Evaluation of Particle Beam Fourier Transform Infrared Spectrometry for the Analysis of Globular...

Evaluation of Particle Beam Fourier Transform Infrared Spectrometry for the Analysis of Globular Proteins: Conformation of -Lactoglobulin and Lysozyme

V I N C E N T E . T U R U L A a n d J A M E S A . d e H A S E T H *

Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556

The efficacy of the particle beam LC/FT-IR interface, in its development as a tool for the determination of dynamic protein structure from experiments such as HPLC separations and folding/refolding intermediate analysis, is presented here. The particle beam apparatus can be used to desolvate proteins rapidly in preparation of IR measurements. Several experiments have been designed to determine whether the operation of the particle beam apparatus causes alteration to the complex structural features of globular proteins, and whether it produces a solid-state spectrum representative of protein solution structure. It is shown here that the structural integrity of/~-lactoglobulin is maintained when nebulized, desolvated from solution, and deposited onto the IR-substrate. Since enzyme activity is dependent upon the maintenance of higher-order structure, a complementary series of spectrophotometric-activity experiments with lysozyme collected from the particle beam were performed to determine the state of the tertiary and quaternary structures. The lysozyme particle beam deposit not only produced a secondary structure estimate similar to that of solution; it also retained its biological activity. It is demonstrated that the particle beam can induce structural changes in proteins with a carrier-liquid concentration gradient; this characteristic is useful for band assignment. Index Headings: Particle beam LC/FT-IR spectrometry; Desolvation; B-Lactoglobulin (BLG); Lysozyme; Micrococcus lysodeikticus.

I N T R O D U C T I O N

Infrared spectrometry has been used extensively to ex- amine protein solution structure. The position and intensity of infrared (IR) amide absorption bands are sen- sitive to globular protein secondary structure content. 1,2 Information about the protein secondary structure is con- tained in the amide I, II, and III regions. These bands arise from delocalized vibrations of the peptide linkages? The band that is recognized as the most informative for structure correlation is the amide I band. 3-5 Attempts to determine protein solution structure date back to the 1950s. 6 The desire to obtain protein spectra in an environment as close to the native aqueous state led workers to employ thin transmission cells to minimize the effects of the solvent, since strong absorption of H20 resulted in the loss of large regions of the spectrum, particularly in the amide I region. In an effort to circumvent this situation, deuterium oxide was used as a medium for IR protein structure investigations. 7 Transmission experiments with dispersive spectrophotometers and D:O were difficult because of the inability to remove contaminant HOD, and they had questionable background compen-

Received 26 October 1993; accepted 13 July 1994. * Author to whom correspondence should be sent.

sation, since it was difficult to match cell pathlengths. Byler and Susi found good agreement for the secondary structure estimates of 17 proteins between their O20 Fou- rier transform infrared (FT-IR) spectra and corresponding X-ray crystallographic determinations, s Jakobsen and co-workers have used attenuated total reflectance (ATR) FT-IR spectrometry to study globular protein structural changes induced with nonaqueous solvents. 9-11 The proteins were adsorbed from H20 solution onto a germanium ATR element. The resultant spectral changes led to conformational assignments. Condensed-phase spectra of proteins by these analyses yield intrinsically broad band contours. In both transmission and ATR techniques, spectral subtraction of water was required to reveal the amide absorptions. To allow extraction of important spectral features, spectra must undergo rigorous mathematical manipulations such as deconvolution, differen- tiation, and curve fitting. These techniques are prone to produce errors, since they rely heavily on human judge- ment. An approach has been undertaken in this laboratory that is aimed at the development of a technique with particle beam/FT-IR spectrometry that will allow for estimation of protein solution structure without the mask- ing water absorptions in the measured spectrum. Since the particle beam can prepare samples in such a state rapidly, we believe it can be used not only for the IR characterization of proteins separated by high-perfor- mance liquid chromatography (HPLC) but also to study in vitro folding mechanisms for globular proteins.

Particle beam LC/FT-IR spectrometry is an aerosol method originally developed as an HPLC interface for mass spectrometry 12 and, later, infrared spectrometry. ~3 The aerosol apparatus eliminates the more volatile solvent (mobile phase) by evaporation from the analyte molecules. In normal operation the protein solution is pumped through a small orifice and exits as a liquid jet. This column of liquid is then nebulized with a stream of helium. The resultant protein/water droplets are directed into a cylindrical glass chamber where the water molecules are stripped by evaporation and removed. This process is known as desolvation. After the desolvation process, the protein molecules are dried to an extent that can be controlled by certain parameters. The protein molecules are subsequently impacted against an infrared- transparent substrate, which is later removed and analyzed by off-line FT-IR microscopy. The desolvation process is endothermic and occurs in a few milliseconds, so that the low-energy solution conformers are trapped as the aerosol particles cool and eventually freeze; internal

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rotations, restricted by desolvation, are not likely to occur. Therefore, this rapid sampling of a protein in solution to produce a dried particle beam deposit can be spectro- scopically analyzed and can yield a solid-state spectrum highly representative of the protein's solution structure.

