Anal. Chem. 1994,66, 3908-3915
Conformational Changes in the Reversed Phase Liquid Chromatography of Recombinant Human Growth Hormone as a Function of Organic Solvent: The Molten Globule State S. Wicar, M. G. Mulkerrin,? G. Bathory, L. H. Khundkar, and B. L. Karger' Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02 1 15
As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Ckm. 1992,64 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the cY-helicityand possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two a-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of >30% (v/v) 1-PrOH. In the case of CHJCN, no significant conformational changes are observed up to 40% (v/v) and at temperatures up to 40 "C. This study provides insight into the mechanism of reversed phase chromatographic retention of rhGH and the general role of organic solvents on protein structure. In a previous paper,' the reversed phase liquid chromatographic (RPLC) behavior of recombinant human growth hormone (rhGH) and its closely related N-methionyl variant (Met-hGH) was studied. It was shown that conformational changes in the proteins, resulting from addition of organic modifiers [ 1-propanol ( 1-PrOH) and acetonitrile] to aqueous buffer solutions and/or by a hydrophobic adsorbent surface, significantly influenced the retention behavior. Indeed, it was further suggested that a conformational change was a necessary Dept. of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080. (1) Oroszlan, P.; Wicar, S.; Teshima, G.; Wu, S . L.; Hancwk, W. S.;Karger, B. L.Anal. Cfiem. 1992,64, 1623-1631.
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Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
Flgure 1. Illustration of the helical structure of human growth h ~ r m o n e . ~
ingredient to create a hydrophobic difference in the two closely related species, leading to separation. Both in solution and on the adsorbent surface, changes in protein structure were studied by evaluating shifts in the intrinsic fluorescence emission spectra of the single tryptophan (Trp) found in these proteins. The Trp fluorescence emission maximum red shift for rhGH and Met-hGH increased in proportion to the CH3CN concentration. However, in solutions containing 1-PrOH, the red shift of Trp emission for both proteins varied sigmoidally within a limited 1-PrOH concentration range (1 525% v/v). Moreover, at higher protein concentrations (>0.3 mg/mL) and above room temperature, the spectral shifts were accompanied upon standing with protein precipitation. Both the sigmoidal red shifts of the fluorescence emission and the fact of protein precipitation at 1-PrOH concentrations near the inflection point of the fluorescence emission curve suggested a conformational change of rhGH under the influence of significant concentrations of 1-PrOH. The results of static fluorescence spectroscopy, however, were unable to describe the specific changes in native structure in any detail. Indeed, from earlier studies on the influence of aliphatic alcohols2 and more recently triflu~roethanol~ on protein structure, it was likely that the fully denatured state did not result, and some conservation of a-helicity of rhGH in the new conformation remained. Structurally, hGH is a relatively stable protein, with a four helix bundle in an unusual antiparallel arrangement," see Figure 1. Along with the four a-helices there are three additional short a-helical segments in the connecting loops. As proposed from results of UV absorption spectroscopy5 and (2) Tanford, C. Adu. Prorein Cfiem. 1968,23, 12 1.
0003-2700/94/0366-3908804.5010
0 1994 American Chemical Society
confirmed by recent X-ray structure a n a l y ~ i s Asp , ~ 169 in helix IV is hydrogen bonded to the single Trp 86 in helix 11. In the folded protein, this hydrogen-bonded Trp is buried within the hydrophobic core of the protein. Related studies of the unfolding of bovine growth hormone (bGH) by guanidine hydrochloride (Gdn HC1) demonstrated the presence of a stable intermediate.G8 This intermediate has recently been suggested to be the molten globule state for bGH.9 The molten globule state of bGH was characterized as (a) largely a-helical; (b) retaining a compact hydrodynamic radius; (c) having the aromatic side chains randomly oriented, yielding a near-UV CD spectrum similar to that of the unfolded state; and (d) possessing a solvent-exposed hydrophobic surface, leading to protein association. On the other hand, unfolding experiments on human growth hormone utilizing Gdn HCl were originally reported to be a two-state process.lOJ1 Interestingly, very recently the formation of a folding intermediate in the course of Gdn HCl denaturation of hGH at higher protein concentrations was observed,12as found for bGH, suggesting similar behavior for hGH. The goal of this paper is to provide a more detailed spectroscopic characterization of the changes in rhGH structure resulting from the addition of 1-PrOH and CH3CN. Not only would such a study permit a deeper understanding of the retention behavior of rhGH in RPLC, but it would also provide insight into the behavior of relatively structured proteins in reversed phase chromatography. Farand near-UV CD spectra, second-derivative UV absorption spectra, dynamic fluorescence anisotropy, fluorescence