structures (Nb and U,,). behavior. The thermodynamic, S,and log k,values for pentaL-phenylalanine were previously' found to be constant over these experimental conditions. Collectively, these data for Accurate quantitation of the more complex effects assopenta-L-phenylalanine are consistent with the behavior of a ciated with small shifts in conformational equilibria that may noninterconverting solute. be induced by interaction with the nonpolar surfaces employed in hydrophobicinteraction and reversed-phase HPLC requires The large variations in bandwidth with temperature for more sophisticated approaches to the analysis of chromatobombesin were quantitatively characterized in terms of the graphic data than those described above. Since variations in maximal change in bandwidth (ACT,,) and the chromatopeak shapes are characteristic of secondary equilibria when graphic relaxation time (t112). The ACT,, value corresponds the rates of isomerization, conformational change, or aggreto the difference between the maximal experimental bandgation are comparable to the time scale of the chromatographic width and the value extrapolated on the assumption that a process, analysis of peak shape changes can be used to noninterconverting solute can be accurately described by small characterize these effects.22-25 Detailed mathematical treatmolecule band-broadening theory139n-25 (e.g., similar to pentament of the dynamic interconversion shown in Figure 1has L-phenylalanine as shown in Figure 3). The method used for been carried out by Horviith and c o - ~ o r k e r s . Analysis ~ ~ * ~ ~ ~ ~ ~the derivation of the corresponding value of til, a t which of the nonlinear dependencies of the observed peak second Anma was found is illustrated in Figure 4. The ACT" values moments, or comparison of experimentally obtained chroobtained a t each gradient time for bombesin chromatographed matograms with simulated peak profiles, allows16the respecon the C18 sorbent are displayed in Figure 5, with the tive apparent rate constants described in Figure 1 to be temperatures at which each ACT,, value was obtained calculated. indicated in brackets. Generally, the results indicated that larger Auma values were observed a t the longer gradient times The first-order kinetic approaches used by Hearn et al.15 as the temperature was decreased. More specifically, for and Cohen et al.5 or the more sophisticated theoretical gradient times of 60 and 75min maximal changes in bandwidth approaches of Horviith and co-~orkers*6~26~27 are limited if (ACT" = 0.05 and 0.07, respectively) were observed a t 55 "C. peaks representing the individual species considered by the A t a gradient time of 90 min the maximal change in bandwidth model cannot be discriminated in the chromatogram or if the (ACT" = 0.10) was observed a t 45 "C, while at gradient times system involves interconversion between a number of conof 120 and 150 min, the band-broadening maxima (ACT,, = formational states with relaxation times equivalent to the 0.15 and 0.30, respectively) were observed a t 25 "C. Finally, chromatographic time scale. In order to gain further insight at agradient time of 180min the maximal change in bandwidth into these more subtle kinetic situations involving the (ACT,, = 0.55) was observed at 15"C. As noted in the previous conformational interconversions of polypeptides in RPsection, such bandwidth behavior is representative of conHPLC, the dependencies of the experimental bandwidth upon formational interconversion. temperature and column residence time for three polypepAs the gradient time increased from 60 to 180 min, three tides, bombesin, glucagon, and @-endorphin,were consedistinct trends were apparent for bombesin chromatographed quently investigated. on the C18 sorbent. Interaction of Bombesin with the n-Octadecylsilica (i) As the gradient duration was increased (i.e., increased Sorbent. Bombesin is a tetradecapeptide first isolated from column residence time), larger increases in bandwidth were observed at a particular temperature. (21) Hearn, M. T. W.;Aguilar, M. I.;Nguyen, T.; Fridman, M. J. (ii) For a particular gradient time, significant changes in Chromtogr. 1988,435, 271-284. (22) Giddings, J. C. In Dynamics in Chromatography;Giddings, J. C., bandwidth occurred only over a discrete temperature range. Keller, R. A., Eds.; Marcel Dekker: New York, 1965; pp 12-91. (iii) Smaller gradient times were required for maximal band (23) Delisi, C.; Hethcote, H. W.; Brettler, J. W. J. Chromatogr. 1982, 240, 283-295. spreading to be achieved as the column temperature was (24) Giddings, J. C. J. Chromatogr. 1960,3,443-453. elevated. (25) Cysewski,P.; Jaulmes, A.; Lemque, R.; Sebille, B.; Vidal-Madjar, ~~

~

C.; JilgB, G. J. Chromatogr. 1991,548,61-80. (26) Henderson, D. E.; Horvhth, Cs. J. Chromatogr. 1986,368, 203213. (27) Jacobson, J.; Melander, W.;Vaisnys, G.; Horvhth, Cs. J. Phys. Chem. 1984,88,4536-4542.

