254
Langmuir 1991, 7, 254-261
Protein-Metal Interactions in Protein-Colloid Conjugates Probed by Surface-Enhanced Raman Spectroscopy Angela M. Ahernt and Robin L. Garrell* Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 Received May 3, 1989. I n Final Form: July 13, 1990 Surface-enhanced Raman (SER) spectroscopy has been used to assess protein-metal interactions in colloidal immunoprobes. The absence of a detectable signal from conjugates of colloidal gold with antirabbit IgG, albumin-biotin, or protein A suggests that the proteins are not chemisorbed on the metal particles. Similarly, albumin, albumin-biotin, and protein A are not covalently bound to colloidal silver, but rather adsorb on silver initially with the protein hydration shell intact. Over a period of days, new SER bands appear in the albumin spectrum that are attributed to vibrations of aromatic amino acids. This implies that conformational changes are occurring in the protein near the interface. The time scale for these changes is consistent with what has been observed by circular dichroism and UV-visible spectroscopy. The effect of avidin on the binding of biotin to colloidal metal probes has also been characterized. The SERS results indicate that biotin binds to a cleft or pocketlike structure in avidin, restricting biotin-surface interactions. This study demonstrates the utility of SER spectroscopy for characterizing protein-metal interactions and the availability of adsorbed biomolecules for complexation. Protein-colloid conjugates are widely used to help locate and quantitate antibody-antigen binding sites in cells and tissues by light and electron microscopy.ls2 The probes are prepared by mixing silver or gold colloid particles, typically 5-20 nm in diameter, with a solution of the antibody. While adsorption takes place almost immediately,2 little is known about how the protein binds to the metal. Because the bioactivity is usually not destroyed, it has been assumed that the proteins do not bind covalently to the colloid particle surface.' At the very least, the retention of bioactivity suggests that the metal surface does not significantly perturb the protein structure in the vicinity of the antigen binding site. We have used surface-enhanced Raman spectroscopy (SERS) to characterize the interactions between proteins and colloidal metal surfaces. The aims were to determine (1) whether proteins directly adsorb on the colloid surface, (2) whether proteins covalently bind to colloidal noble metals, and (3) how bioactivity is retained in adsorbed proteins. These questions have broader significance in relation to the biocompatibility of prosthetic devices and the development of enzyme-based sensors and analyzers.384 SERS is an extremely sensitive technique for monitoring the adsorption of species from aqueous solution and for characterizing the adsorbate orientation and strength of interactions with noble metal substrates. The mechanisms for the enhancement effect, the surface selection rule for determining adsorbate orientation, and applications of the technique have been reviewed.596 In SERS, the intensity of Raman bands for small molecules and ions adsorbed on certain rough metal surfaces may be enhanced
* Author to whom correspondence should be addressed. Present address: Aluminum Company of America, Alcoa Technical Center, Alcoa Center, PA 15069. (1)Horisberger, M. In Preparation of Biological Specimens for Scanning Electron Microscopy; Murphy, J. A., Roomans, G. M., Eds.; Scanning Electron Microscopy, Inc.: Chicago, IL, 1984; pp 315-336. (2) Roth, J. In Techniques in Immunocytochemistry, Bullock, G. R., Petrusz, P., Eds.; Academic Press, London, 1983; Vol. 2, pp 216-284. (3) Jakobsen, R. J.; Wasacz, F. M.; Smith, K. B. In Chemical,Biological and Industrial Applications oflnfraredSpectroscopy;Durig, J . R., Ed.; John Wiley and Sons: Chichester, 1985; pp 199-213. (4) Frew, J. E.; Hill, H. A. 0. Anal. Chem. 1987,59, 933A-944A. (5) Moskovits, M. Reu. Mod. Phys. 1985, 57, 783. (6) Koglin, E.; SBquaris, J.-M. Top. Curr. Chem. 1986, 234, 1. +
up to 6 orders of magnitude compared to the same modes for species in ~ o l u t i o n . The ~ electromagnetic fields responsible for most of the enhancement are localized near the surface. SERS is therefore most sensitive to species 0-10 A from the ~ u r f a c e .An ~ advantage of SERS as a probe of solution/metal interfaces, particularly for biological applications, is that water is a weak Raman scatterer and, under most experimental conditions, will not exhibit enhanced Raman ~ c a t t e r i n g . ~ ? ~ The utility of SERS and surface-enhanced resonance Raman spectroscopy (SERRS) for characterizing the interactions between biological molecules and metal surfaces has been demonstrated for amino proteins,6J5nucleosides, nucleotides and DNA,lG flavins,17J8 and p o r p h y r i n ~ . ~Results 9 ~ ~ ~ of SERRS investigations suggest that adsorption of flavoproteins on silver electrodes leads to denaturation and/or to dissociation of the chromophores from the proteins.l7J8 Very few nonresonant SERS studies of proteins have been reported; however, both Koglin and SBquaris6 and Nabiev and Chumanovll obtained SER spectra of bovine serum albumin (BSA) and lysozyme. A number of spectral features were assigned to vibrations of the aromatic amino acid side chains. Recently a SER spectrum of avidin was reported in a study of the binding of avidin to 2-((4'-hydroxyphenyl)azo)(7) Koglin, E.; Lewinsky, H. H.; SBquaris, J. M. Surf. Sci. 1985, 258, 370. (8) Chen, T. T.; Owen, J. F.; Chang, R. K.; Laube, B. L. Chem. Phys. Lett. 1982, 89, 356. (9) Saski, Y.; Iwasaki, N.; Nishina, Y . Surf. Sci. 1988, 198, 541. (10) Nabiev, I. R.; Savchenko, V. A.; Efremov, E. S. J. Raman Spectrosc. 1983, 14, 375. (11)Nabiev, I. R.; Chumanov, G. D. Biophysics 1986, 32, 199. (12) Suh, J. S.; Moskovits, M. J. Am. Chem. SOC.1986, 208,4711. (13) Kim, S. K.; Kim, M. S.; Suh, S. W. J.Raman Spectrosc. 1987,18, 171. (14) Lee, H. I.; Suh, S. W.; Kim, M. S. J.Raman Spectrosc. 1988,29, 491. (15) Cotton, T. M.; Kaddi, D.; Iorga, D. J. Am. Chem. SOC.1983,105, 7462. (16) Otto, C.; deMul, F. F. M.; Huizinga, A.; Greve, J. J. Phys. Chem. 1988., 92. 1239. --, ~ - - (17) Lee, N.-S.; Hsieh, Y.-2.; Morris, M. D.; Schopfer, L. M. J. Am. Chem. Soc. 1987. - - - , -109. - - , 13.58. ---(18) Holt, R. E.; Cotton, T. M. J. Am. Chem. S O ~1987, . 109,1841. (19) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986,90,6017. (20) deGroot, J.; Hester, R. E. J . Phys. Chem. 1987, 92, 1693. ~~~~