quenching, and frequency-domain lifetime measurements have been utilized to study the changes of rhGH as a function of concentration of 1-PrOH and CH3CN at two different temperatures, 20 and 45 OC. For organic solvent concentrations of >30% (v/v) 1-PrOH at both temperatures, a molten globule state is proposed to occur. The molten globule state has been viewed as an intermediate in the unfolding pathway of a globular protein,13 consisting of a secondary structure close to that of the native state and a somewhat unfolded tertiary structure. Because of the unfolded tertiary structure, the molten globule can provide additional hydrophobic surfaces for interaction in RPLC, while at the same time being fully reversible to the native state when the stress (in this case 1-PrOH) is removed. On the other hand, in the case of the examined concentration range of CH3CN, the spectral shifts are interpreted in terms of simple solvation effects and not a conformational change. (3) Nelson, J. W.; Kallenbach, N. R. Proteins: Struct.. Funct., Genet. 1986, I , 211-217. (4) deVos, A. M.; Ultsch, M.; Kossiakoff, A. A. Science 1992, 255, 306-312. (5) Bewley, T. A.; Hao-Li, C. Biochem. Biophys. 1984, 233, 227. (6) Havel, H. A.; Kauffman, E. W.; Plaisted, S. M.; Brems, D. N. Biochemistry 1986, 25.65334538. (7) Brems, D. N.; Plaisted, S. M.; Kauffman, E. W.; Havel, H. A. Biochemistry 1986, 25, 6539-6543. (8) Brems, D. N.; Plaisted, S. M.; Dougherty, J. J., Jr.; Holzman, T. F. J . Biol. Chem. 1987, 262, 2590-2596. (9) Brems, D. N.; Havel, H. A. Proteins; Struct.. Funct., Genet. 1989,5,93-94. (10) Brems, D. N.; Plaisted, S. M.; Havel, H. A,; Kauffman, E. W.; Stodola, J. D.; Eaton, L. C.; White, R. D. Biochemistry 1985, 24, 7662-7668. (11) Brems, D. N.; Plaisted, S. M.; Havel, H. A,; Tomich, C. C. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 3367-3371. (12) DeFelippis, M. R.; Alter, L. A.; Pekar, A. H.; Havel, H. A,; Brems, D. N. Biochemistry 1993, 32, 1555-1562. (13) (a) Ptitsyn, 0. B. J. Protein Chem. 1987, 6, 272-293. (b) Kuwajima, K. Proteins: Struct., Funct., Genet. 1989, 6, 87-103.
This study provides useful information into potential structural changes of proteins in RPLC as a function of organic solvent and column temperature and thus insight into the mechanism of retention and separation in RPLC. Furthermore, the study should aid in protein purification processes using recombinant technology where mixed aqueous+xganic solvent mixtures are often employed.
EXPERIMENTAL SECTION Instrumentation. Circular dichroism (CD) spectra were measured on an Aviv 62 DS spectropolarimeter (Aviv and Associates, Lakewood, NJ). Near-UV CD spectra were measured over the wavelength range 250-350 nm at 0.5 nm intervals. The far-UV CD spectra were obtained in the wavelength range of 190-250 nm at 0.2 nm intervals. Each spectrum was the result of the sum of five scans, with a 2 s integration time for each data point. The temperature was maintained constant in a thermostatically controlled cylindrical quartz cell (Hellma Cells, Jamaica, NY). Absorption spectra were collected from 250 to 350 nm at intervals of 0.1 nm with a 0.1 nm bandpass on an Aviv 14 DS absorption spectrometer. Each spectrum was the sum of 10 scans with a 5 s integration time for each data point. The excitation source for Trp polarization measurements was a cavity-dumped dye laser R6G 702-2 (Coherent, Palo Alto, CA) synchronously pumped by the second harmonic output of a mode-locked Nd-YAG Antares laser (Coherent). The output of the dye laser at 600 nm was frequency doubled with a 10 mm LiIO3 crystal, and the residual visible light was filtered out by a Corning 7-54 filter (Corning, NY). Fluorescence was collected with f / l optics, dispersed with a 0.25 m Heath monochromator and detected with a red-enhanced microchannel-plate photomultiplier R2809U (Hamamatsu, Middlesex, NJ). Fluorescence decay was recorded by timecorrelated single-photon counting, with constant fraction discriminators, a time-to-amplitude converter, and multichannel analyzer cards TC 455, TC 934, PCIIA (Tennelec/ Nucleus, Oak Ridge, TN) installed in a 16 MHz IBMcompatible PC. Polarized fluorescence (both parallel and perpendicular) was selected with a dichroic sheet polarizer (Oriel, Stratford, CT). Typical instrumental response functions for these measurements were approximately 100 ps full width at half-height. The steady-state fluorescence quenching data as well as the frequency domain fluorescence decay data were obtained on an SLM 48000 S spectrofluorometer (SLM Aminco, Urbana, IL). The quenching results were collected in the spectral data acquisition mode. When trichlorethanol was used as quenching agent, the data were corrected for the background fluorescence of solution. The excitation wavelength was 295 nm with a bandpass width of 1 nm. Fluorescence emission was collected at 350 nm with a bandpass of 4 nm. All data were further corrected for the inner filter effect according to ref 14. The frequency-domain decay data were recorded in the dynamic acquisition mode. A mercury-xenon arc lamp (UXM-501MA, Ushio Inc., Japan) served as the light source. Samples were excited at 300 nm with a bandpass of 2 nm, and
-
(14) Lakowicz, J. R. In Principles of Fluorescence Specfroscopy;Plenum Press: New York, 1983.