The experiments presented in this study examined the three processes in particle beam operation that were considered to be possible sources of structural degradation. These are the formation of the liquid jet for monodisperse aerosol generation, desolvation, and protein impact onto the substrate. Experiments were designed to isolate these processes in such a way that the cause of any structural degradation to the protein, if it occurred, was recognized. The protein chosen for this work was fl-lactoglobulin (flLG--approximately 35,000 Daltons)--a structurally stable milk protein that has been well characterized by infrared spectrometry. Not only can flLG be examined in denatured conditions, '4 but its IR solution spectral bands are similar to the corresponding solid-state bands which permit spectral comparisons between phases. Comparisons of reference IR data of native and denatured flLG have led to its conformational elucidation. The amide I and II IR bands of/3LG solutions are similar to the same bands in the ~LG solid-film spectra. 3 Susi and co- workers examined native flLG in D20 solutions in various pH denatured states and found that a mixture of /F-sheets and a-helix, with some disordered segments, comprised the native structure. 4,5 The conclusion of each of our experiments was based on comparisons of/3LG IR spectra with reference data generated in our laboratory by parallel sampling techniques (i.e., ATR CIRCLE ® cell and thin solid films). Since structural changes can be dis- cerned by frequency shifts and intensity changes in the IR amide I, II, and III bands, spectral comparisons were made with the original band contours. Deconvolution and curve fitting were used only as a supplement. Band assignments were made first by the use of literature data as a benchmark; then more exact assignments were made by following band contour changes that were induced by the exposure of flLG to structure-altering solvents (e.g., methanol or ethylene glycol).

In a procedure to determine the effect of the particle beam on the tertiary and quaternary structure of a protein, a model enzyme (lysozyme) was chosen. A visible spectrophotometric assay based on the lyric action of lysozyme with bacteria ~5,~6 was employed to determine biological activity, and thus, the state of the higher-order structure of lysozyme passed through the particle beam. Lysozyme is an enzyme from chicken egg-white that pos- sesses a high percentage ofa-helicity with some/3-sheets; the helical region forms a deep and defined crevice that is the active site? 7

It is the purpose of this paper to argue that preliminary studies indicate that the structural integrity of globular proteins is maintained when they are prepared for spectroscopic analysis by particle beam analysis.

EXPERIMENTAL

~-lactoglobulin (L-2506) and lysozyme (L-6876) were purchased from the Sigma Chemical Company (St. Louis, MO) and used without further purification. Protein solutions were prepared in an aqueous solution of 18 M~2

deionized water, 0.15 M sodium chloride, and a phosphate buffer that consisted of 5.75 mM potassium dihy- drogen phosphate (KH2PO4) and 31.4 mM anhydrous disodium hydrogen phosphate (NazHPO4). Although it was difficult to subtract the phosphate buffer bands in thin-film spectra completely, and hence, the structurally important amide III band was occasionally masked, the presence of the buffer eliminated the potential for structural changes induced by ambient pH fluctuations and enabled close emulation of physiological pH. In the spec- ified proportions, the buffer maintained an approximate pH of 7.4; the pH varied slightly with different protein concentrations. For experiments with pH denatured protein, the phosphate buffer was not included in solution preparation. Denaturation was accomplished through subjection of the protein to pH extremes in saline solution with 0.1 M sodium hydroxide (NaOH) and 0.1 M hy- drochloric acid. Unless otherwise stated, /~LG was in- cubated in these environments at room temperature for at least 24 h before the spectra were recorded. The concentration of the protein solutions used varied and de- pended upon the sensitivity of the experiment: for ATR, 1% w/v (10 mg/mL); solution transmission, 10% w/v (100 mg/mL); particle beam, < 1% w/v. A sandwich cell con- structed with polished calcium fluoride (CaF2) windows (25 mm dia. x 4 mm, International Crystal Inc., Garfield, N J, Cat. # 0002D-181) with a 6-#m-thick Teflon ® spacer (Harrick Scientific, Ossining, NY, Cat. # PSS-M25) was used to record transmission solution spectra. Reference spectra of solid films were prepared by the deposition of protein solution onto a CaF2 window and evaporation to dryness in an ambient temperature vacuum desiccator over dryrite. All transmission spectra were collected on a Perkin-Elmer 1725X FT-IR spectrometer (DTGS detector) with 1000-2000 single-beam scans coadded at a resolution of 4 cm- ' (Perkin-Elmer, Norwalk, CT). At- tenuated total reflectance measurements were made with a sealed ATR micro-CIRCLE ® cell (200-tzL volume) and a zinc selenide (ZnSe) element (Spectra-Tech, Shelton, CT). The cell was mounted in the sample compartment o fa Bio-Rad FTS-60 FT-IR spectrometer (Bio-Rad Lab- oratories, Digilab Division, Cambridge, MA) in such a way that the loading of buffer or protein solution could be done externally while the purge integrity of the instrument was maintained. A Lure-Lok ® syringe with a volume capacity of 3 mL was used to load the CIRCLE ® cell. Spectra were collected at regular time intervals so that protein adsorption onto the element could be followed. A mercury cadmium teluride (MCT) detector was used for the CIRCLE ® cell measurements. Two thousand scans at 4-cm-~ resolution with boxcar truncation were the ATR collection conditions. The ZnSe element was cleaned by a soap solution scrub, sonication, and, lastly, solvent rinses. The empty cell was scanned prior to analysis so that any residual protein was detected and recorded. The particle beam operation is described else- where,'3 but a brief description follows. A 10-#L injection loop was used to introduce the protein solution into the flowing stream. The liquid was pumped at 0.3 mL/min into the apparatus to produce a Rayleigh jet which was dispersed at approximately 20 psig by helium (Fig. 1). Impacts were made onto either potassium chloride (KC1), zinc selenide, or calcium fluoride windows. Deposit di-

1256 Volume 48, Number 10, 1994

Aerosol Generator 0 o

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FiG. 1. Experimental setup for particle beam LC/FT-IR spectrometry for the analysis of/3-1actoglobulin. Protein solutions were introduced into the flowing stream by the six-port injector. A ffit-filter eliminated any solid particles that might clog the fused-silica tube. The dispersion gas interacts orthogonally with the liquid jet in the aerosol generator. Evaporation occurs in the glass desolvation chamber, and an IR-transparent substrate that collected the protein deposits was held by a rotating mount.

ameter varied and was dependent upon the horizontal position of the substrate collector. Particle beam deposit spectra were acquired with a Spectra-Tech IR Plan@ microscope enclosed in a laboratory-designed Plexiglas@ purge compartment, and interfaced to the Perkin-Elmer 1725X FT-IR spectrometer. The microscope was config- ured with an MCT detector, and 1.5-mm upper and 1.0- mm lower circular apertures were used. This configura- tion scales to a 100-um-diameter field of view. Spectral manipulations such as digital subtraction, deconvolution, and curve fitting were calculated with LabCalc@ and GRAMS@ software (Galactic Industries, Salem, NH).

