2044
J. Phys. Chem. 1988, 92, 2044-2048
Functional Actlvlty of Hemogloblns Adsorbed on Colloidal Silver: A Surface-Enhanced Resonance Raman Spectroscopy Study J. de Groot, R. E. Hester,* Department of Chemistry, University of York, Heslington, York YO1 5DD, England
S. Kaminaka, and T. Kitagawa Institute for Molecular Science, Okaraki National Research Institutes, Myodaiji, Okazaki, 444 Japan (Received: July 30, 1987; In Final Form: November 2, 1987)
Surface-enhanced resonance Raman spectra from submicromolar concentrations of human and carp hemoglobins adsorbed on colloidal silver are reported. The adsorbed hemoglobins show reversible dioxygen and carbon monoxide binding and undergo a reversible R- to T-state transition. The Fe-NHi, stretching vibration is unperturbed by adsorption at the silver surface, indicating that the intact heme environment is preserved and the tetrameric structure is retained, even at submicromolar concentrations. Molecular graphics representationsof hemoglobins indicate that the propionate groups attached to the porphyrin macrocycle provide suitable binding groups for adsorption of the hemoglobin at the positively charged silver surface.
Introduction It is well-established that adsorption of molecules to silver surfaces (roughened electrodes, colloidal dispersions, island films) commonly results in greatly increased sensitivity in Raman light scattering.’-j In addition, such adsorption is highly effective in eliminating fluorescence as a spectral interference in Raman spectroscopy! When the excitation wavelength (within the range associated with large surface-enhancement effects with silver, Le., ca. 450-850 nm5l6) is tuned into a strong absorption band of the adsorbed molecules, further sensitization results.2 The combined processes of surface-enhanced resonance Raman spectroscopy (SERRS) have been used to good effect in obtaining vibrational spectra from a variety of molecules, including biological systems, at concentrations in the micromolar to nanomolar range. For recent reviews see ref 7 and 8. When the SERRS technique is used with biological systems, it is important to ask whether the forces involved in adsorption of the biomolecule at the SERS-active surface denature or otherwise modify its structure such that its physiological functions are destroyed or impaired. The literature on SERRS of heme proteins is particularly interesting in this context. It has been reported that the heme groups in both cytochrome c (cyt c) and hemoglobin are separated from their proteins to form p-oxo dimer complexes on silver sols prepared by borohydride reduction of silver ions.g However, others have shown that cyt c is unchanged by adsorption to silver (both electrodes and sols).1o We recently reported our finding that the more sensitive hemoglobin molecule also is stable and remains structurally intact on citrate-reduced silver sols.” In the present paper we develop more fully our study of hemoglobin and its functional/structural stability when adsorbed on citrate-reduced silver sols. The prosthetic group in hemoglobin is the iron porphyrin complex, which is common to a wide variety of heme proteins. The physiological function of this heme group in hemoglobin obviously is directed by the protein to which it is attached. The (1) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163. (2) Albrecht, M. G.; Creighton, J. A. J. A m . Chem. SOC.1977, 99, 5215. (3) Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. Interfacial Electrochem. 1977, 84, I . (4) Lippitsch, M. E. Chem. Phys. Lett. 1981, 79, 224. (5) Creighton, J. A,; Blatchford, C. G.; Albrecht, M. G . J . Chem. SOC., Faraday Trans. 2 1979, 75, 790. (6) Creighton, J . A. In Surface Enhanced Raman Scattering; Chang, R. K., Furtak, T. E., Eds.; Plenum: New York, 1982; p 315. (7) Koglin, E.; Sequaris, J.-M. In Top. Curr. Chem. 1986, 134, 1. (8) Cotton, T. M. In Advances in Spectroscopy; Clark, R. J. H., Hester, R. E., Eds.; Wiley: New York, 1988; Vol. 16, Chapter 3. (9) Smulevich, G.; Spiro, T. G. J. Phys. Chem. 1985, 89, 5168. (10) Hildebrandt, P.; Stockburger, M. J. Phys. Chem. 1986, 90, 6017. (11) de Groot, J.; Hester, R. E. J. Phys. Chem. 1987, 91, 1693.