(28) Anastasi, A.; Erspamer, V.; Bucci, M. Experientia 1971,27,166167. (29) Carver, J. A.; Collins, J. G. Eur. J. Biochem. 1990,187,615-650. (30) Erne, D.; Schwyzer, R. Biochemistry 1987,26,6316-6319.

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Table I. Physical Data for Solutes Studied

solute penta-L-phenylalanine bombesin @endorphin

glucagon 0

sequence

MW

HzN-FFFFF-OH H2N-XQRLGNQWAVGHLM-OH0 H2N-YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE-OH HzN-HSQGTFTSDYSKYLDSRRAQDFVQWLMNT-OH

1640 3470

760

3520

X = 6-oxoproline (pyroglutamic acid),

-.

2 0.4 5

h

2.

0.3

z (0

0.2 0.1

0.0

Figure 3. Dependence of experimental bandwidth on both gradient tlme and temperature for the control solute penta+phenylalanlne chromatographed on the C18 Stationary phase. For explanation of the plot see legend in Figure 2A. 0.6

r

0.4

-

0.1

-

h

VI C

.E

U

5

0

10

20

30

40

50

I

I

I

I

60

70

80

90

Temperature ("C)

Flgure 2. (A) Dependence of experimental bandwidth (mln) on the gradient time (mln) and column temperature ("C) for bombesln chromatographed on the C18 sorbent. The plot presents a threedimensional mesh constructed from an XY plane of gradient tlme vs column temperature extended intothe Zaxis by experimentalbandwidth. The XYplane reflectsthedependenceof experimentalbandwidthupon temperature for a range of gradient times. The YZplane represents the dependence of experimental bandwidth on gradient tlme for a giventemperatwe. (6)Relationshipbetweenthe Svalues (a parameter closely related to the Chromatographic contact area) and column temperature for bombesln chromatographed on the C18 n-alkylsllica support. Thls plot was reproduced from data in ref 2.

These results can be rationalized by considering a system whereby the conformational interconversion of the solute during column migration is characterized by a chromatographic relaxation time (t1p) for a given temperature. The t 1 p values for each temperature were determined as the retention time at which the Au- value was observed (as illustrated in Figure 4) and are tabulated in Table 11. If the chromatographic residence time (t,) is much less than t112 (i.e, trw> t&the bandwidthbehaviorwillagainreflect the interactive behavior of a single chromatographic species. In contrast, when the column residence time of the peptide is commensurate with the relaxation time for the conformational interconversion (i.e., t, = tip), the stationary phase will act as a topographic probe for the different conformational

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993 0.6

-

0.5

I

[l5*]

P

Y)

.-CE

0.4

v

x

2'

0.3

O2

I II

t

/

(5.)

150

100

50

0

200

Gradient Time (mins)

Relationship between AU- and gradient time for bombesin chromatographed on both the C18 and C4 rralkylsilica sorbents. Each data point represents a Aumx value derived for each gradient time at the temperature marked in brackets for the C18 sorbent and parentheses for the C4 sorbent. Figure 5.