The lysozyme activity assay was based on the decrease in absorbance over time during the reaction of a bacterial substrate, Micrococcus lysodeikticus (Sigma Chemical Company, Cat. # M-3770), with lysozyme. ~5,16 A stock solution of crystalline lysozyme at a concentration of 1 mg/mL was prepared in 0.1 M phosphate buffer [10.37 g of sodium phosphate monobasic monohydra te (NaHEPO4" H20) and 3.12 g of sodium phosphate dibasic anhydrous (Na2HPO4) diluted to one liter with deionized water], which maintained a pH of 6.27-6.30 during one week. Analogous stock solutions of lysozyme collected from both the particle beam and from an evaporated film substrate were also prepared in the phosphate buffer. In an effort to ensure that a large quantity of lysozyme was recovered from the particle beam substrate, a 50-uL loop was installed into the Rheodyne injector valve. Dilutions of the enzyme to a concentration of 1.37 #g/mL and preparation of the substrate solution to a concentration of 0.1 mg/mL were performed just prior to analysis. The absorbance was monitored with a Hewlett-Packard HP 8452A diode array spectrophotometer (San Jose, CA) scanned from 400 to 600 nm with a 1-cm-pathlength quartz cell. After the addition of 100/~L of lysozyme to 3.00 mL of substrate, measurements were made once a minute for five minutes.

All 2-D Cartesian graphs were made with SigmaPlot~ (Jandel Scientific, San Rafael, CA). The first-order regression lines illustrated were produced by least-squares fits.

RESULTS AND DISCUSSION

Aerosol Generation. For operation of the particle beam apparatus, the aerosol formed must be close to monodisperse; that is, it must be an aerosol with a narrow distri- bution of particle sizes. This condition ensures identical desolvation for each particle. The aerosol generation technique employed requires the formation ofa Rayleigh liquid jet. The jet can be formed at HPLC flow rates if the flowing liquid passes through a small orifice. This ar- rangement satisfies the condition of high pressure for jet formation. The small orifice used in the particle beam apparatus is an inert polyimide-coated fused-silica tube with an inner diameter of 25 #m. Since globular proteins with molecular weights in the tens of thousands of Dal- tons have average radii of gyration that may, depending upon the state of folding, approach the inner diameter of the fused silica, it was considered important to determine whether shear degradation occurs, and whether the structure was affected. To determine whether shear degradation had occurred, we isolated the jet formation process from the particle beam apparatus. Instead of passing BLG through the entire particle beam apparatus, we passed it through the 25-#m fused-silica tube only, and collected it for subsequent analysis. A bored brass cylinder (probe) that houses the stainless-steel fused-silica tubing union was clamped above a collection flask. The height was fixed so that enough air resistance slowed down particle velocity after jet breakup to ensure that a harsh impact with the flask or solution, which might cause structural dam- age, was avoided. The phosphate buffer/saline solution was pumped at 0.3 mL/min while a ~LG solution of an initial concentration of 10% w/v was introduced by way of a Rheodyne 6-port injector with a 100-~L loop. A previous timing experiment with a UV-visible detector and organic dye (Erythrosin B, fw 879.9 g/mole) determined the optimal collection time after injection. The absorption pattern of the dye indicated a laminar flow profile at this flow rate. ~LG did not appear to move through the fused-silica tube in a compact slug, as did the dye. It is speculated that this behavior was caused by some protein that had adhered to the stainless steel tubing. Since the sample eluted from the fused silica over a longer period of time, the initial protein solution was more diluted than the dye sample. The collected /~LG solution was analyzed by both solid film transmission and ATR CIRCLE ® cell FT-IR spectrometry. The original spectra are shown in Fig. 2. Shear degradation col- lections were compared to reference spectra run by the same method. Clearly, both solid- and liquid-state experimental spectra are nearly identical to their corresponding reference spectra. The amide I band exhibits a contour indicative of the mixture of ~-sheet and a-helical content of BLG. A slight difference between the set of transmission spectra and those of the ATR exists in the shape of the amide II bands. This is well known and is caused by protein adherence onto the CIRCLE ® cell ATR element. 11

APPLIED SPECTROSCOPY 1257

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0

1636

1860 1760 1660 1560 1460 1360

Wavenumber / cm ~

Attenuated Total Reflectance 1633 c/2

1630 D //~ 1548

1800 1660 14;0 1200

Wavenumber / cm 1

FIG. 2. Spectra of the amide I, II, and III regions of 3-1actoglobulin collected after elution, from the fused-silica tube. The upper two traces are transmission spectra recorded as thin-films: (A) 3LG pumped through fused silica; (B) flLG reference solution. The lower two traces are ATR CIRCLE ® cell spectra: (C) 3LG pumped through fused silica; (D) flLG reference solution.