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
3909
the emission was collected using a squirrel cage R928 photomultiplier tube (Hamamatsu) with a WG 320 nm long pass filter (Schott Glass Technologies Inc., Duryea, PA). To eliminate the effect of protein molecular rotational diffusion on the intensity decay kinetics, Glen-Thompson calcite prism polarizers were utilized in the magic angle 0rientati0n.l~The cell temperatures were maintained at 20 and 45 OC by a Polyscience thermostatic bath (Preston Industries, Inc., Niles, IL) and a Neslab flow-through cooler (Neslab Instruments, Inc., Newington, NH). Both the quenching and the decay data of the proteins were measured in a 1 cm optical path spectroscopic quartz cuvette. Scattered light from a Ludox suspension (Du Pont Co., Wilmington, DE) served as a reference solution. Chemicals. rhGH samples were obtained in vials containing 5 mg of protein and -50 mg of excipients, mainly mannitol. Fluorescence measurements were performed on rhGH preparations that were buffer-exchanged by gel permeation chromatography using PD- 10 columns packed with Sephadex G-25 M (Pharmacia LKB, Piscataway, NJ). CD and UV absorption spectra were obtained from protein samples exchanged with 50 mM phosphate buffer (pH 6.5) using a Centricon 10 microconcentrator (Amicon, Beverly, MA). HPLC grade water was obtained from J. T. Baker, Inc. (Phillipsburg, NJ), and 1-PrOH and acetonitrile were purchased from Burdick & Jackson (Muskegon, MI). Trichlorethanol (TCE) was obtained from Sigma Chemicals Co. (St. Louis, MO). Acetonitrile, water, and 1-propanol were essentially free from fluorescence trace impurities and showed very low UV absorbance (below 0.005 AU) in the range of 295-400 nm. Disodium hydrogen phosphate, sodium dihydrogen phosphate, and potassium iodide were analytical grade.
RESULTS AND DISCUSSION Circular Dichroism Spectra. Significant information regarding the structure of a protein in solution is provided by the far-UV region (190-250 nm) of the circular dichroism spectrum where absorption is mainly due to peptide bonds and is largely determined by the spatial distribution of these bonds, Le., by the conformation of the polypeptide backbone. Consequently, the far-UV spectra reflect the secondary structural content of the protein. Since rhGH in aqueous solution is -55% a-helical? this portion of the CD spectrum is thus dominated by the contributions of the a-helical elements. To quantify the effect of the addition of organic solvents on the peptide far-UV CD spectrum, the mean residue weight ellipticity [e]MRW at 208 nm was chosen: B,,MRW 9 [= 1 ,,,
(deg cmz dmol-')
(1)
where cobs is the observed ellipticity in degrees, MRW is the mean residue weight (for rhGH, 112), 1 is the path length (in cm), and C is protein concentration (in mg/mL). Samples of rhGH containing -0.2 mg/mL in 50 mM phosphate buffer (pH 6.5), or in a buffer-organic solvent mixture, were placed in a thermostated cell of the spectropolarimeter. The precise 3910
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
24000
E,
1
22000
m
0
c? L
0
E
73
NE --
=z
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20000
p
18000
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16000
-I I
\ I
I
I
I
1
10
20
30
40
50
temperature ['c]
Figure 2. Mean residue weight ellipticity ([e],,]) of rhGH in 50 mM phosphate buffer, pH 6.5, at 208 nm as a function of temperature and 1-PrOH concentratlon: (m) 10, (0)20, (0)and 40% (v/v) 1-PrOH.
sample concentrations were determined from absorption spectra using the known extinction coefficient at 277 nm = 0.82 cm-I). A . Fur-UV CD Spectra. Figure 2 illustrates the rhGH dependence at 208 nm of [e]MRW on temperature for 10,20, and 40% (v/v) 1-PrOH. It should first be noted that no change in [O]MRWwas observed for rhGH in aqueous solution over the temperature range examined (not shown). Furthermore, at 10 OC, [e]MRW is found to be practically independent of 1-PrOH concentration. The stability of the native protein at low temperature evidently is sufficient to prevent the organic solvent from influencing the protein structure in the studied composition range. With increasing temperature, in solutions containing 10% and 40% (v/v) 1-PrOH, the [e]MRW values decrease monotonically, and in analogy to the static fluorescence emission shifts,' the more pronounced decrease is observed at the highest 1-PrOH concentration. Interestingly, however, at 20% (v/v) 1-PrOH, the [e]MRW values first increase, pass through a maximum at -25 "C, and finally decreaseclose to the value for 10% 1-PrOH at 45 OC. Finally, the maximum decrease of [e]MRW is observed at 45 OC and 40% (v/v) 1-PrOH; however, the absolute value of [e]MRW still remained quite large (16 000 deg cm2 dmol-I). The observed decrease in [e]MRW (10-20%) corresponds to only a 10% loss in a-helicity; i.e., the secondary structure remains largely intact. Therefore, the new conformation above 30% (v/v) observed in ref 1 cannot be a fully denatured protein. It is interesting to note that under the intermediate solvent conditions of 20% (v/v) 1-PrOH there is a small increase in a-helical structure as the temperature is raised from 10 to 25 OC. Either there is a rearrangement in the four-helix bundleL5 or more residues are involved in a-helical structures resulting in an increase in the intensity of the CD signal. Any rearrangement in the four-helix bundle could be assumed to arise from a decrease in the free energy of the folded protein, possibly in response to a change in the dielectric constant of the solvent or from a change in the solvation of specific residues in the protein. On the other hand, it is possible that additional residues are involved in an a-helical conformation. From the
-
(15) Cooper, T.M.; Woody,
R.W . Biopolymers 1990, 30, 657-676.