0743-~463/91/2407-0254$02.50/0 0 1991 American Chemical Society
Protein-Metal Interactions benzoic acid (HABA).21The avidin SER spectrum exhibits one broad band centered at 1603 cm-l, which was not assigned. The authors were able to determine, however, that adsorption of the HABA-avidin complex on silver did not result in denaturation of the protein. In this paper, we show that SER spectroscopy of colloidal probes indicates that albumin, albumin-biotin, and protein A adsorb on silver with the protein hydration shell intact. Over a period of time, bands appear in the albumin SER spectrum that are attributable to vibrations ofthe aromatic amino acids Phe, Tyr, and T r p near the metal surface. The time dependence of the SER spectrum of albumin adsorbed on silver colloid has not previously been reported. The time scale for the albumin spectral changes (1week) is consistent with the results of UV-vis and circular dichroism (CD)spectroscopic studies, in which it was found that changes in the conformation of adsorbed albumin occur over a period of days.22123From our SER spectra we deduce that albumin, albumin-biotin, and protein A are not covalently bound to colloidal silver. We have also used SERS to investigate the binding of biotin (vitamin H) to avidin and find that biotin binds to avidin in a cleft or pocketlike structure that encases the biotin molecule, consistent with results of electron spin resonance studies.24 This study demonstrates the utility of SERS for obtaining information about the surface orientation and availability for complexation of adsorbed biomolecules.
Materials and Methods The following chemicals were used as received from Sigma Chemical Co.: bovine serum albumin fraction V powder (9899'"r), albumin-biotin (95? protein), anti-rabbit IgG (whole molecule),mannan from Saccharomycescereuisiae,poly(Asp.Na, GluaNa) (5000-15000 MW), and Trizma Base (reagent grade). The following were also used as received: protein A from Staphylococcus aureus and avidin (Polysciences,Inc.), glacial acetic acid (EM Sciences, reagent grade), NaOH (Mallinckrodt, analytical reagent), K2C03(EM Sciences, reagent grade), AgNO3 (Mallinckrodt, analytical grade), NaBH4(Aldrich,99+ %), chlorobutanol (1,l,l-trichloro-2-methyl-2-propanol, Aldrich, 98%), 2-methyltetrahydrothiophene (Alfa, 98Y0),DzO (Aldrich, 99.8 atom p( D), and colloidal gold (SPI, 10 nm and 20 nm). Biotin (Aldrich, 99", ) was decolorized with carbon black and recrystallized from doubly distilled water prior to use. 2-Imidazolidone (Aldrich, 96%) was recrystallized twice from chloroform. The following protein-colloidal conjugateswere used as received: anti-rabbit IgG (wholemolecule)-10 nm colloidal gold conjugate (Sigma Chemical Co.), albumin-biotin-10 nm colloidal gold' conjugate (Sigma Chemical Co.), protein A-colloidal silver conjugate (Polysciences, Inc.), and protein A-20 nm colloidal gold conjugate (Polysciences, Inc.). Doubly distilled water was used throughout. Silver colloids were prepared by dissolving 3.5 mg of NaBH4 in 75 mL of degassed 4 "C water. To this, 7.0 mL of 2.2 X M AgNO3 was added dropwise with stirring in the dark. The resulting sols were deep yellow, characteristic of silver colloids containing particles 10 nm in diameter.25These colloids were stable for at least 1 month without the addition of stabilizers but were prepared fresh and aged for only 1 day prior to use. The colloidal probes were prepared by using procedures similar to
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(2!) (a) Ni. F.; Cotton, T. M. J. Raman Spectrosc. 1988, 19,429. (b) In NI and Cotton's investigation, the avidin Raman spectrum was not resonantly enhanced with 363.3-, 457.9-, or 488.0-nmradiation;however, HABA is resonantly enhanced in this wavelength region. Ni and Cotton thus obtained a SER spectrum of avidin but a SERR spectrum of HABA. (22) Andrade, J. D.; Hlady, V. L.; Van Wagenen, R. A. Pure Appl. Chem. 1984,56, 1345. (23) Soderquist, M. E.; Walton, A. G. J . Colloid Interface Sci. 1980, 75. 386. (24) Chignell, C. F.; Starkweather, D. K.; Sinha, B. K. J . Biol. Chem. 1975,250, 5622. (25) Garrell, R. L. Ph.D. Thesis, University of Michigan, 1984, pp 3761.
Langmuir, Vol. 7, No. 2, 1991 255 those described elsewhere.'V2 The concentration of proteins used was similar to that used by others for colloidal probes,l,z20 pg/ mL (equivalent to about M for albumin, molecular weight 68 000). The pH of the colloids was adjusted to the isoelectric point (pl) of each protein unless otherwise specified, using CH3COOH, NaOH, KzC03, or Trizma base. None of these species gave rise to detectable SERS features in our experiments. Protein-colloid conjugates prepared in our laboratory were not stabilized with additives such as polyethylene glycol to prevent aggregation;they were allowed to equilibrate for at least 30 min followingthe addition of protein to the sol and prior to spectral acquisition. Samples that were equilibrated for more than 30 min were stored at 0-5 "C during the equilibration. In some experiments, a rotating disk silver electrode was used as the substrate for SERS. The preparation of the electrode and the instrumentation involved are described elsewhere.26 The electrode was roughened in 0.1 M KCl by successive oxidationreduction cycles in the absence of the analyte. The roughened electrodewas then placed in a cell with fresh electrolytecontaining the potential adsorbate. A saturated calomel electrode (SCE) was used as the reference electrode and a platinum wire served as the auxiliary electrode. All Raman spectra were obtained with a Spex 1403 double monochromator equipped with 1800 grooves/" holographic gratings, an RCA C31034 photomultiplier tube detector, and a Spex DMlB computer. A Cooper Lasersonics Model 150 argon ion laser (X = 514.5 nm) was used as the excitation source for the experiments withsilver substrates. The argon ion laser was used to pump an Aurora 600 DCM dye laser at 647 nm for the experiments with gold. Unless otherwise specified, all spectra were obtained with 100 mW of radiation at the sample, 200-pm slit widths, and 2-cm-l resolution for 514.5-nm excitation (270pm slit widths and 1.9-cm-l resolution for 647-nm excitation) and were single scans at a rate of 2 cm-1/5 s. A five-point polynomialsmoothingfunction was applied to all Raman spectra. UV-visible absorption spectra were recorded on an IBM 9420 scanning spectrophotometer.