Table 11. Chromatographic Relaxation Times (min) bombesin temp P-endorphin glucagon c4 C18 c4 c4 (OC) 5 15 25 37 45 55 65 75 85

66 70 60 40 39 32 29 86 >82 79 73 49 32

>97 >94 >92 >90 >85 >82 75 49 27

intermediates present as the peptide migrates along the column. Under these conditions a significant degree of band broadening will arise, since the molecular composition and binding kinetics of the interactive surface presented to the probing stationary-phase ligands will be different for each conformational intermediate of the solute. The presence of such conformational intermediates would either be associated with broadening of Gaussian peaks, as observed in this study, or the emergence of asymmetrical or multiple peaks, depending on the intrinsic efficiency of the column to resolve the different molecular species present during solute chromatographic migration. While changes in the hydrodynamic dimensions of the peptide associated with conformational interconversions would influence the diffusional properties of the peptide, the variation in the adsorption and/or desorption kinetics of the solute as the secondary structure (and hence the chromatographic contact area) varies would contribute more strongly to the observed band broadening. The work of Horvgth and colleagues with proline-containing dipeptides undergoing cis-trans isomerization26-27and the work of Gesquiere et al.31and Lebl et al.32 on the cis-trans isomerization of small proline- or N-methyltryptophancontaining peptides are consistent with this conclusion. Similarly, the band broadening observed2,6J5.3=6 with various (31) Gesquiere,J. C.; Diesis, E.; Cung, M. T.; Tartar, A. J . Chromatogr. 1989,478, 121-129. (32) Lebl, M.; Fang, S.; Hruby, V. J. J.Chromatogr. 1991,586,145-148. (33) Miller, N. T.; Karger, B. L. J . Chromatogr. 1985, 326, 45-61. Mutter, M. J . Chromatogr. (34) Steiner, V.; Schir, M.; Bornsen, K. 0.; 1991,586, 43-50.

enzymes and other proteins chromatographed on n-alkylsilicas and hydrophobic interaction chromatographic sorbentswould also be consistent with this interpretation. It would also be anticipated that the relaxation time for the interconversion should decrease as the column temperature is elevated. Thus, changes in band-broadening behavior should become apparent at progressively smaller gradient times as the column temperature was raised. In this investigation with bombesin this behavior is clearly evident from Figure 2A, where anomalous bandwidth behavior was observed at decreasing gradient times as the temperature rose from 15 to 65 "C (see Table I1 for t1/2 values). Similar band-broadening dependencies on temperature have been observed for the rates of unfolding of a-lactalbumin on hydrophobic interaction chromatographic supports by Karger and co-workers.36 The conformational changes which gave rise to these variations in the experimental bandwidths were also manifested as transitions in other retention parameters. For example, the S values (reproduced in Figure 2B) and log k, values of bombesin chromatographed on the C18 sorbent decreased' over the temperature range 5-37 "C, indicative of a change in the interactive topography from a structure with a high contact area to a structurewith a lower chromatographic contact area and lower affinity. In addition, changes in bandbroadening behavior also correlated with the change in thermodynamic parameters found when bombesin was chromatographed on the C18 sorbent. In particular, transitions in the ASo,,,, values1 were observed over the same temperature range (e.g., 5-37 "C) where significant changes in band broadening were evident. The changes in retention and thermodynamic parameters for bombesin chromatographed on the C18 sorbent were previously attributed' to the formation of a partial amphipathic a-helix. Hydropathy profiles and Edmundson wheel projections suggest that the C-terminal region of bombesin from Asn-6 to Met-14 can exist as a short hydrophobic helix.1° Moreover, circular dichroism (CD) investigations have i n d i ~ a t e d lthat ~ , ~bomb~ esin exists as an extended flexible chain in aqueous buffers but assumes a partially ordered structure in lipid suspension, e.g., in the presence of a 200-fold excess of lysolecithin. Other solution spectroscopic studies,1°as30notably Fourier transform infrared and two-dimensional nuclear magnetic resonance spectroscopy and time-resolved fluorescence measurements, are also consistent with the conformation of bombesin changing from a random coil structure in solely aqueous buffers to a partial helical structure in more hydrophobic lipid environments. The mechanism of the stabilization of bombesin by the C18 ligand may thus be similar to the process involving the induction of a C-terminal helix found with lysolecithin micelles and dimyristoylphosphatidylserine vesicles.'* Overall, these observations suggest that conformational changes occurring during the chromatographic process not only disrupted the interactive structure of the solute but also affected the adsorption-desorption kinetics over discrete temperature ranges. The fact that bombesin demonstrated a pronounced column residency effect suggests that the conformational changes involved the combined influence of the ligand in addition to the effect of hydrogen bond and other dipole-dipole interactions mediated by the bulk aquoorganic solvent.7J8 Recent studies of proteins adsorbed onto (35) Kunitani, M. G.; Cunico, R. L.; Staats, S.J. J . Chromatogr. 1988, 443, 205-220. (36) Oroszlan, P.; Blanco, R.; Lu, X.-M.; Yarmush, D.; Karger, B. L. J . Chromatogr. 1990, 500, 481-502. (37) Purcell,A. W.; Aguilar, M. I.;Hearn, M. T. W., unpublishedresults. (38)Wilce, M. C. J.; Aguilar, M. I.; Hearn, M. T. W. J . Chromatogr. 1991,548, 105-116.