50
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8
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' 250
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260
270
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290
300
310
320
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Wavelength (nm)
I
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270
280
290
300
310
320
Wavelength (nm)
Flgure 3. Near-UV CD spectrum of rhGH in increasing 1-propanol at 25 OC.Shown is the CD spectrum of hGH in the absence of 1-propanol (-) and with lo(- -), 20(- -), 30(- -), and 40% 1-propanol (.e.).
limited data, it is not possible to decide which of the above two explanations is the correct one in this case. In contrast to 1-PrOH, the results obtained for rhGH in aqueous CH3CN solutions do not display any significant loss of [6]MRW at 10,20, and 30% (v/v) CH3CN for temperatures from 10 to 35 "C (data not shown). Only at 45 "C and 30% (v/v) CH3CN is [ ~ ] M Rreduced W (-15%), and this loss in a-helicity may signal the beginning of a change in rhGH conformation, as suggested in our earlier paper.' B. Near-UV CD Spectra. The signal in the near-UV portion of the CD spectrum (250-320 nm) reflects the asymmetry of the environment of the aromatic residues (tryptophan, tyrosine, phenylalanine) and thus provides information on the packing of the side chains and their exposure to the solvent. Consequently, the near-UV CD spectrum is related to the tertiary structure of the protein. The spectra of rhGH in increasing concentrations of 1-PrOH at 25 OC are shown in Figure 3. Since there are 8 tyrosines, 13phenylalanines, and 1 tryptophan, it is not possible to assign the observed dichroism to any specific aromatic residue, except for the single Trp. Nevertheless, for Tyr and Phe residues, overall trends can be observed. The region of the spectrum from 270 to 280 nm is primarily a result of the contribution of tyrosine residues to the near-UV CD spectrum. While there is some negative dichroism, expected from the tyrosine residues in this region, there is little spectral fine structure. The three negative peaks at 255-256, 261-262, and 268-269 nm have been assigned to ~henylalanine.~ These negative peaks appear over the whole 1-PrOH concentration range studied, suggesting that at least some portion of the tertiary structure of rhGH remains after the conformational change has occurred. This conclusion agrees with the previous results of the far-UV CD studies. Turning next to the peakat 294 nm,which has been assigned to the IL,:lLb transitions of the single tryptophan residue in the m o l e c ~ l eit, ~can be seen that there is a decrease in signal with increasing 1-PrOH concentration, such that the tryptophan signal is eliminated and only the effects of the negative peaks of tyrosine are observed. This 1-PrOH concentration range is identical to that found for change in the fluorescence properties of the Trp residue at 25 OC1 (see also Figure 7).
Flgure 4. Near-UV CD spectrum of rhGH in increasing acetonitrile at 25 OC.Shown is the CD spectrum of hGH in the absence of acetonitrile (-) and with lo(- -), 20 (- -), 30 (- -), and 40% acetonitrile (-).
In order for the Trp CD spectrum to be so perturbed, the internal packing of rhGH must have been disrupted, because the Trp residue is buried within the four-helix bundlee4 Interestingly, this significant disruption of local structure appears to be temperature dependent, as also found in the fluorescence studies. At higher temperature, the significant change in [e]MRW at 294 nm appears closer to 20% (v/v) 1-PrOH, whereas at subambient temperatures, the change appears at somewhat higher 1-PrOH concentrations (results not shown). In contrast to 1-PrOH, there is little disruption of the nearUV CD spectra of rhGH with increasing CH3CN at 25 OC (Figure 4) independent of the temperature studied. It is clear that in no case from 10 to 40% organic modifier is the CH3C N as disruptive to the internal structure of rhGH as 1-PrOH. Second-Derivative UV Absorption Spectra. As shown in the studies of hGH (Figure l), the relative positions of two helices of the four-helix bundle (helices I1 and IV) are not independent. These helices are constrained by a hydrogen bond between Trp 86 and Asp 169.4 As long as the hydrogen bond is conserved, the distance between the respective helices is relatively fixed. According to ref 16, the hydrogen bond manifests itself in the second-derivative absorption spectrum as a red shift (- 12 nm) of the Trp 'La [O-O] band. This peak, first observed in ref 5, can thus serve as a sensitive indicator of the mutual displacement of both helices in the hGH fourhelix b ~ n d 1 e . l ~In order to follow the possible structural changes in the altered conformational state resulting from addition of 1-PrOH to an aqueous buffer solution of the protein, we focused our attention on the 290-310 nm portion of the Trp UV absorption spectrum. Calculation of the second derivative from the absorption spectrum of rhGH in aqueous solution revealed the position of the 'La band to be at 303 nm. As expected, addition of CH3CN up to 40% (v/v) caused essentially no change in this band, indicating that the H-bond remains intact. This fact is in accordance with the conclusions of the static fluorescence' (16) Strickland, E. H.; Billups, C.; Kay, E. Biochemistry 1972, 11. 3657. (17) Mulkerrin, M. G.; Cunningham, B. C. In Protein Folding: In Vivo and In Vitro; Cleland, J. L., Ed.; ACS Symposium Series 526; American Chemical Society: Washington, DC, 1993; pp 240-253.