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Results and Discussion A. Absorption Spectra. UV-visible absorption spectrophotometry was used as a n indirect probe of the adsorption of proteins from solution onto the surface of the metal colloid particles. Adsorption can lead to aggregation and precipitation of the which is manifested in changes in the absorption spectrum. An unaggregated silver colloid comprised of 10 nm diameter 400 nm with a full width at half particles has A,, maximum (fwhm) of -84 nm, while a similar gold colloid (average particle diameter of 10 nm) has A,, 520 nm. Adsorbate-induced aggregation leads to increased absorption at longer wavelengths and broadening of the primary absorption band, and often to the appearance of a secondary absorption peak that is red-shifted relative to the primary absorption band.28 The molecular weights and p l values of the proteins studied here are given in Table I.2929 At the pl, a protein has zero net charge. Protein-water interactions are less favorable than when the protein is charged and solubility in water is at a minimum. Repulsive electrostatic interactions between protein molecules are also at a minimum a t the PI.~O These factors should combine to favor adsorption on metal surfaces near the pl. Because metal colloid particles are slightly positively charged in aqueous
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(26) Garrell, R. L.; Beer, K. D. Spectrochim. Acta 1988,43B, 617. (27) von Raben, K. U.; Chang, R. K.; Laube, B. L.; Barber, P. W. J. Phys. Chem. 1984,88, 5290. (28) Creighton,J. A.; Blatchford, C. G.;Albrecht, M. G. J.Chem.Soc., Faraday Trans. 2 1979, 75, 790. (29) Horisberger, M.; Rosset, J. J. Histochem. Cytochem. 1977, 25, 295. (30) Kinsella, J. E.;Whitehead, D. M. In Proteins at Interfaces: Physicochemical and Biochemical Studies; Brash, J. L.; Horbett, T. A,, Eds.; American Chemical Society: Washington, DC, 1987; pp 629-646.
Ahern and Garrell
256 Langmuir, Vol. 7, No. 2, 1991 Table I. Protein Molecular Weights a n d Isoelectric Points protein albumin (fraction V) albumin-biotin biotin anti-rabbit IgG protein A avidin mannan
mol w t 68 000 68 241 241 150 000 42 000 67 000 60 000
PI0 5.2-5.5 5.2-5.5 not applicable 7.6 5.9-6.2 10.0-10.6 7.0
n
U J
P 0
From ref 2.
v
1
1.0
t
E WAVELENGTH (nm)
Figure 1. UV-vis absorption spectra: (-) silver colloid; (- - -) silver colloid albumin at PI; ( 0 )silver colloid + albumin a t p H = 8.0; (A)silver colloid + albumin-biotin; (w) silver colloid + 9.0 X M biotin.
+
solution, protein adsorption is maximized at pH values a t or slightly basic of the ~ 1 . ~ 3 3 ~ The UV-visible absorption spectrum of colloidal gold without protein did not vary with pH. Spectra of the colloid with added albumin, albumin-biotin, poly(Asp-Na, Glu-Na),and biotin showed only the single band at -520 nm, with no evidence of extensive aggregation of the colloid. The absorption spectra of gold conjugates with protein A, anti-rabbit IgG, and albumin-biotin also showed no evidence of colloidal aggregation. These data indicate that the degree of aggregation of the colloid was unaffected by the presence of protein or by changes in pH. The absorption spectra of a silver colloid and silver colloid with albumin, albumin-biotin, and biotin (vitamin H) added are shown in Figure 1. The silver colloid spectrum (solid line) has the characteristic band centered at 396 nm. As was the case for gold, adjusting the pH to the various p l values listed in Table I with Trizma base, acetic acid, sodium hydroxide, or potassium carbonate did not alter the absorption spectrum. The same spectrum is obtained if avidin, anti-rabbit IgG, poly(Asp.Na, GlwNa), or mannan (a polysaccharide) is added, indicating that these macromolecules do not cause substantial aggregation of the colloid. On the basis of their absorption spectra, these macromolecules will be described as belonging to class I. Albumin, albumin-biotin, and biotin all cause aggregation of the colloid as evidenced by the shift in the 396-nm band to longer wavelengths and an increase in the fwhm. These adsorbates comprise class 11. As will be shown below, SER spectra could only be obtained from molecules in class 11. B. Protein a n d Biotin Raman Spectra. The Raman spectra of biotin, albumin-biotin, albumin, and antirabbit IgG are shown in Figure 2. Because biotin is not a protein, its spectrum (Figure 2a) is very different from that of albumin (Figure 2c), whose spectrum is very similar to that of the albumin-biotin complex (Figure 2b). Comparison of the Raman spectrum of albumin (Figure 2c) with that of anti-rabbit IgG (Figure 2d) reveals a number of similarities. These include the bands at 1002 ~~
(31)Wetzel, H.; Gerischer, H. Chem. Phys. Lett. 1980, 76, 460.