Desolvation. The glass desolvation chamber of the particle beam apparatus is wrapped with heating tape so that the chamber is maintained at ambient temperature during aerosol evaporation. Since evaporation is an endothermic process, the desolvation chamber cools during solvent evaporation; if the chamber is warmed to 25-30°C, con- densation of the solvent vapor is prevented. To what degree over-heating affects protein structure, if any, was investigated by operation of the particle beam at a dras- tically elevated temperature. A test was made with the desolvation temperature raised and maintained at 160°C, through which a/~LG deposit onto a ZnSe window was made. The resulting spectrum is illustrated in Fig. 3D and is identical to the other particle beam spectra. On the basis of velocity measurements made with identical op- erating parameters, the time a protein molecule is resident in the 30-cm-length desolvation chamber is approximately 1-5 milliseconds. Since no spectral change was observed in the/~LG deposit collected through the desolvation chamber at 160°C, we concluded that excessive interface heat did not transfer to the aerosol, nor did it alter the structure of the traversing protein; it is unlikely that the temperature of the aerosol approaches 160°C, since the brief time the particles spend in the chamber is not long enough for equilibration to occur. Interestingly, a slight decrease in the amount of phosphate buffer that was collected was observed (at approximately 1100-1000 c m - 1).

Deposition. The effect of the IR-transparent substrate material was examined (Fig. 3). Originally it was thought that, to prevent salt exchange between/~LG and the substrate, one should use ZnSe substrates for particle beam

A

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Wavenumber / cm 1

FiG. 3. /~-lactoglobulin particle beam deposits on various substrate materials. (A) ~LG reference solution evaporated on ZnSe; (B) particle beam deposit on ZnSe; (C) particle beam deposit on KC1; (D) /3LG passed through desolvation chamber at 160°C on ZnSe. (Note the decrease in absorbance around 1000 cm ~ attributed to volatilization and removal of buffer.)

studies. An evaporated film deposit was made onto ZnSe (Fig. 3A) for reference. Deposits onto both CaF 2 (spectrum not illustrated) and KC1 (Fig. 3C) were made, and spectra were found to be identical to that from ZnSe deposit (Fig. 3B). The deposit on KC1 was stored in a desiccator and rerun a week later, and it was found that there was no spectral alteration. The spectrum of the deposit with the desolvation chamber heated to 160°C (Fig. 3D) shows no discernible difference from the other spectra, except that the absorbance around 1000 cm-1 is lower because of a more efficient removal of the buffer. Overall, we found that protein deposits were completely dried, and spectra were identical to each other, regardless of the substrate material and duration of storage (Fig. 3).

During the solvent elimination process in the particle beam apparatus, aqueous protein aerosol particles cool as the ambient water evaporates. Towards the later stages

1258 Volume 48, Number 10, 1994

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FiG. 4. Comparison of the amide I region of a reference solution of BLG. Solid line: (A) original contour; (C) deconvolved; (E) second derivative, to that of a particle beam /~LG deposit redissolved into solution. Dashed line: (B) original contour; (D) deconvolved; (F) second derivative.

of the desolvation process, the temperature of the particles drops further and causes the small amount of residual water, trapped with the protein, to freeze, so that a low- temperature protein/water "comet" or particle is produced. The low-temperature beam of particles then impacts an IR transparent substrate. The substrate is later removed from the interface and placed into an FT-IR microscope for spectral measurement. No deleterious effects on structure are believed to occur as a result of temperature reduction; however, the massive protein particle moves rapidly and strikes the substrate with consid- erable kinetic energy or momentum. If denaturation does occur, it is very probable that it is a result of this high- energy impact. Two spectral comparisons (again, one liquid and one solid state) were made to determine whether impact with the substrate causes protein conformation changes. Immediately after a buffered ~LG solution was run through the particle beam apparatus and deposited

onto a CaF2 window, it was redissolved with a measured volume, that is, 10 uL of buffer/saline solution directly on the window (Fig. 4). An identical CaF2 window was placed above the ~LG solution with a 6-um-thick Teflon spacer to make the cell. Spectra of the redissolved deposit (original, deconvolved, second derivative) are shown in Fig. 4B, 4D, and 4F. Since isolation from solvent vapor is based on momentum separation, throughput is high for massive macromolecules. 18 Yet it was difficult to determine the concentration of this solution, since transport efficiency of the particle beam varies slightly with respect to aerosol generator alignment. The reference solution, 10% w/v (Fig. 4A, 4C, and 4E), was originally believed to be more concentrated than the deposition solution. The original contours of the amide I region, as well as the deconvolved and second-derivative spectra, are identical for these/~LG solution transmission spectra (Fig. 4). Both spectra exhibit amide I maxima at 1632 cm- ' that correspond directly to literature values offlLG in H20? ,8 A greater absorbance was measured with the particle beam rinsed deposit than with that of the reference solution (the absorbance scale is not illustrated in Fig. 4, but the intensity of the reference deposit was 65% of that of the rinsed deposit). This observation indicated that the transport efficiency (quantity injected/quantity recovered) was somewhat greater than 20%. Slight differences in intensity of some component bands illustrate the difficulty inherent in quantitative measurements with the use of this sampling technique.

In the solid state, similar results for/3LG are observed (Fig. 5A and 5B) since the amide I band maximum is around 1639 cm- ' for both vacuum-evaporated reference and particle beam deposits. Deconvolution and curve fitting revealed a slight difference between the two sets of spectra. The amide I absorption maximum at 1639 cm- ' was definitively assigned to the/F-sheet. Beta structure is known to be exhibited as several component bands in IR spectra. 1,2 An amide band at 1639 cm-1 has been previously assigned to a disordered conformation in solution spectra of alkaline denatured/~LG (in isopropanol-d,, pD = 12). 19 Protein spectra acquired in deuterium oxide environments, however, cannot be related directly to corresponding H20 spectra, and wavenumber shifts of several bands are often observed. 2°,21 In our observations, no concomitant amide II or III disordered band intensity increases were observed in the raw data; the amide III disordered 1261-cm-1 band decreases in intensity in the particle beam spectrum. Therefore, structural disordering as a result of impact was ruled out. The assignment of the band at 1639 cm-1 to intramolecular/F-sheet, as solid- film, was confirmed by a series of flLG spectra run in various methanol/water environments: a substantial decrease in this band intensity was observed with a change in medium from aqueous to methanolic, which is unfa- vorable for the stability of the r-sheet structure (this con- sideration is discussed in more detail in the "Solvent Perturbation" section). Additional work with/3LG/ethylene glycol/water solutions envinced the beta assignment. Wasacz et al. found that v-globulin, a globular protein similar in structure to/~LG (in that it has a large /3-sheet content), forms an intermolecular disordered /3-sheet structure as an ATR film in the presence of ethylene glycol, t° The amide I maximum at 1629 cm -t that