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various reversed-phaseand hydrophobic interaction sorbents in the presence of aqueous-organicsolutionsand salt solutions using fluorescence,"8~3Dcircular dichroism spe~troscopy,~~41 and diffuse reflectance Fourier transform infrared' and lowangle laser light scatterind2 spectroscopy provide further evidence that solvent- and surface-induced changes in polypeptide or protein conformation can occur under these chromatographic conditions. In addition, this investigation has indicated that conformational interconversions can be monitored by manipulation of experimental conditions, such as gradient time or column temperature, and analysis of the derived kinetic parameters. The 3D-mesh surface presented in Figure 2A therefore represents a map of chromatographic conditions under which conformationaltransitions, as evident from band-broadening behavior, can occur on the chromatographic time scale. Several alternative explanations for the observed changes in band-broadening behavior can be largely excluded. Selfassociation of bombesin is unlikely under the reversed-phase chromatographic conditions and the peptide loading concentration employed. For example, bombesin is known to be monomeric at concentrations below 300 pMIO in aqueous solutions. It is also possible that the discrete changes in experimental bandwidth might be due to a phase transition in the stationary-phase ligands. This possibility was previously discounted1 on the basis that no such transition was observed for the control solutes (e.g., penta-L-phenylalanine). In addition, as discussed below, different patterns of retention and bandwidth behavior were observed for the three different peptides investigated, which again suggests that the bandbroadening transitions observed with the same sorbents primarily reflected differencesin the solute structure. Finally, although electrostatic and other polar effects are also known2 to occur under some conditions in RP-HPLC, experimental data obtained with peptides of identical composition yet different sequence or peptides involving D-amino acid rep l a c e m e n t s ~suggest ~ ~ that these effects do not significantly contribute to the observed changes in band-broadening behavior with bombesin under the conditions employed in this study. Interaction of Bombesin with the n-Butylsilica Sorbent. Figure 6A shows the dependence of bandwidth on temperature for bombesin chromatographed on the C4sorbent for gradient times varying between 15 and 180 min. For gradient times between 15 and 45 min and above 90 min, no significant change in bandwidth was detected over the temperature range investigated. With a gradient time of 60 min, however, a maximal change in band broadening (Aumu = 0.09) was observed a t 30 "C, while for gradient times of 75 and 90min, significant increases in bandwidth were observed between 5 and 25 "C. These findings with the C4 ligands can be contrasted with the results obtained with the C18 ligands, where much larger changes in bandwidth were observed over the same temperature range but a t longer gradient times (Le., 75-180 min). However, the trend with the t1p values decreasing with increasing temperature (see Table 11) was also observed for bombesin with the C4 sorbent. The differences in the band broadening observed on the C18 and C4 sorbents can be directly compared in Figure 5. (39)saavedra,5.S.;Worth-Grubin,A.;Lochtiller, C. H. Anal. Chem. 1988,60,2166-2168. (40)Heinitz, M.L.; Flanigan, E.; Orlowski, R. C.; Regnier, F. E. J. Chromatogr. 1988,443,229-246. (41)Zhou, N.E.;Mant,C. T.; Hodges, R. 5.Pept. Res. 1990,3,8-20. (42)Griubem, N.;Blanco, R.; Yarmueh, D. M.;Karger, B. L. Anal. Chem. 1989,Sl;514-1520. (43)Hanson, M.;Unger, K. K.; Mant, C. T.; Hodges, R. S. J. Chromtogr. 1992,599,77-86. (44)Aauilar, M.I.; Mowoe, 5.;Boublik, J.: Rivier, J.; Hearn, M.T. W. J. ChroGtogr., in press. -