1~0.251
la00
300
RAMAN SHIFT (cm-') Figure 2. Raman spectra of (a) biotin (solid), obtained with 15 mW of 514.5 nm incident radiation, 1 cm-I resolution, scan rate of 1 cm-l/s; (b) albumin-biotin complex (solid), obtained with 100 mW of 514.5 nm incident radiation, 2 cm-l resolution, scan rate of 1 cm-'/5 s; (c) aqueous albumin (500 mg/mL), obtained with 66 mW of 514.5-nm incident radiation, 1.5 cm-I resolution, scan rate of 1 cm-'/s; (d) anti-rabbit IgG (solid), obtained with 100 mW of 514.5-nm incident radiation, 2 cm-l resolution, scan rate of 1 cm-'/5 s.
cm-I (Phe), 1011cm-I (Trp), and 1033 cm-l (Phe) assigned to aromatic amino acid side chain vibrations, the amide I and amide I11 modes a t 1650-1670 and 1239 cm-l, respectively, and the CH and CH2 deformation modes at 1337 and 1450 cm-1.32*33The similarities between the Raman spectra of different proteins (Figure 2c,d) suggest that the SER spectra of different proteins may also be very similar and t h a t globular proteins may not be readily distinguishable by nonresonant SERS. C. S E R S of Colloidal Gold Probes. We have been unable to obtain SER spectra of albumin, albumin-biotin, poly(Asp.Na, GluNa), biotin, anti-rabbit IgG, or protein A adsorbed on colloidal gold, whether the conjugates are obtained commercially or prepared in our laboratory. The commercial gold probes are supplied as 50% glycerol solutions; for these materials we obtain only normal (unenhanced) Raman spectra of glycerol with no other spectral features that could be attributed to enhanced Raman scattering from adsorbed proteins. As indicated above, for gold conjugates prepared in our laboratory we observe no protein-induced aggregation, even at the protein isoelectric points. The lack of enhanced scattering could be due either to a lack of sufficiently extensive adsorption of protein on the colloid particle surface or to a lack of sufficient aggregation of the colloid particles. Although colloid aggregation is not required for observing enhanced Raman scattering from adsorbates, aggregates do contribute to the SER scattered intensity disproportionately to their c o n ~ e n t r a t i o n .For ~ ~ ~adsorbates ~~ with low Raman scattering cross sections, such as the proteins inves(32) Bellocq, A. M.; Lord, R. C.; Mendelsohn, R. Biochim. Biophys. Acta 1972,257, 280. (33) Chen, M. C.; Lord, R. C. J. Am. Chem. SOC.1976, 98,990. (34) Garrell, R. L.; Shaw, K. D.; Krimm, S. Surf. Sci. 1983, 124, 613.
Protein-Me tal Interact ions
Langmuir, Vol. 7, No. 2, 1991 257
tion, applied potential, or pH. Hsieh et al.41were also unable to obtain a SER spectrum of albumin on a silver electrode (concentration -68 pg/mL (lo4 M), pH = 7.4). Koglin and SBquaris6 reported a vibrationally rich SER spectrum of 20pg/mL albumin M) adsorbed on silver a t -900 mV VN SCE, pH = 8.0. Nabiev and Chumanovll also obtained a SER spectrum of albumin on silver, at -670 mV and a concentration of -68 pg/mL (10-6 M). There are significant differences between their spectrum and the spectrum reported by Koglin and SBquaris that may be due to differences in the roughening procedures employed, the details of which were not given in either work. Furthermore, those roughening procedures may have differed from those employed in the present work where no SER spectrum could be obtained. Hsieh et al., who used a roughening procedure very similar to the 550 1750 procedure employed here, were also unable to obtain a RAMAN SHIFT (cm -9 SER spectrum of albumin. Figure 3. S E R and Raman spectra of albumin: (a) SERspectrum The major feature in the SER spectra of albumin (Figure of50mg/mL (7.4 X 10-4M)albumin,pH = 8.0; (b) SERspectrum 3b) and protein A (not shown) and in the spectrum of of 20 pcg/mL (2.9 X lo-' M) albumin, p H = p l or p H = 8.0; (c) albumin-biotin (to be discussed below) is a broad band Raman spectrum of 20 pg/mL (2.9 X 10-7 M) albumin, p H = p l or p H = 8.0. centered a t about 1640 cm-l. In Raman spectra of concentrated aqueous proteins (40 mg/mL) an intense broad band centered a t 1650 cm-l has been assigned to tigated here,35more extensive aggregation may be required the amide I mode plus the water bending mode.33 The in order to observe enhanced Raman scattering. The fact potential energy distribution of the amide I mode is that gold surfaces provide 200 times lower enhancement approximately 80 % carbonyl stretching vibration, with of Raman scattering than do silver surfaces may also much of the remainder due to the N-H in-plane bending partially account for the difficulty in obtaining SER spectra vibration. In very dilute protein solutions (20 pg/mL), a of the proteins on colloidal gold.36 As noted below, even band at 1650 cm-' is observed that is due to the u2 bending the SER spectra of proteins adsorbed on colloidal silver mode of water, as shown in Figure 3c. We originally are weak. believed that the 1640-cm-l band of the SER spectrum of D. Colloidal Silver Probes. 1. Albumin and Proalbumin (Figure 3b) was due to enhanced scattering from tein A SERS. Albumin is a large globular protein the amide I mode. No one has yet observed preferential containing 578 amino acid residues. It has a repeating loop structure due to the presence of 17disulfide bonds.3'~~~ enhancement solely of the amide I mode, however,6 and the intensity enhancement of the 1640 cm-' band is only The loops have high a-helical content, and the repeating a factor of 2-4 relative to the 1650-cm-lband in the aqueous loop structure can be divided into three domains that can albumin spectrum (Figure 3c). We therefore attribute be divided further into six subdomains. Albumin in its the increased intensity of the 1640-cm-' band to surface hydrated form is a prolate ellipsoid with dimensions of 14 enhancement of the u2 bending mode of structured water X 4 X 4 nm39 that adsorbs on surfaces in a side-on associated with the protein. NMR studies have shown 0rientation.