Analytical Chemistry. Vol. 66, No. 22, November 15, 1994
3911
and the far- and near-UV CD measurements, Le., no significant red shift in the emission first moment. Similarly, little if any blue shift in this band is observed for 1-PrOH until high 1-PrOH concentrations. Surprisingly, even in a solution of 40% PrOH, the Trp ' L a band is shifted to the blue by only 4 nm, to 299 nm. This limited blue shift may indicate that the hydrogen bond distance and/or angle has been altered, although the 4 nm shift suggests that the indole is undoubtedly still hydrogen bonded. Therefore, helices I1 and IV are likely still within hydrogen-bonding distance of each other. It is unusual that the dichroic effect on a chromophore would be eliminated, as suggested from Figure 4, when the chromophore remains held in a hydrogen bond. This effect has, however, been observed previously for growth hormone. In an analysisof the role of site-directed mutants on the activity and structure of human growth h ~ r m o n e , ~ Ja *mutation, isoleucine (58) to alanine (I58A), was found to eliminate the dichroism of the tryptophan. This was also true of the sitedirected mutant I58V (valine substitution for i~oleucine).'~ The dichroism of Trp 86 is thus sensitive to local structure, and it appears that it is easily eliminated. In summary, the data from circular dichroism and from second-derivative absorption spectroscopy indicate that the rhGH molecule is largely intact, but there appears to have been an alteration in the packing of the interior of the four-helix bundle. Polarization of Trytophan Fluorescence. To examine further the properties of the new state of rhGH formed under the influence of 1-PrOH composition, we explored the overall size changes that occurred with the conformational change. Measurements of Trp dynamic fluorescence polarization allow determination of the rotational relaxation time, T , of Trp movement. For spherical globular proteins, a simple relationship exists between the relaxation time, T , and the mean radius of the protein, R
where k is the Boltzmann constant, T is the absolute temperature, and 71 is the viscosity of the solution.20 In this work, the mean rotational relaxation times were obtained from the classical Wahl method20.21for rhGH in phosphate buffer (pH 6.5) and in the buffer-1-PrOH mixture. The data obtained for rhGH at 45 "C revealed that the mean relaxation time increased from 5 ns in aqueous solution to 13 ns after the completion of the conformational change at 45% (v/v) 1-PrOH and at 45 OC. The viscosities of the modified buffer solutions, determined by falling ball type viscometer, increased simultaneously with the relaxation times (from 0.63 CP for aqueous buffer to 1.43 CP for buffer containing 45% 1-PrOH). The hGH molecule is known to be slightly a~ymmetric;~ however, assuming a spherical shape, eq 2 permits the estimation of the mean radius of the protein molecule. In aqueous buffer solution, the radius of rhGH, was calculated to be 20 A; in solutions modified by the addition of 1 5 4 5 % (v/v) 1-PrOH, the protein radius increased to 23
-
(18) Cunningham, B. C.; Wells, J. A. Science 1989, 244, 1081-1085. (19) Mulkerrin, M. G., unpublished results. (20) Wahl, P.; Lami, H. Biochim. Biophys. Acto 1967, 133, 233-242. (21) Wahl, P.; Timasheff, S.N. Biochemistry 1969, 8, 2945-2949.
3912
Analytical Chemistty, Vol. 66, No. 22, November 15, 1994
A.
As in the case of bGH, where an increase in radius from 18 A in the native to 26 A in the molten globule state was ~ b s e r v e d ,the ~ hGH molecule in the new conformation is slightly expanded (by 10%)but significantly retains much of its compact shape. It should be noted that anisotropy decays constructed from the measured polarized fluorescence decays showed evidence for two exponential constants, as expected for a single tryptophan-containing protein.22 With no organic modifier, a single-exponential function adequately fit the data, but the resulting initial value of the anisotropy was quite small. With addition of increasing amounts of 1-PrOH, an additional faster component was observed. However, the error associated with the extraction and fitting procedure was comparable to the changes weobtained on adding theorganicmodifier. (A more sophisticated analysis of the data using global fitting methods could lead to extraction of the two rotational time constants, but this was beyond the scope of this work.) We therefore performed the analysis with the classical method of Wahl.20,2* Fluorescence Quenching. Quenching of the intrinsic fluorescence of proteins by extrinsic quenching agents is controlled by the ability of the reagent to be within the van der Waals contact region of the fluorophore. Since the accessibility of the fluorophore to a quenching reagent is restricted by chemical and steric e x c l ~ s i o n , *quenching ~.~~~~~ can be used as an indirect means to study protein structure alteration. The chemical and steric barriers preventing the quencher from penetrating into the protein structure are specific. This fact leads to the possibility of studying the fluorophore environment with probes of different hydrophobic and polar properties. In the present work, TCE, up to a concentration of 0.05 M, was used as a nonpolar and potassium iodide, up to 0.3 M, as an ionic quenching probe. Fluorescence quenching measurements have been performed for rhGH in 50 mM phosphate buffer, pH 6.5, at 0, 15, and 45% (v/v) 1-PrOH or CH3CN concentrations and at 20 and 45 OC. Quenching results were evaluated according to the Stern-Volmer equation for dynamic quenching14
-
where FOand F a r e the corrected emission intensities in the absence and in the presence of a quencher, KSVis the SternVolmer quenching constant, [Q]is thequencher concentration, k, is the bimolecular quenching rate constant, and TO is the fluorescence lifetime in the absence of the quencher, discussed in the following section on fluorescence intensity decay. Figure 5 summarizes the results of TCE quenching in the Stern-Volmer coordinate system. In aqueous solution and at 15% (v/v) CHjCN, the plots of fluorescence quenching of rhGH by TCE in phosphate buffer (pH 6.5) at 20 OC resulted in straight lines with correlation coefficients of 0.992 and 0.995, respectively. The slopes KSVand k, are summarized in Table 1. It is interesting to note that the slopes, KSV,and (22) Silva, N., Jr.; Gratton, E.; Mei, G.; Rosato, N.; Rusch, R.;Finazzo-Agro, A. Biophys. Chem. 1993, 48, 171-182. (23) Robbins, D. J.; Dcibel, M. R., Jr.; Barklcy, M. D. Biochemistry 1985, 24, 7250.