0022-3654/88/2092-2044$01.50/0
primary role of hemoglobin is oxygen transport. This is accomplished by a change of its quaternary structure.I2 The proximal histidine residue provides the direct link to the heme group and the nature of the Fe-Nm bond is closely related to the quaternary structure, but the overall conformation of the protein environment clearly is important a1s0.I~ Hence, any major changes in the protein structure in hemoglobin upon adsorption at a SERS-active surface would be expected to result in a loss of its physiological activity. Heme proteins have been well-characterized by R R spectroscopy in terms of metal oxidation and spin states,I4 ligation state,I5 heme environment,I6-l8 protein and excitation wavelengthZ2and by normal-coordinate analysis.23 Since an intact protein is required for specific physiological activity, heme proteins provide an excellent set of model biological systems with which to address the question of functional activity after adsorption at a SERS-active surface. In our earlier short report on the SERRS of oxyhemoglobin (Hb02), we concluded that the H b 0 2 did not undergo drastic perturbation upon adsorption at the silver surface.” This conclusion was based on the similarities in the bonding properties of the adsorbed H b 0 2 with those of unadsorbed native oxyhemoglobin and from the respective Raman excitation profiles. We also mentioned briefly our finding that adsorbed hemoglobin would reversibly bind dioxygen and carbon monoxide. We now present evidence in support of that claim.
Experimental Section Silver colloids were prepared by the methods of Lee and M e i ~ e l , ’F~a b r i k a n ~ sand , ~ ~C r e i g h t ~ n .The ~ pH’s of the silver sols were adjusted with 2 mM Tris acetate buffer (Sigma (12) Perutz, M. F. Nature (London) 1970, 228, 726. (1 3) Nagai, K. In Optical Properties and Structure of Tetrapyrroles. Blauer, G., Sund, H., Eds.; de Gruyter: Berlin, New York, 1985; p 157. (14) Spiro, T. G.; Strekas, T. C. J. A m . Chem. SOC.1974, 96, 338. (15) Scholler, D. M.; Hoffman, B. M.; Schriver, D. F. J . Am. Chem. SOC. 1976, 98, 7866. (16) Spiro, T. G.; Stong, J. D.; Stein, P. J . A m . Chem. SOC.1979, 101, 2648. (17) Rimai, L.; Salmeen, I.; Petering, D. H. Biochemistry 1975, Z4, 378. (18) Scholler, D. M.; Hoffman, B. M. J. Am. Chem. SOC.1979,101, 1655. (19) Nagai, K.; Enoki, Y.; Kitagawa, T. Biochim. Biophys. Acta 1980, 624, 304. (20) Rousseau, D. L.; Ondrias, M. R. Annu. Reo. Biophys. Bioeng. 1983, 12, 357. (21) Rousseau, D. L.; Ondrias, M. R.; Shelnutt, J. A.; Simon, S. R. Biochemistry 1982, 21, 3428. (22) Strekas, T. C.; Spiro, T. G. J. Raman Specfrosc. 1973, I , 387. (23) Abe, M.; Kitagawa, T.; Kyogoku, Y. J . Chem. Phys. 1978,69,4526. (24) Lee, P. C.; Meisel, D. J . Phys. Chem. 1982, 86, 3391. (25) Fabrikanos, A,; Athanassiou, S.; Lieser, K. H. Z. Naturforsch. B: Anorg. Chem., Org. Chem., Biochem., Biophys., Biol. 1963, 18B, 612.