~3 Although the protein is slightly larger that the reorientational dynamics of bulk water are than the 10 nm diameter silver colloid particles, adsorption significantly reduced in the two water layers close to the of albumin only requires that a small area of the protein protein and that translational diffusion is decreased 5-10adhere to the s u r f a ~ e . This ~~,~ anchor ~ is strong enough fold within 10 A of the protein surface.42 to bind albumin to the surface. The actual contact area It is unlikely that the increased intensity of the 1640-cm-' of adsorbed albumin is very difficult to e~timate.~O band in the SER spectrum compared with the aqueous SER spectra for two different solution concentrations albumin spectrum is attributable to SER scattering from of albumin adsorbed on colloidal silver at pH = 8.0 and free water or to water bound to the colloid surface and not an unenhanced Raman spectrum of aqueous albumin are associated with protein. Under conditions of ionic strength shown in Figure 3. At 20 pg/mL (lo-' M), only one SER and applied (or surface) potential similar to our experiband is observed, centered at 1640 cm-l (Figure 3b). A ments, water molecules do not exhibit enhanced Raman similar SER spectrum is obtained at pH 5.5. If the albumin ~ c a t t e r i n g . ~SERS ~ of water and, in particular, the concentration is increased 3 orders of magnitude to 50 enhancement of the u2 mode have only been observed a t mg/mL (Figure 3a), the spectrum is richer in vibrational a silver electrode surface in the presence of 21 M salts8 information but shows irreproducible features attributable or at applied potentials more negative than-1.3 V vs SCE.9 to Rayleigh scattering from the suspended protein molThese conditions do not exist in our experiments. ecules. It was not possible to obtain a SER spectrum of In order to test the validity of attributing the increased albumin adsorbed on a silver electrode at any concentraintensity of the 1640-cm-1band to enhancement of the u2 mode of water rather than to enhancement of the amide (35) Thomas, G. J., Jr. In Vibrational Spectra and Structure; Durig, I mode, we prepared colloid and albumin solutions in 50/ J. R., Ed.; Marcel Dekker, Inc.: New York, 1975, Vol. 3; pp 239-315. (36) von Raben, K. U.; Chang, R. K.; Laube, B. L. Chem. Phys. Lett. 50 (v/v) H20/D20. If the increased intensity is attrib1981, 79, 465. utable to enhanced Raman scattering from the u2 bending (37) Brown, J. R. In Albumin: Structure, Function, and Uses; Rose-
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noer, V. M. Oratz, M., Rothchild, M. A., Eds.; Pergamon Press: New York, 1977; pp 27-51. (38) Young, P. R.; Tilghman, S. M. In Multidomain Proteins: Structure and Eoolution; Hardie, D. G., Coggins, J. R., Eds.; Elsevier Science Publishers: Amsterdam, 1986; pp 55-83. (39) Bendedouch, D.; Chen, S.-H. J. Phys. Chem. 1983,87, 1473. (40) MacRitchie, F. Adu. Protein Chem. 1987, 32, 283.
(41) Hsieh, Y.-Z.; Lee, N.-S.; Sheng, R.-S.; Morris, M. D. Langmuir 1987, 3, 1141.
(42) Ahlstrom, P.; Teleman, 0.;Jonsson, B. J.Am. Chem. SOC.1988, 110,4198. (43) Blatchford, C. G.; Kerker, M.; Wang, D.4. Chem. Phys. Lett. 1983, 100, 230.
Ahern and Garrell
258 Langmuir, Vol. 7, No. 2, 1991
- I
I
1700
900 RAMAN SHIFT (cm -1)
Figure 4. SER and Raman spectra of albumin in 50/50 (v/v) H*O/DzO: (a) SER spectrum of 20 pg/mL albumin; (b) Raman spectrum of 20 pg/mL albumin. mode of water, we would expect to observe the same magnitude of enhancement for the u2 modes of HDO and D2O. Three bands should be observed a t approximately 1650, 1440, and 1210 cm-l 44 in a 1:2:1 intensity ratio, in addition to the bulk (unenhanced) scattering from aqueous albumin a t 1650 cm-1. If instead the increased intensity of the band at -1650 cm-1 is attributable to enhanced Raman scattering from the amide I mode of albumin, upon deuteration we would expect to observe a broadening of the 1650-cm-' band on the low frequency 1.e. . an increase in intensity at about 1630-1640 cm-', because of the contribution from the amide I' mode (the deuterated analogue of amide I), and a concomitant decrease in peak height at 1650 cm-'. The area of the combined SER scattering for amide I and amide 1', plus the bulk aqueous albumin Raman scattering a t 1650 cm-l, would be expected to be constant. Figure 4 shows the Raman and SER spectra of albumin in H20/D20. Three peaks are evident in both spectra at 1210,1438,and 1638 cm-', which are assigned to the DzO, HDO, and HzO bending modes, respectively. These bands are all enhanced by a factor of 2-4 when albumin is adsorbed on the colloid surface. Absorption spectra (not shown) of the albumin-silver colloid H20/D20 mixtures are similar to the spectrum shown in Figure 1 (- - -), indicating adsorbate-induced aggregation. The aggregation of the colloid, along with the similar magnitude of enhancement for the DzO, HDO, and H20 bending modes, suggest that the increased intensity of the mode at 1640 cm-l in the albumin SER spectrum (Figure 3b) is attributable to enhanced scattering from water in the hydration shell of the protein. The OH stretching mode of bound H2O is not significantly enhanced. Determining whether it is weakly enhanced is not possible because of appreciable scattering from bulk water. In addition to the 1640-cm-' band, the SER spectrum of protein A (not shown) exhibits a weak band centered a t 1390 cm-l that has not yet been assigned. In other respects, the SER spectra of albumin and protein A are quite similar, providing evidence that globular proteins may not be readily distinguishable by nonresonant SER spectroscopy. A SER spectrum of avidin2I adsorbed on a silver electrode also exhibits a single broad band centered at 1603 cm-'. Our data suggest that this band may be
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(44) Califano, S. Vibrational States; John Wiley and Sons: London, 1976;p 242. (45)Tu,A.T. In Spectroscopy ofBiological Systems; Clark, R. J. H., Hester, R. E., Eds.; John Wiley and Sons: Chicester, 1986;pp 47-112.