1639

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1800 1600 1400 1200

Wavenumber / c m 1

Fio. 5. Solid films and effects of film thickness on ~LG amide I, II, and III regions. (A) BLG particle beam deposit on ZnSe; (B) thin, uniform BLG evaporated deposit on ZnSe; (C) nonuniform BLG evaporated deposit on ZnSe; (D) ~LG film evaporated from H20/ethylene glycol medium. The deconvolved amide III region has been expanded.

they observed was ascribed to this structure. Given the similar secondary structure of/~LG to v-globulin, we prepared a /~LG/50% ethylene glycol/50% water film and found an identical contour of the amide I, II, and III regions for both the impacted sample from the particle beam and that of an evaporated film from water (Fig. 5D). Deconvolution revealed two underlying bands that composed the/3-sheet low-frequency component absorptions: 1631 and 1639 cm -~. Through curve fitting of the amide I region of/3LG of a particle beam deposit and an evaporated film, it is shown that the/3-sheet bands shift in intensity maximum (Fig. 6), although the net integrated areas of these bands remain fairly constant. (Evaporated film: a-helix, 44%;/3-sheet, 37%. Particle beam: a-helix, 55%,/3-sheet 30%.) This behavior is not fully understood,

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Wavenumber / cm ~ Fic. 6. Deconvolved and curve-fit spectra of the amide I region of the evaporated film and particle beam/3LG IR spectra. Estimates for secondary structure based on areas of component bands. (A) Evaporated film spectrum ofj3LG on ZnSe window: 44% a-helix, 37%/3-sheet, 17% turns. (B) Particle beam deposit on ZnSe substrate: 55% a-helix, 30% /3-sheet, 14% turns.

but we speculate that the particle beam deposition of the protein onto the substrate may alter the packing, or con- nectivity, or may cause twisting of the/3-sheets. These changes in the "super-secondary structure" show up in the IR spectra. The curve-fit results for/3LG in solution, measured with the CaF2 cell, are very similar to those from the particle beam (a-helix, 57%; B-sheet, 30%). The amide I contour in Fig. 4 was used for this estimation and was not re-illustrated with the curve-fit bands. Slow evaporation of/3LG in aqueous media at room temperature slightly alters the intensity of the secondary structure bands relative to the/3LG solution spectra. Similar tests with lysozyme, however, show it also retains its natural solution conformational state as an evaporated film; particle beam, evaporated-film, and solution spectra for lysozyme are nearly identical. We did not observe intensity changes in the lysozyme particle beam/evaporated film spectra. In addition, we endeavor to explain why secondary structure estimates for lysozyme are much closer to each other than for those of/3LG, but can only, at this time, conjecture that the effects of packing are not as drastic in proteins with a higher helical content as they are with high/3-sheet proteins. We are investigating this issue further. These spectral comparisons strongly suggest that the rapid sampling with particle beam provides a deposit closer to solution spectra than a vacuum-evaporated film.

1260 Volume 48, Number 10, 1994

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1700 1680 1660 1640

Wavenumber/cm -~

0.40 -

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FIG. 7. Deconvolved and curve-fit spectra of the amide I region of the evaporated film and particle beam lysozyme IR spectra. Estimates for secondary structure based on areas of component bands. (A) Evaporated film spectrum oflysozyme on ZnSe window: 60% a-helix, 12% B-sheet, 28% turns. (B) Particle beam deposit on ZnSe substrate: 66% a-helix, 9%/3-sheet, 22% turns.

The authors acknowledge here that the above determinations of the percentage ofa-helicity are high relative to that of the literature for flLG. 8 With the BLG spectra it was difficult to distinguish the disordered amide I band from an a-helix since their band centers coincide. More- over, we have made no compensation for the differences in molar absorptivities of the different amide bands as is necessary to produce accurate estimations. However, for the purpose of these experiments, our approximate estimations served to allow structural comparisons.

This work, with rather small deposits (< 100-um diameter), has led to a valuable insight: slight wavenumber shifts can occur as a result of nonuniform deposit thickness, which is known as "wedging". 22,23 Lack of distortion of the IR beam in FT-IR microscopy is critical. A thick sample can cause distortion of the image at the detector, which results in wavenumber shifts and quantitative errors. This problem is more pronounced, due to the re- fractive index, in strong absorption bands, which in this case is the amide I band. We investigated this effect by the measurement of several films each of different thick- nesses. This was done by varying the number of times the protein solution was placed over existing dried deposits and re-evaporated--both particle beam deposits and films. Figure 5 shows a A5 cm-~ shift between a thin sample (spectrum B: max. 1639 cm-t) and a thick deposit (spectrum C: max. 1634 cm-0. The overall contour of

the deconvolved amide III region changes little (expanded plot, Fig. 5). With individual deposits on separate IR- substrates (e.g., 13-mm diameter), we found the wavenumber shift was alleviated by spreading the deposit with buffer to uniform thickness while on the substrate, and then proceeding with reevaporation of the buffer and measurement of the resultant film. This procedure is not necessary with the normal operation of the particle beam. Thicker deposits cause shifts to a lower wavenumber in film spectra measured with a microscope; we have observed this behavior in proteins with different conformation content as well. 24 Particle beam deposit thickness depends upon lateral placement of the substrate. With judicious positioning of the substrate and nozzle-substrate distance, small, thin, and uniform deposits are produced and reliable IR spectra obtained.