(24) Havel, H. A.; Kauffman, E. W.; Elzinga, P. A. Biochim. Biophys. Acto 1988, 955, 154-163.
2.2,
-
2.0
1.81.6-
k 1.41.21.0-
0.81 0.00
,
I
0.01
I
I
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I
0.02
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0.03
I
0.04
.
I
1
0.05
0.06
TCE concentration (M)
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
KI concentration (M)
Flgure 5. Stern-Volmer plot of rhGH fluorescence quenching by TCE: sample, 0.5 mg/mL rhGH in 50 mM phosphate buffer (pH 6.5); temperature, 20 "C; (0)aqueous buffer, (0)15% (v/v) CH3CN, (e) 45% (v/v) CH3CN, (0)15% (v/v) 1-PrOH, and (0)45% (v/v) 1-PrOH.
Flgure 8. Stern-Volmer plot of rhGH fluorescence quenching by KI: sample, 0.5 mg/mL rhGH In 50 mM phosphate buffer (pH 6.5); temperature 20 OC; (0)aqueous buffer and (0)45% (v/v) 1-PrOH.
Table 1. Quenchlng of Tryptophan Fluorescence of rhGH In Aqueous Solutlons Contalning Organic Modltiers: Parameters of the Stern-Volmer Equatlon'
at this temperature and protein concentration.) The resulting KSVvalue exceeded the respective value for aqueous solution by a factor of 3 (expressed in k,, the factor is 1.5). In contrast, the addition of CH3CN up to 45% (v/v) merely decreased the slope of the linear Stern-Volmer plot (Figure 5). This significant difference in TCE accessibility to Trp in rhGH is in agreement with the results obtained by static fluorescence measurements1 and by near-UV CD measurements; Le., 1-PrOH results in an elimination of the Trp CD signal while CH3CN does not. The KI quenching measurements at both 20 and 45 "C in aqueous buffer or with CH3CN displayed small values of the Stern-Volmer quenching constants; see Table 1. The KSV and k, values for the aqueous rhGH were lower than those found for bGH at pH 8.5 (k, = 0.8 X lo9 M-l s-l 24), possibly due to the overall negative charge of bGH at pH values far above the isoelectric point. Only at extreme conditions (45% (v/v) CH&N, 45 "C) was a higher value of the Stern-Volmer constant found (Table 1). As noted earlier, these conditions may represent the beginning of a conformational change. In the presence of 45% (v/v) 1-PrOH, iodide quenching of tryptophan fluorescence rapidly increased and then approached a constant value (Figure 6). The observed rise of Fo/F proceeded so rapidly with KI concentration that the usual explanation of two populations of f l ~ o r o p h o r eiss ~likely ~ incorrect. The increase may possibly be the result of an interaction between negatively charged iodide anions and some positively charged residues in the neighborhood of Trp. Saturation of this ion pair binding may cause a reduction in fluorescence emission to a constant value. The positive residue(s), buried in the core of the protein in the native state, may have become less restricted in the new conformation. It is unclear at the present time what these residues may be. It is interesting to compare the Fo/F values found at 2 0 "C for the protein in the aqueous buffer and in the presence of 45% (v/v) 1-PrOH at 0.02 M KI. In aqueous solution, the ratio was close to 1, suggesting no quenching, while in 45% 1-PrOH, Trp was substantially quenched, as the ratio exceeded the value of 1.7. As for TCE quenching, the addition of 45% 1-PrOH substantially changed the accessibility of Trp to KI
quencher
KI
TCE organic modifier (%, v/v) A. 20 OC HzO CH3CN (1 5) CH3CN (45) 1-PrOH (1 5) 1-PrOH (45) B. 45 'C H20 CH&N (15) CHlCN (45) a
Ksv (M-I) 6.3 6.7 5.2 b 20.8
10"k (M-l
s-j)
2.4 2.2 1.3
Ksv (M-I) 0.50 0.36 0.19 0.23
1.9 1.2 0.5 0.7
0.30 0.29 1.10
1.7 1.4 3.1
3.5
*
Buffer, 50 mM phosphate, pH 6.5. Nonlinear plot.
the respective quenching constants, k,, for aqueous rhGH at pH 6.5and for rhGH at 15% CH3CN at 20 "C are 2-3 times larger than the respective values for native bovine growth hormone (bGH) and for the bGH molten globule intermediate formed in the presence of 3.7 M Gdn HCl at pH 8.5 (the respective k,values were 0.75 X lo9 and 1.0 X lo9M-l s-1 24 suggesting increased TCE accessibility to Trp in rhGH at pH 6.5, as compared to bGH at pH 8.5. Nevertheless, the quenching constants remained low as Trp is buried in the four a-helix bundle. Further addition of CH3CN to 45% (v/v) results in a lower k, value, suggesting that CH3CN effectively protects Trp from TCE penetration. Interestingly, systematic positive deviations from linearity were found at 15% (v/v) 1-PrOH. Such deviations may be due to protein heterogeneity arising during the conformational change under the influence of 1-PrOH. Positive deviations, interpreted as static quenching, were found previously for the monomeric and associated intermediates of both bGH and hGH which occurred in the course of guanidine hydrochloride unfolding of both species.12,24 At 45% (v/v) 1-PrOH, a linear Stern-Volmer plot is again obtained, suggesting the completion of the protein conformational change. (Note, that no precipitation was observed 1 7
Analytical Chemistry, Vol. 66, No. 22, November 15, 1994
3913
Table 2. Effect of 1-PrOH on the Tryptophan Fluorescence Intensity Decay in rhGH* 1-PrOH(%, v/v) 7i (ns) A ( 7 ) (ns) X2
6-
5-
A. 20 OC
0 15
30 45 B. 45 OC 0 15
30 45 I?