Figure 5. UV-vis absorption spectra of albumin as a function of time: (-) silver colloid; (- - -) silver colloid + albumin, time = 0.5 h; (@) silver colloid + albumin, time = 24 h; (A)silver colloid + albumin. time = 170 h. attributed to the u2 bending mode of H2O in the hydration shell of avidin. Saski et al.9 have shown that the frequency of the u2 mode of water depends strongly on the experimental conditions, which may account for the 37-cm-l difference between the peak positions for the band in the avidin SER spectrum and the band in our albumin SER spectrum. Koglin and SBquaris6 and Nabiev and Chumanovll obtained SER spectra of albumin on silver electrodes that differ from each other and from the colloidal SER spectra presented here. Koglin and SBquarisassigned SER bands to the aromatic amino acids Phe and Tyr, to CH2 and CH3 deformation modes, and to the amide I and amide I11 modes. Nabiev and Chumanov concurred with the assignments of Koglin and S6quaris and assigned additional bands in their spectrum to the S-S stretching vibration of cystine and some of the aromatic ring modes of Trp. The origins of the differences between the albumin SER spectra obtained with colloids and with electrodes are being investigated in our laboratory. The time dependence of the absorption spectrum of albumin in a silver colloid is shown in Figure 5. Absorption spectra were recorded 0.5,24, and 170 h (1week) after the addition of albumin. As the elapsed time increases, the absorption maximum shifts to the red and the peak broadens, indicating an increase in the degree of aggregation of the colloid.27 The SER spectra of albumin as a function of time and pH are shown in Figure 6. Using the method of Curley and Siiman and the intensity of the 1004-cm-' band, we calculate that the magnitude of the surface enhancement effect has increased to -lo3 (from 2 to 4) after 1week.46 The increased intensity is probably due to a combination of increased adsorption and increased aggregation. The spectrum evolves over time to contain bands that we attribute to amide modes and to side chain vibrations in the aromatic amino acid residues, based on SER spectra of homodipeptides (Tyr-Tyr, Phe-Phe, and T r p - T r ~ ) . ~ ' The bands in the spectra shown in Figure 6a,b are attributed solely to the aromatic amino acids even though aliphatic amino acids comprise 92% of the protein. (Albumin has 26 Phe, 18Tyr and 2 Trp residues.32~37)Two factors may account for this apparent anomaly. First, the large Raman scattering cross sections of the aromatic ring modes of Phe, Tyr, and T r p may dominate SER spectral (46)Curley, D.;Siiman, 0. Langmuir 1988,4, 1021. (47)(a) Herne, T.M.; Ahern, A. M.; Garrell, R. L. J . Am. Chem. SOC. 1991,113, 846-854.(b) Garrell, R. L.; Herne, T. M.; Ahern, A. M.; Sullenberger, E. In Progress in Biomedical Optics: Optical Fibers in Medicine V [SPIEProceedings 12011;Katzir, A., Ed.;SPIE Bellingham, WA, 1990;pp 451-460.
Protein-Metal Interactions
Langmuir, Vol. 7, No. 2, 1991 259
point, we observe enhanced Raman scattering from water molecules associated with the protein. These water molecules may be immobilized near charged groups or may simply exhibit reduced radial diffusion as suggested by recent molecular dynamics simulation^.^^ Gradually, over a period of days, this structured water a t the proteinmetal interface is displaced and the protein adsorbs directly on the metal. When this occurs, we observe enhanced Raman scattering from aromatic amino acid residues near the surface. The adsorption of albumin on colloidal silver as monitored by SERS shows a time dependence similar to that for albumin adsorbed on other hydrophobic surfaces such as silicone. On the basis of studies with UV-vis spectrophotometry and CD spectroscopy, Soderquist and Walton have proposed a three-step adsorption/desorption process.23 In step 1,BSA rapidly ( 1min) and reversibly adsorbs in a side-on conformation. Step 2, which occurs over a period of days or weeks, involves protein conformational changes a t the surface that make desorption energetically unfavorable. I t is believed that hydrophobic interactions are the principal driving force for the adsorption of BSA and that the conformational changes that take place after adsorption serve to increase these interactions. These changes may include the unfolding of the protein to make portions of the albumin molecule's hydrophobic interior available for binding to the surface. In step 3, denatured BSA very slowly desorbs in a pseudoirreversible manner. Our results are consistent with the adsorption model of Soderquist and Walton and also provide more detailed structural information about the conformational changes taking place in step 2. The SER spectra in Figure 6 were obtained for albumincolloid samples containing no antimicrobial agent. In order to establish that the changes in the albumin SER spectra were not due to microbial growth, we attempted to obtain SER spectra of protein solutions containing antimicrobial agents. In the commercial colloidal probe preparations, 0.02 % sodium azide is added and the probes can be used and stored for about 1year.2 When 0.0270 sodium azide was added to our albumin-colloid mixture, we obtained a SER spectrum of azide that was similar to the published SER s p e c t r ~ m , with ~ g no features attributable to adsorbed albumin. When 0.01% or 0.02 % chlorobutanolw was added as a preservative to the albumin silver colloids, the albumin spectrum was enhanced but did not show time dependence. A spectrum similar to that in Figure 6c was obtained regardless of the time elapsed, up to 2 weeks, between the addition of albumin to colloid and the acquisition of a spectrum. These data suggest that the time dependence of the albumin SER spectra might have resulted from bacterial growth during the 1-week equilibration period. To test for the presence of bacteria, Sterigel (Carolina Biological Supply Co.) plates were streaked with silver colloid-albumin mixtures with and without chlorobutanol added. After 10 days, no bacterial growth was evident in either samples with or without chlorobutanol. These results indicate that the growth of bacteria is not a problem for samples that are allowed to equilibrate for 1 week. It is not clear why the spectral changes were not observed for albumin/silver colloid samples containing chlorobutanol. One reason may be that the time scale for albumin conformational changes a t the surface in the presence of N
- 0 950
1750
RAMAN SHIFT (cm -1)
Figure 6. SER spectra of albumin as a function of time: (a) 20 pg/mL albumin a t p H = p l = 5.5, time = 170 h; (b) 20 pg/mL albumin a t p H = 8.0, time = 170 h; ( c ) 20 pg/mL albumin a t pZ or p H = 8.0, time = 0.5 h. propin
Day 1: SERS
Of
H20
Day 7 : SERS of
Phe,Trp,Tyr
Figure 7. Schematic representation of the conformation changes taking place in albumin on a colloidal silver surface as a function of time.