On the basis of the excellent comparison (both wavenumber and intensity) between the contours of particle beam deposits to evaporated thin films and solution spectra, we are confident that the portions of secondary structure do not change upon high-energy impact.

Since infrared spectrometry does not convey information with regard to higher-order (tertiary, quaternary) conformational structure, a biological activity assay was employed to ascertain whether the overall structure was affected by the particle beam. When the decrease in absorbance of bacteria cells as they dissolve is monitored spectrophotometrically, the activity of lysozyme can be determined. As with BLG previously, the overall secondary structure was rigorously examined by various IR sampling techniques. Figure 7 illustrates the high similarity between the amide I regions of both particle beam and film spectra, as well as exhibiting the high degree ofa-helicity that lysozyme contains. Since the structural do- mains of particle beam lysozyme were determined by the IR measurements to remain intact, the next step was to analyze the higher-order structure with the activity assay. The convenience of the diode array instrument enabled two analytical wavelengths to be monitored simulta- neously. Three forms of lysozyme were assayed for activity: crystalline lysozyme, lysozyme evaporated in a vacuum desiccator on a ZnSe plate, and lysozyme collected from particle beam deposits. Pure substrate was scanned before addition of lysozyme in each case. In ac- cordance with the amounts used in our assay, the reac- tants were consumed in the first five minutes. As can be seen in Fig. 8 (assay at 450 nm), each form of lysozyme causes a similar absorbance drop as time progresses (similar negative slopes in regression lines). Lysozyme activity was calculated from the absorbance behavior for the first three minutes, since this approach provides the most accurate results; that is, the reaction closely approximates first-order kinetics.t5 One activity unit was defined as the amount that would cause an absorbance change, or drop in optical density (OD), of 100 in one minute in a 1-mL volume at 450 nm, as outlined by Gorin et al. for this enzyme. ~6 The results are presented in Table I. The activity measured for the crystalline (out ofsupplier's bottle) lysozyme was 21.3 units/mg, while the evaporated-dried lysozyme yielded an activity of 19.9 units/mg; that is, an approximate 7% activity loss occurred as a result of the dehydration. The activity loss attributed to dehydration (7%) and the loss estimated by particle beam operation


o

<

o Z

0.98

0.96

0.94

0.92

0.90

0.88

0.86

0.84

0.82

0.80

I I I I I I

©

I I I I I I

0 1 2 3 4 5

Time / minutes

FIG. 8. Lysozyme activity assay monitored with a visible diode array spectrophotometer. Absorbance behavior at 450-nm wavelength for 5 rain; 0.1 mg/mL Micrococcus lysodeikticus substrate reacted with crystalline, evaporated-from-water, and particle-beam lysozyme. (- - --O-- - -) Sub- strate and crystalline lysozyme; ( - - - Q - - - ) substrate and evaporated lysozyme; ( - - - - £ 3 - - - ) substrate and lysozyme collected from particle beam.

(19%) constitute a portion of the difference between crystalline and particle beam lysozyme. These results suggest that a majority (approximately 74%) of the lysozyme that has passed through particle beam apparatus retains its activity. For a majority of the lysozyme injected into particle beam, we therefore conclude that the higher-order structural features, particularly the active-site cleft, were not altered.

Solvent Perturbation. Figure 9 shows the spectra of structurally altered ~LG that were used as a means of comparison for assignments from experimental data. Sol- vents and strong acidic/alkaline solutions were used so that we could obtain spectra of denatured ~LG./3-sheet separations induced by ethylene glycol (Fig. 9A) result from a decrease in the hydrophobic interaction which is partially responsible for the ordering in the native conformation, z5 The 100% methanol spectrum shows a marked increase in the intensity of the o~-helix contri- bution at 1652 cm -~ (Fig. 9B). A globular protein in methanol will tend towards a structure predominated by a-helicity, since the hydrogen-bonding capacity of proteins is different in methanol than it is in HzO. The similarity of the amide II contour in each spectrum (at ap-

TABLE I. Spectrophotometric activity assay of lysozyme.

Specific activity

(Units- mg- ~)

Form of Absorbance behavior (A.U.) based450_nmOn

lysozyme AA45o n m , 3 m i n ~ k a 5 7 0 n m , 3 m i n absorbance

Crystalline 0.094 0.064 21.3 Evaporated film 0.088 0.061 19.9 Particle beam 0.071 0.048 16.1

1262 Volume 48, Number 10, 1994

A

1655 1639

1536

B

1( 1634

1539 1412

1530

C

W 1640

D

laoo 1600 1400 1200 Wavenumber / cm

FIe. 9. Solid thin-film spectra of denatured ¢~-lactoglobulin, amide I, H, and Ill regions. These spectra represented the extremes of structural degradation against which experimental spectra were compared. (A) 50% ethylene glycol/50% water; (B) BLG evaporated from 100% methanol; (C) ~LG denatured to a pH of 1.0 with dilute HC1; (D) ¢~LG denatured to high pH of 13.0 with dilute NaOH.

proximately 1530 cm-~) illustrates the insensitivity of the amide II band towards conformation changes. The 1412- cm-~ band is related to the methanol solvent. Exposure of 3LG to pH extremes causes it to dissociate from a dimer into two monomers. Although/~LG was exposed to very acidic conditions, that is, close to pH = l (Fig. 9C), there was very little change in the native conformation, as evidenced by the spectrum. At low pH, the /3LG dissociation occurs with no other accompanying conformational change? a This spectrum is very similar to the native buffer ~LG film spectrum, with the exception of the increased 1721-cm- l absorption (Fig. 9C), which is related to excess salt. At high pH (Fig. 9D), the dissociation appears in concert with a conformational alteration, or denaturation; much of the ordered secondary structure composition is lost. In the spectrum at pH 13, many of the conformation bands are changed. This spec-

Original Deconvolved Deconvolved & Curvefit Amide I, I1, & II1 Amide I & II Amide III

1310 cm"

B ~ 1239 cm'~.