3.09 0.81 3.61 1.06 5.60 1.74 6.22 1.40
0.81 0.19 0.84 0.16 0.88 0.12 0.94 0.06
2.36 0.95 2.86 0.87 4.70 1.05 4.85 0.99
0.61 0.39 0.76 0.24 0.93 0.07 0.94 0.06
2.66
0.72
3.21
0.57
h
5.14
0.42
5.95
0.78
1.81
0.30
2.38
0.61
u)
c
V
cv
4.
3-
2-
4.43 4.62
0
I
1
I
I
I
10
20
30
40
50
1-PrOH (%v/v)
0.97 0.81
Buffer, 50 m M phosphate, p H 6.5.
Flgure 7. Effect of 1-PrOH on fluorescence mean decay time of rhGH: sample, 0.5 mg/mL r h W in 50 mM phosphate buffer (pH 6.5); temperature, 20 (0)and 45 OC (0). 1
and, along with the near-UV CD spectra, again suggested an alteration of the environment of the Trp residue. Fluorescence Intensity Decay. The measurements of the fluorescence emission decay of Trp served as an additional tool for studying the conformational behavior of rhGH under the influence of 1-PrOH and CH3CN. Moreover, mean lifetime values of the excited state, (7),provided a measure of the lifetime rate constant without quencher, 70, for the calculation of the quenching rate constants, k , (eq 3). In the absence of organic solvents, the fluorescence intensity decay in 50 mM phosphate buffer (pH 6.5) at 20 OC was fitted to a double-exponential equation, with the mean decay time of 2.7 ns (Table 2). Similar values were obtained for hGH by Kauffmann et a1.25(2.8 ns at pH 8.0). It is interesting to note that multiexponential intensity decays are often found for proteins having a single Trp2"29 and are explained in terms of either the intrinsic heterogeneous decay of tryptophan, the interaction of Trp with charged amino acid groups within the particular protein matrix, or the conformational heterogeneity of the protein in solution. The effect of 1-PrOH on the frequency-domain intensity decays of the Trp emission of rhGH was studied at 20 and 45 OC. The results are summarized in Figure 7, and in Table 2. There is a close similarity between the sigmoidal curves of the dependence of the mean decay timeon 1-PrOH concentration at 45 OC (Figure 7) and the position of the first moment of fluorescence emission on 1-PrOH concentration at the same temperature (Figure 9 of ref 1). In both curves, the inflection points of the sigmoidal curves were close to 20% (v/v) 1-PrOH, with the most significant changes proceeding between 15 and 25% (v/v) 1-PrOH. A correspondence between the spectral shifts and mean decay times was also observed at 20 "C, where the inflection point was shifted to -25% (v/v) 1-PrOH with (25) Kauffman, E. W . ; Thamann, T.J.; Havel, H. A. J. Am. Chem. Soc. 1989,llI , 5449-5456. (26) Grinvald, A.; Stainberg, Z . Biochim. Biophys. Acta 1976,427, 663478. (27) Ross, J. A. B.; Rousslang, K. W.; Brand, L. Biochemistry 1981,20, 43614369. (28) Lakowicz, J. R.; Laczko, G.; Gruczynski, I.; Cherek, H. J . Biol. Chem. 1986, 261, 2240-2245. (29) Vincent, M.; Brochon, J. C.; Merola, F.; Jordi, W.; Gallay, J. Biochemistry 1988,27,8752.
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Analytical Chemistry, Vol. 66,No. 22, November 15, 1994
1.5
3 , 0 40 50 10
20
30
CH,CN (%v/v) Figure 8. Effect of CH3CNon fluorescence mean decay time of rhGH: sample, 0.5 mg/mL rhGH in 50 mM phosphate buffer (pH 6.5); temperature, 20 (0)and 45 OC (0).
the slope of the transition curve shallower; see also ref 1. The temperature change from 20 to 45 OC not only influenced the position of the inflection point and the slope of the transition curve but also decreased the absolutevalues of the mean decay time, as noted earlier.30 In the course of the static measurements of the fluorescence emission shifts of rhGH under the influence of l-PrOH,' an increase in spectral data variance was found at 1-PrOH concentrations around the inflection point of the transition curve, suggesting the heterogeneity of the Trp environment. A similar variance increase in the same 1-PrOH concentration range was also observed with the intensity decay data, as expressed by the respective x2 values. The effect of CH3CN on the emission intensity decay of Trp in rhGH at 20 and 45 OC is presented in Figure 8 and in Table 3. At both temperatures, the mean decay time increased monotonically with increasing CH3CN concentration in the range 0 4 5 % (v/v); at 20 OC, the increase was almost linear. The intensity decay was adequately described by a double-exponential equation, and the corresponding x2 (30) Gruczynski, I.; Eftink, M.; Lakowicz, J . R. Biochim. Biophys. Acta 1988,955, 244-252.