features from the aliphatic amino a ~ i d s . ~ Second, ~ * ~ 8 the aromatic and/or hydrophobic nature of these amino acids may lead to their preferential adsorption on the metal surface compared to the aliphatic amino acids. The small differences between the spectra in parts a and b of Figure 6 are a manifestation of the pH dependence of proteinsurface interactions, described earlier. We have used dipeptide rather than amino acid SER spectra to make the assignments because dipeptides contain an amide bond and are therefore better vibrational spectroscopic models for proteins. Indeed, we find that the SER bands assigned to Trp, Phe, and Tyr in our albumin spectrum agree more closely with bands in the homodipeptide SER spectra of Trp, Phe, and Tyr4' than with the amino acid SER spectra reported by Nabiev et al.1° The bands in the published SER spectra of lysozyme and albumin6J1also agree more closely with the SER bands of Phe-Phe, Tyr-Tyr, and Trp-Trp than with SER vibrations of the amino acids. We therefore advocate the use of dipeptide (or longer peptide) SER spectra rather than the analogous amino acid spectra to aid in the assignment of bands in protein SER spectra. The time-dependent SER spectra lead us to conclude that a reorganization occurs in the adsorbed portion of the protein. Figure 7 shows a schematic representation of the reorganization that may be occurring. Initially, the protein is adsorbed with its hydration shell intact. A t this (48)Asher, S.A.; Ludwig, M.; Johnson, C. R. J.Am. Chem. SOC.1986, 108, 5186.
(49) Kunz, R. E.; Gordon, J. R., II; Philpott, M. R.; Girlando, A. J. Electroanal. Chem. 1980,112, 391. (50)Fischer, L. In Laboratory Techniques in Biochemistry and Molecular Biology; Work, T. S., Burdon, R. H., Eds.; Elsevier Science Publishers: Amsterdam, 1980;pp 94-101.
Ahern and Garrell
260 Langmuir, Vol. 7, No. 2, 1991
Y
!\
cn z w
z I-
I'
01
150
1750
550 RAMAN SHIFT (cm -')
M biotin, 66 mW of Figure 8. (a) SER spectrum of 9.0 X 514.5 nm incident radiation, 2 cm-l resolution, scan rate of 1 cm-l/s; (b) SER spectrum of 20 Wg/mL albumin-biotin.
chlorobutanol or impurities in chlorobutanol is greater than 2 weeks, the period of our observations. 2. Albumin-Biotin SERS. Colloidal probes labeled with albumin-biotin complex are commercially available. It is known from transmission electron microscopy that the albumin is adsorbed on the metal surface in such a manner as to leave the biotin free to bind t ~ a v i d i nFigure .~~ 8 shows the SER spectra of biotin and albumin-biotin taken 0.5 h after their addition to silver colloids. The spectrum of albumin-biotin (Figure 8b) is the same as the spectrum of albumin (Figure 3b) and bears no resemblance to the biotin spectrum (Figure 8a). From this we conclude that the biotin portion of the complex is not adsorbed on the colloid surface, in agreement with the microscopy results. As was the case for albumin, the SER spectral features of the albumin-biotin complex are attributable to structured water at the surface of the protein. 3. Avidin-Biotin Complexation. The goal of using SERS to probe the avidin-biotin complex was to determine whether the biotin binding site in the adsorbed complex is on the surface of the avidin molecule or located in a hydrophobic pocket. Green et al. found, using a series of bis(biotiny1) diamines, that biotin residues separated by more than 12 bonds could form intermolecular bridges upon complexation with avidin, resulting in linear polymers of avidin.52 Their results suggested that the entire biotin molecule is buried in a cleft. Chignell et al. corroborated the results of Green et al. and proposed a detailed model for biotin binding to avidin based on electron spin resonance studies.24If indeed avidin totally encases biotin in a deep cleft, we would not expect to observe a SER spectrum of biotin. If, on the other hand, biotin is encased in a shallow depression, and if this depression is on the same side of the avidin molecule that binds to the surface, we would expect to observe features due to biotin in the SER spectrum of the adsorbed complex. It was first necessary to establish whether the order of addition of analytes to the silver colloid influences the adsorption properties of the avidin-biotin complex. SER spectra were obtained of silver colloids to which biotin and avidin were added either sequentially or as the preformed complex. The procedure was carried out with ~
(51) Bonnard, C.; Papermaster, D. S.; Kraehenbuhl, J.-P. In Immunolabeling for Electron Microscopy; Polak, J. M., Varndell, I. M., Eds.; Elsevier Science Publishers: Amsterdam, 1984; pp 95-111.
(52) Green, N. M.; Konieczny, L.; Toms, E. J.; Valentine, R. C. Bio-
chem.
J. 1971, 125, 781.
a
I
I
975
1800
RAMAN SHIFT (cm -1)
M biotin, scan rate Figure 9. (a) SER spectrum of 9.0 X M) avidin of 1 cm-l/s. (b) Spectrum of 20 mg/mL (3.0 X in silver colloid, scan rate of 1 cm-l/s. The spectrum shows no evidence of surface enhancement.