<

? ~ _ _ ~ ~ 1313 cm.t 1240 era" 0.011 A.U. 0.012 A.U.

1800 1600 1400 1200 1750 1700 1650 1600 1550 1500 1320 1280 1240

Wavenumber / cm t Wavenumber / cm -~ Wavenumber / cm"

FIG. 10. ~-lactoglobulin particle beam deposi ts f rom an aqueous mobile phase with an increasing me thano l concentrat ion. Original contours, deconvolved, and curve-fit amide I and II, and the deconvolved amide III. (A) Gradient at beginning o f acquisit ion, 100% water; (B) 50% water/ 50% methanol; (C) 25% water/75% methanol. The intensity o f the B-sheet 1635-cm- ' decreases as the 1655-cm- ' a-helix increases as the me thano l content increases.

trum was collected immediately after the addition of NaOH.

Much information about protein structure can be obtained from spectra of proteins that have been exposed to structure-altering nonaqueous solvents; these were introduced into the particle beam by means of a gradient elution HPLC program. The resulting spectral changes were followed by IR spectrometry and provided reference for band assignments. Although nonaqueous solvent systems often irreversibly perturb protein structural integrity, we demonstrate the worth of this experiment and show IR band assignment definitiveness within the complex protein spectrum. Timasheff et al. measured the effect of the intensity change of amide I IR bands as the structure transition from predominant ~-sheet to a-helix structure occurred with a series of D20/CH3OD mixtures? A trend of increased amide I a-helix intensity was observed with increased CH3OD concentration. In similar fashion, a final experiment was performed in this study to determine whether the particle beam could be operated in this mode and could successfully desolvate proteins in changing solvent environments. Solvents that cause known structural changes were used, e.g., methanol for the formation of a-helices and ethylene glycol for the separation of S-sheets. 25 This experiment was performed by a con- tinuous particle beam acquisition of five injections of 1% (w/v)/~LG each in a different solvent environment that started with 100% water; the percentage methanol was increased by 25% after the previous collection was made. In this manner,/~LG was exposed to greater concentrations of methanol with each successive injection, and caused more/F-sheet to a-helix conversion. A large 32- mm-diameter CaF 2 window was used as the collection substrate and was mounted onto the holder in such a way that the shaft was rotated approximately 70 ° after each deposit; thus a clean portion of the window was moved into the beam path. Although irregular, a single collection track of five deposits was easily made in this configura-

o r~o

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o Z

1 . 9 ¸

1.8 1.7 1.6

1.5 1.4

1.3 1.2

1.1

1.0 0.9

0.8

0.7 0.6 0.5

% (v/v) H20 100 75 50 25

I I I I [].

' . . ......... No buffer

........ ~. Amide III " ' " " . . .

Amide I ............

0

I I I I

0 25 50 75 100

% (v/v) Methanol Fzo. 11. Intensi ty changes in amide bands of/3-1actoglobulin induced by cartier liquid gradient. ( - - - - O - - - - ) Amide I A~_..oo, 1635 cm ~/ A.~ho.x 1657 cm ~; ( - - - ~ - - - ) amide II Ae_s.=, 1516 cm '/A._he.x 1531 cm ~; ( - - - - f l Y - - - ) a m i d e III A,_.,oc, 1244 cm ~/A._.e,~ 1315 c m - L

tion. A separate experiment to observe/3LG amide III bands was made with only three different solvent concentrations and without the phosphate buffer, since it masks the amide III region. The original contours of the particle beam spectra show the intensity changes that occur in the amide I and II bands with increasing methanol concentration. Representative spectra are illustrated in Fig. 10. Again, the most noticeable change occurs with the amide I bands. Clearly, the intensity of the a-helix component (approximately 1660 cm -~) increases with a simultaneous decrease in the intensity of the/3-sheet component (approximately 1635 cm- ~). Band positions were confirmed by deconvolution. Also in Fig. 10 are the deconvolved and curve-fit contours of these spectra, which illustrate, to a greater extent than the original spectra, the trends with increasing methanol concentration. These results are displayed in Fig. 11 as ratios of r-sheet to a-helix band intensities for the amide I, II, and III regions. For the amide II region the curve-fit/3-sheet band is located at 1516 cm -~, and the curve-fit a-helix band is at 1531 cm -~. The deconvolved band positions in the amide III are 1244 cm -1 for the/3-sheet band and 1315 cm -~ for the a-helix band. Spectra were normalized to 0.5 absorbance units before band intensities were measured. The amide I and III bands have steep slopes indicative of the conformational sensitivity of these bands. A moderate slope of the amide II follows previous findings that this backbone band, for the most part, does not change with conformation displacements. Moreover, evaporated films of the methanol/water concentrations that correspond to those used in the particle beam acquisition were measured, and no significant spectral intensity changes were observed. Apparently, as the methanol evaporated in the vacuum desiccator, the protein crystallized on the win-


dow to a conformation very close to its native conformation. With the particle beam deposits, the dynamic conformation was maintained upon impact. An attempt was made to pass a low-concentration ethylene glycol and water solvent with BLG through the particle beam apparatus. It was impossible, however, to desolvate BLG from the nonvolatile ethylene glycol, and no deposits were collected. The use of other structurally altering substances in the particle beam apparatus is presently under investigation. With variations in the composition of the mobile phase or carrier liquid, real-time protein structural changes can be induced and detected with the particle beam apparatus in a single study.