Table 3. Effect of CH&N on the Tryptophan Fluorescence Intensity Decay In rhGH CH$N (Wv/v) q(ns) A (7)(ns)
A. 20 O 0
X2
C
15 30 45
3.09 0.81 3.38 0.87 3.86 0.88 4.35 1.10
0.81 0.19 0.88 0.12 0.90 0.10 0.91 0.09
2.36 0.95 2.82 1.21 3.44 1.50 4.38 1.82
0.61 0.39 0.54 0.46 0.57 0.43 0.66 0.34
2.66
0.72
3.08
0.65
3.56
0.67
4.06
0.76
1.81
0.30
2.09
1.06
2.61
1.16
3.50
1.21
B.45 'C 0 15 30 45
values were relatively small over the whole concentration range and at both temperatures; see Table 3. Based on these results, the observed changes in the fluorescence lifetime again mainly reflected solvation of the protein when CH3CN was added. MoltenGlobuleState. 1-PrOH. The results in the previous sections reveal that the new conformational state of rhGH in the presence of >30% (v/v) 1-PrOH maintains much of the secondary structure of the native state, while unfolding with respect to the tertiary structure. Moreover, the new state, while slightly expanded, was still relatively compact. These characteristics are consistent with the description of the molten globule state which is proposed as an intermediate in the unfolding pathway of globular protein^.*^,^^ In particular, the molten globule state is envisioned to consist of a protein structure with the overall fold of the native state in which the secondary structural elements remain largely intact, but these elements are no longer tightly packed. The looseness of the structure can lead to solvent penetration, an increase in the dynamics of the protein, and enhanced side-chain mobility. Indeed, there may be some relationship between the new rhGH conformation induced by 1-PrOH and the molten globule state of bGH.9 The existence of a high degree of secondary structure in the new state of rhGH may not be surprising given the high level of helicity in the native state and the known ability of alcohols to induce a-helical structure.32 Indeed, alcohols often lead to a state with significant secondary structure and some loss in tertiary structure.33 The altered state may be important in the study of the molten globule state, since it is a state that can be maintained over a range of conditions. It is evident that this solution-driven conformational change played a significant role in the retention behavior of rhGH in RPLC. In analogy to bGH,4,9the new state of rhGH likely exposed a hydrophobic surface to be presented to the hydrophobic stationary phase for binding and thus retention. It is also possible that during the conformational transition, an equilibrium existed between the native folded form of rhGH (31) Goto, Y.; Fink, A. L. Biochemisfry 1989, 28, 945-952. (32) Nelson, J. W.; Kallenbach, N. R. Biochemistry 1989, 28, 5256-5261. (33) Fink, A. L.; Painter, B. Biochemistry 1987, 26, 1665-1671. (34) Sadler, A. J.: Micanovic, R.; Katzenstein, G. E.; Lewis, R. V.: Middaugh, C. R.J . Chromafogr. 1984, 317, 93.
and the new conformation state. Further addition of I-PrOH would increase the amount of conformationally changed protein, but simultaneously, the alcohol may also solvate hydrophobic domains of the protein. This solvation may be sufficient to cause elution above -30% (v/v) 1-PrOH concentration. The above solution-driven process for reversed phase retention and elution of rhGH with 1-PrOH is in agreement with earlier studies of others for standard proteins.34 A molten globule or related state of proteins, stabilized by the addition of 1-PrOH, may play an important role in selective protein separation by reversed phase liquid chromatography (RPLC). As was shown in our previous work in the reversed phase chromatography of two variants of human growth hormone (Met-hGH, rhGH),' these species could not be separated on the hydrophobic stationary phase in the native state. The necessary differences in hydrophobicity resulted only during the conformational changes between the native and molten globule states. Thus, such conformational changes leading to molecular differences can be an important ingredient in the successful separation of related species. In addition, since a molten globule state will possess significant secondary structure, separation can occur in the desired conformational state followed by reversion back to the native state upon collection and removal of the alcohol. In the case of acetonitrile, solvent-induced structural effects in rhGH are minor in the region studied. Conformational changes, if they occur, must be surface driven, by virtue of the highly hydrophobic surface. This conclusion was made previously.' The less destabilizing character of acetonitrile relative to 1-PrOH is likely related to the weaker solvent strength in reversed phase chromatography of the former organic modifier. Finally, such spectroscopic/structural analyses, as presented in this paper, are important tools in providing insight with respect to the manipulation of structure for separation purposes. It is often the case that protein separation in liquid chromatography takes place via selective alteration in conformation, leading to a state with different interaction properties. This is especially so in RPLC and hydrophobic interaction chromatography. If the forces causing change are not excessive, the conformational alterations will be reversible, leading to the return to the native state. Moreover, when analysis and not purification is the desired goal, irreversibly altered protein structure can also be successfully employed.
ACKNOWLEDGMENT B.L.K. gratefully acknowledges NIH GM-15847 for support of this work. The authors further acknowledge discussions with Dr. W. Hancock, Hewlett Packard, on this project. Contribution 572 from the Barnett Institute. Received for review April 14, 1994. Accepted August 18, 1994." @Abstractpublished in Aduunce ACS Absrrucrs, October 1, 1994.
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