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For the C4 sorbent, changes in band broadening were evident at gradient times between 60 and 90 min, while for the C18 sorbent, these changes were observed at gradient times greater than 90 min. This behavior reflects a significantlyfaster rate of interconversionon the C4 sorbent relative to that observed on the C18 sorbent and suggests that the longer n-alkyl chains conferred a significant degree of conformationalstabilization on the bombesin molecule. Previous studies on the retention behavior of bombesin chromatographed on the C18 and C4 sorbents have demonstrated' significantlydifferent temperature dependencies for the parameters S and log k,. With the C4 ligand, a transition in S values occurs around 50 "C, corresponding to a change from an initially low contact area structure to a more extended structure (see Figure 6B). Similarly,transitions in the values of ASo,, and AHo,, were also observed near to this temperature. Thus, the conformational changes which gave rise to the larger S values at ca. 50 "C with the C4 sorbent appear to be associated with rapid kinetics as there were no apparent changes in band broadening a t this temperature. Conversely, the structural changes which result in large bandwidth changes at the lower temperatures (i.e., 5-30 OC) correspond to only very small changes in the interactive contact area between bombesin and the C4 ligands. Overall, the results suggest that bombesin adopts a significant degree

3044

ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993

of secondary structure in the presence of both n-alkyl ligands, with the relative stability of the peptide structure being different with each sorbent. Comparison of the van't HofP and bandwidth data for bombesin with the C4 and C18 sorbents indicates a shift toward greater stabilization of the bombesin secondary structure with the C18 ligand compared to the C4 ligand under water-acetonitrile elution conditions. This stabilization was particularly reflected in the present study as a slower rate of interconversion (i.e., larger tip value) during chromatographic migration with the C18 sorbent. Interaction of 8-Endorphinwith the n-Octadecylsilica and n-Butylsilica Sorbents. @-Endorphinis an endogenous neuropeptide that binds to opiate receptors and is involved in the intrinsic regulation of pain perception. The human form of the 31 amino acid residue peptide has the sequence depicted in Table I. The C-terminal 19 amino acid segment of @-endorphinhas a high propensity to form an amphipathic helix in hydrophobic environments and at hydrophobic surfaces.4536 The effect of n-alkyl ligands on the bandbroadening behavior of @-endorphin was consequently investigated at different temperatures and gradient times with both C18 and C4 sorbents. The dependence of bandwidth on temperature observed for @-endorphinchromatographed on the C18 sorbent is displayed in Figure 7A. The bandwidth data for @-endorphinexhibited a relatively planar temperature dependence for all the gradient times employed in this study. These data correlate well with the previously reported temperature dependencies of the S (shown in Figure 7B) and log k , values observed1 for @-endorphin under the same chromatographic conditions. Both of these retention-derived parameters exhibited relatively constant values over the entire temperature range. However, changes in the value of AS",,, for the interaction of @-endorphin with the C18 sorbent indicated a transition occurred around 50 "C.I The absence of significant changes in band broadening in Figure 7A suggests that the previously observed change in AS",,,, a t this temperature may be associated with a rapid transition between the structure which exists at low temperatures and the high-temperaturestructure. Since @-endorphinhas been shown6 by CD studies to exist as an extended random coil structure in aqueous buffers but assumes a defined helical secondary structure in trifluoroethanol and methanol solutions and in the presence of lipids such as dodecyl sulfate, the chromatographic behavior below 50 "C may be a consequence of the induction of this amphipathic a-helical structure of the peptide6 by the nonpolar ligand. It has previously been demonstrated45 that similar band-broadening behavior occurs for other peptides related to the human @-endorphinmolecule. @-Endorphinpeptides with intact C-terminal sequences, i.e., @-endorphin,13,31 displayed a greater propensity for conditiondependent (hydrophobic ligand density, gradient time, and flow rate) changes in band-broadening behavior than smaller peptides encompassing only the N-terminal sequences, e.g., @-endorphin,1-16again reflecting the ability of the n-alkyl ligand to stabilize the C-terminal amphipathic helical region of @endorphin. The temperature dependence of the bandwidths for @-endorphin chromatographed on the C4 sorbent for each gradient length investigated is depicted in Figure 8A. For gradient times exceeding 60 min, nonlinear dependencies of bandwidth on temperature were apparent. At a gradient time of 75 min, the maximal change in bandwidths (Aum, = 0.031) was observed a t 85 "C. Bandwidth maxima were also observed a t 75 OC for gradient times of 90 and 120 min (Aumm= 0.021 and 0.250, respectively) and at 65 "C for gradient lengths of 150 and 180 min (Aum= = 0.050 and 0.145, respectively). The (45) Hearn, M. T. W.; Aguilar, M. I. J.Chromatogr. 1986,352,3546. (46) Taylor, J. W.; Kaiser, E. T. Pharmacol. Reu. 1986,38 (4), 291-319.