either biotin or avidin present in excess for 4:l complexation, and also with a 4:l ratio of biotin to avidin (the exact binding ratio). The results obtained were independent of avidin and biotin concentrations. The SER spectrum of biotin and the spectrum of avidin plus silver colloid are shown in Figure 9. The normal Raman spectrum of biotin is shown in Figure 2. The poorer resolution and signal-to-noise ratio of the biotin SER spectrum (Figure 9a) compared with Figure 8a and with previously reported biotin SER spectra53 is due to the higher incident laser power which in this case results in some degradation of biotin a t the surface to graphitic carbon.54 If biotin is added to the colloid first, the spectrum in Figure 9a is obtained. If avidin is then added, the spectroscopic features characteristic of biotin are no longer evident, and an unenhanced Raman spectrum similar to the avidin spectrum in Figure 9b is obtained. If avidin is added to the colloid first followed by biotin, an unenhanced spectrum similar to the avidin spectrum Figure 9b again is obtained. If biotin and avidin are mixed together, allowed to complex, and then the complex is added to the colloid, we once again observe an avidin-like spectrum (Figure 9b). The lack of biotin features in the spectrum of any sample containing both biotin and avidin suggests that the avidin-biotin complex forms readily and that once it forms, biotin cannot interact with the silver surface. This observation is consistent with two possible scenarios. One is that avidin displaces biotin from the surface because it has a higher surface affinity. This is unlikely because avidin does not interact with the surface to the extent that a SER spectrum is observed and that significant aggregation of the silver colloid is induced (described in section A). (We cannot completely exclude the possibility that avidin adsorbs on colloidal silver; however, the avidinsurface interactions are either absent or too weak or too sparse to be observed by SERS.) Alternatively, the portion of the biotinmolecule that in the absence of avidin interacts with the metal surface may be the same portion that is involved in avidin binding. Due to the extremely high avidin/biotin complexation constant (KD lO-l5 M), it is likely that avidin abstracts biotin from the silver colloid surface. Avidin may then envelop biotin, making interactions of biotin with the surface sterically impossible. We have attempted to determine the orientation of biotin on colloidal silver by obtaining SER spectra of the
-
(53)Ahern, A. M.; Garrell, R. L. Anal. Chem. 1987,59, 2813. (54) Mernaugh, T. P.; Cooney, R. P.; Turner, K. E. Chem. Phys. Lett. 1984, 110, 536.
Protein-Metal Interactions
Langmuir, Vol. 7, No. 2, 1991 261
model compounds 2-imidazolidone (11) and 2-methyltetrahydrothiophene (III), whose structures are shown below along with the structure of biotin (I). Unfortunately, the
.
.
HC-CH
I \ H2CS ,C , H(CH2)4COOH I
\i"
HN
H2C-C'
I1
I11
SER spectra of 2-imidazolidone and 2-methyltetrahydrothiophene do not resemble the SER spectrum of biotin and so do not provide insight into the binding of biotin to silver. We therefore cannot exclude either of the scenarios presented above. Although the SERS results do not provide definitive structural information about the binding of biotin to avidin, they are consistent with the model of Chignell et al. in which the binding site for biotin is a cleft located within a hydrophobic depression in the surface of avidin, with the carboxyl group of biotin located about 9 8, below the avidin ~ u r f a c e . ~ ~ ? ~ ~ Recently, it has been shown that the depth of a protein's binding site and the tautomeric form of a molecule that binds to a protein can be obtained directly by SERS or SERRS. Results of a SERS investigation of the bilirubinalbumin complex confirmed earlier conclusions that bilirubin is encased by albumin.41 Using SERRS, Ni and Cotton were able to determine that it is the hydrazone form of HABA that binds to avidin and that the complex does not dissociate upon adsorption on a silver surface.21 We have been unable to obtain a SER spectrum of the avidin-biotin complex. By contrast, Ni and Cotton were able to obtain enhanced Raman spectra of the HABAavidin complex even though they contend that HABA is not in direct contact with the surface.21b (The SER spectrum of the HABA-avidin complex is very similar to the SERR spectrum of HABA. The only contribution from avidin is a weak, broad band centered at 1603 cm-l.) Since HABA occupies the same deep binding sites as does bi0tin,~5it seems likely that Ni and Cotton were able to obtain a spectrum of the complex because of resonance enhancement. This resonance enhancement allows portions of the molecule to be observed that are further from the surface than could be detected with surface enhancement alone, typically 160 %, for SERRS compared to a few tens of angstroms for SERS.6!56 HABA has much less affinity for avidin than does biotin, K D = 5.8~X lo4~ (55) Green, N. M. Biochem. J. 1965, 94, 23c. (56) Cotton, T. M.; Uphaus, R.A.; Mobius, D.J . Phys. Chem. 1986, 90,6071.
-
M a t pH = 6.8 vs K D ~ M a~t pH = ~ 7.0,55157 ~ which ~ may contribute to the ability to acquire an enhanced spectrum of the HABA-avidin complex. Conclusions The biomolecules investigated here fall into two categories based on their absorption spectra. Class I includes avidin, anti-rabbit IgG, and mannan which do not cause aggregation of silver colloids. Class I1 includes albumin, albumin-biotin, and biotin, which cause the colloid to aggregate. We have determined that only molecules in class I1 give rise to surface-enhanced nonresonant Raman scattering and that aggregation of the colloid (or use of electrode substrates) is essential for the observation of SERS from these biomolecules a t the concentrations used here. Enhanced Raman scattering was not observed for any species adsorbed on gold colloids, due either to the lack of adsorbate-induced colloid aggregation or to interference from normal Raman scattering from stabilizers added to the colloidal probes. As expected based on similarities in their normal Raman spectra, the SER spectra of albumin, albumin-biotin, and protein A are quite similar. The increased intensity of the broad band centered a t -1640 cm-l in all of their SER spectra compared with their aqueous Raman spectra is attributed to SER scattering from water molecules in the hydration shell of the adsorbed proteins. With time, the SER spectrum of albumin becomes more intense, and new bands appear that are attributed to vibrations of the side chains of Trp, Phe, and Tyr residues. Accompanying the increase in scattered intensity are changes in the absorption spectra that indicate that colloid aggregation has increased. From the time-dependent SER spectra, we conclude that over a period of time reorganization of the protein takes place near the metal surface. Initially, only the intact hydration shell of the protein adsorbs on the surface. Within 1week, the water molecules are displaced, allowing the amino acids Trp, Phe, and Tyr to adsorb. From this SERS study, we find no evidence for covalent binding of proteins to the metal surface, in support of previous observations. We have also shown that SERS can be used as an in situ probe of the binding of small molecules, such as vitamins, to large globular proteins. The results of the SERS study of the avidin-biotin complex suggest that avidin envelops biotin, consistent with results obtained by other techniques. Acknowledgment. The authors gratefully acknowledge support from the National Science Foundation (DMR-8451962), Eastman Kodak Co., and the PPG Foundation.
~ Registry ~ No.
Ag, 7440-22-4; Au, 7440-57-5; biotin, 58-85-5; mannan, 9036-88-8; 2-imidazolidone, 120-93-4;2-methyltetrahydrothiophene, 1795-09-1; poly(Asp-Na, Glu-Na), 130829-51-5. (57) Green, N. M. Biochem. J . 1963,89, 599.
~