C O N C L U S I O N

We have demonstrated the ability to desolvate proteins from complex solutions with a particle beam system, and have shown that the protein structural integrity is maintained. The processes of liquid jet formation and desolvation cause no degradation to either protein studied. With B-lactoglobulin the harsh impact onto the substrate slightly altered the B-sheet bands; however, no apparent alteration in the secondary structure was observed with lysozyme. Furthermore, through biological activity as- says it was found that the tertiary and quaternary structural integrity of lysozyme was largely maintained. It is presently under investigation whether the given conformational make-up of proteins determines their stability through the particle beam apparatus. Both BLG and lysozyme particle beam spectra produced secondary structure estimates similar to their solution estimates. Similar liquid-solid spectral correlations with a variety of proteins (molecular weights and conformation content) are on-going.

It has been shown that the particle beam apparatus can prepare proteins for IR analysis from very dilute solutions (nanogram quantities injected). Reproducible IR micro- scopic spectra of the protein depositions, with high signal- to-noise ratios, are readily obtainable. Particle beam spectra were obtained at concentrations well below that of any ancillary liquid sampling technique used in this study. Lastly, it was demonstrated that particle beam spectra can be used with HPLC gradient programs to introduce structurally altering substances which enable assignment of the numerous protein IR amide bands to be made. The differences in original band contours caused by methanol were discernible without mathematical manipulations such as deconvolution. With the utilization of the rapid desolvation feature of the particle beam, future work will involve the trapping of folding/refolding intermediates for spectroscopic analysis and adaption of the particle beam to various types of protein chromatography [e.g., hydrophobic interaction chromatography (HIC) and re- versed-phase HPLC].

ACKNOWLEDGMENTS

The authors gratefully acknowledge the Perkin-Elmer Corporation for partial support of this research. We also thank D. M. Byler of the Philadelphia College of Textiles, and R. Mendelsohn of Rutgers Uni- versity for helpful suggestions. This paper was presented in part at the 9th International Conference on Fourier Transform Spectroscopy, Cal- gary, Canada (1993).

1. S. Krimm, J. Mol. Biol. 4, 528 (1962). 2. H. Susi, "Infrared Spectra of Biological Macromolecules and Re-

lated Systems," in Structure and Stability of Biological Molecules, S. N. Timasheff and G. D. Fasman, Eds. (Marcel Dekker, New York, 1969), pp. 575-633.

3. S. N. Timasheffand H. Susi, J. Biol. Chem. 241, 249 (1966). 4. H. Susi, S. N. Timasheff, and L. Stevens, J. Biol. Chem. 242, 5460

(1967). 5. S. N. Timasheff, H. Susi, and L. Stevens, J. Biol. Chem. 242, 5467

(1967). 6. A. Elliot, Proc. Roy. Soc. A221, 104 (1954). 7. P. Dory, A. Wada, J. T. Yang, and E. R. Blout, J. Polymer. Sci. 23,

851 (1957). 8. D. M. Byler and H. Susi, Biopolymers 25, 469 (1986). 9. R. J. Jakobsen, F. M. Wasacz, J. W. Brasch, and K. B. Smith,

Biopolymers 25, 639 (1986). 10. F. M. Wasacz, J. M. Olinger, and R. J. Jakobsen, Biochemistry 26,

1464 (1987). 11. R. J. Jakobsen and F. M. Wasacz, "Effects of the Environment on

the Structure of Adsorbed Proteins: Fourier Transform Infrared Spectroscopic Studies", in Proteins at Interfaces: Physiochemical And Biochemical Studies, J. L. Brash and T. A. Horbett, Eds. (Amer- ican Chemical Society, Washington, D.C., 1987), p. 339.

12. R. C. Willoughby and R. F. Browner, Anal. Chem. 56, 2626 (1984). 13. J. A. de Haseth and R. M. Robertson, Microchem. J. 42, 1365

(1989). 14. n . A. McKenzie, "/3-Lactoglobulins", in Milk Proteins: Chemistry

and Molecular Biology, Vol. II, H. A. McKenzie, Ed. (Academic Press, New York, 1971), p. 257.

15. D. Shugar, Biochim. Biophys. Acta 8, 302 (1952). 16. G. Gorin, S-F. Wang, and L. Papapavlou, Anal. Biochem, 39, 113

(1971). 17. A. Fersht, Enzyme Structure and Mechanism (W. H. Freeman and

Company, New York, 1985), 2nd ed., p. 30. 18. V. E. Turula, J. A. de Haseth, and R. F. Browner, "Estimation of

Globular Protein Secondary Structure by Solvent-Elimination MAGIC/FT-IR", presented at the 19th Annual Meeting of the Fed- eration of Analytical Chemistry and Spectroscopy Societies (FACSS), Philadelphia, Pennsylvania (1992).

19. J. M. Purcell and H. Susi, J. Biochem. Biophys. Methods 9, 193 (1984).

20. P. W. Holloway, Proc. SPIE-Int. Soc. Opt. Eng. 1145, 266 (1989). 21. P. W. Holloway and H. H. Mantsch, Biochemistry 28, 931 (1989). 22. T. Hirschfeld, "Quantitative FF-IR: A Detailed Look at the Prob-

lems Involved", in Fourier Transform Infrared Spectroscopy: Ap- plications to Chemical Systems, J. R. Ferraro and L. J. Basile, Eds. (Academic Press, New York, 1979), Vol. 2, p. 193.

23. P. R. Gritfiths and J. A. de Haseth, Fourier Transform Infrared Spectrometry (John Wiley and Sons, New York, 1986), p. 38.

24. V. E. Turula and J. A. de Haseth, unpublished results, University of Georgia, May, 1993.

25. J. S. Singer, "The Properties of Proteins in Nonaqueous Solvents", in Advances in Protein Chemistry, C. B. Anfinsen, M. L. Anson, K. Bailey, and J. T. Edsall, Eds. (Academic Press, New York, 1962), p . l .

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