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corresponding t l / 2 values for @-endorphin,which are listed in Table 11, decreased with increasing temperature. The differences in band-broadening behavior for @-endorphin chromatographed on the C18 and C4 sorbents in terms of the relationship between Aum= and gradient time are illustrated in Figure 9. While Aum= values did not alter significantly with increased gradient length in the investigations with the C18 sorbent, significant increases in ACT,values were observed for gradient times between 75 to 150 min when @-endorphin was chromatographed on the C4 sorbent. The largest Au,, value was observed a t a gradient time of 120 min a t 75 "C on the C4 sorbent. A transition in the retention (see Figure 8B)and thermodynamic parameters also occurred around 60 "C for @-endorphin on the C4 sorbent,' which correlated with the observed changes in band broadening. The retention data indicated that significant decreases in both Sand log k, values occurred above 60 "C. The thermodynamic parameter, AS"-, indicated a decrease in the order of the solution structure at this temperature. Collectively, these data suggest that a conformational change involving the thermal denaturation of @-endorphinoccurred at approximately 60 "C with the C4 sorbent. Interaction of Glucagon with the n-Octadecylsilica and n-Butylsilica Sorbents. Glucagon is a 29 amino acid

ANALYTICAL CHEMISTRY, VOL. 65, NO. 21, NOVEMBER 1, 1993

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polypeptide hormone secreted by the a-cells of the pancreas in response to low blood sugar levels.4' Glucagon has a molecular weight of approximately 3500, and the amino acid sequence for the mammalian form is listed in Table I. Like bombesin and &endorphin, glucagon has been reported to largely exist as a random structure in solution, but assumes some helical content upon binding to lipids.12*aThe ability of glucagon to adopt a stable eecondary structure in a hydrophobic environment was further investigated through the analysis of the dependence of bandwidth on temperature and column residence time with both the C18 and C4 sorbents. The temperature dependence of bandwidth for each gradient length investigated is depicted in Figure 10A. A planar dependence of bandwidth on temperature was generally observed for all the gradient times investigated, which represented column residence times between 10 and 110min. These small changes in bandwidths were consistent with the previously observed' temperature dependence of the chromatographic contact area (S values) and affinities (log k, values) for glucagon with the C18 sorbent. The S (see Figure 10B) and log k, values remained relatively constant over the (47) Patzelt, C.; Tager, H. S.;Caroll, R.J.; Stainer,D. F. Nature 1979, 282,260-266. (48)Braun, W.; Wider, G.;Lee,K. H.; Wtithrich, K.J. MoZ.BioZ. 1985, 169,921-948.

temperature range investigated. However, the associated thermodynamicparameters' indicated that a transition from a rigid to a more flexible structure occurs around 50 "C during interaction of glucagon with the C18 ligands. Since no significant change in band broadening was observed with the C18 sorbent, this transition would have to be very rapid compared to the time scale of the chromatographic process (Le.,