0 1988 American Chemical Society
Hemoglobins Adsorbed on Colloidal Silver Chemicals, pH 8.5) or 5 mM Bis-Tris acetate acid buffer (Sigma Chemicals, pH 5.8) as desired. Dissolved dioxygen was removed from the silver sol prior to the adsorption of hemoglobin by addition of a trace amount of sodium dithionite. Human oxyhemoglobin was extracted from whole blood by the method of Rossi et a1.26and was stored in carbon monoxy form at 4 "C in 0.05 M phosphate buffer (pH 6.8) at ca. 8 X M concentration. The apoprotein of hemoglobin was prepared as described by Antonini and B r ~ n o r i . ~ ' Carp blood was obtained from live carp [(Cyprinus carpio) anaesthetized with ethyl carbamate] by severing the caudal peduncle and collecting the outflow from the spinal artery into an anticoagulant heparin solution.28 Carp hemoglobin (Hb, ) was extracted according to the method of Gillen and Riggs.2rg N o attempt was made to isolate the three individual hemoglobin components of HbCarp,since these have been shown to be functionally identicaL2* Hb,, was stored at 4 "C in the CO-ligated M concentration. form at ca. 1.5 X Raman measurements were obtained with 90" illumination using a Jobin-Yvon Ramanor HG2 double monochromator, equipped with an RCA 3 1034-A02 cooled photomultiplier tube and photon-counting electronics, controlled by a Nicolet 1074 data processor. SERR spectra excited at 441.6 nm were obtained with 90" illumination using a JEOL-400D Raman spectrometer, equipped with an RCA 3 1034-A02 cooled photomultiplier tube. Powers (at the sample) of up to 100 mW were used from krypton ion (Spectra Physics Model 170), argon ion (Coherent Model 52A), and He/Cd (Kinmon Electronics Model CDR80MGE) lasers. S E R R spectra were recorded from either a rotating cell or a cooled (4 "C) static airtight cell (diameter ca. 5 mm) using a spectral band-pass of 6 cm-' or less. SERR spectra of deoxyhemoglobin (deoxyHb) were obtained by photolysis of adsorbed carbonylhemoglobin (HbCO) using ca. 50 mW of 441.6-nm laser excitation focused at the sample. Molecular graphics representations of H b 0 2 were generated on an Evans and Sutherland PS330 display monitor using the graphics program Hydra.30
Results and Discussion We have found that hemoglobins adsorbed on silver sols prepared using borohydride5 as the reducing agent are much less stable than on those prepared with citrate24or EDTA25 as the reducing agent. Borohydride-prepared silver sols often produced SERR spectra of hemoglobins with Raman bands arising from protein decomposition products and from p o x 0 dimer formation. With this method, even as long as 72 h after the preparation of the silver sol, hydrogen evolution at the container vessel wall could still be observed, arising from the slow decomposition of borohydride with water. It is interesting to note that Creighton et al.31 attributed wavenumber shifts in the S E R bands of pyridine as arising from the strongly reducing environment of the borohydride-prepared sols. The presence of a large excess of such a strong reducing agent in the silver sol obviously raises doubts about the applicability of borohydride-prepared sols in the SERRS of biological systems. Silver sols prepared with the much milder citrate or EDTA reducing agents gave excellent SERR spectra, with no denaturation, from freshly prepared hemoglobin. In all subsequent work citrate reduction was used in the preparation of silver sols, since this gave consistently reliable sols with good SERS activity. (26) Rossi, F. A.; Antonini, E.; Caputo, A . J . Biol. Chem. 1961, 236, 391. (27) p t o n i n i , E.; Brunori, M. Frontiers of Biology: Haemoglobin and Myoglobin in Their Reactions with Ligands; Neuberger, A., Takum, E. L., Eds.; Elsevier: Amsterdam, 1971. (28) Noble, R. W.; Parkhurst, L. J.; Gibson, Q.H. J . Biol. Chem. 1970, 245, 6628. (29) Gillen, R. G.; Riggs, A. J . Biol. Chem. 1972, 247, 6039. (30) Hubbard, R. E. The Representation of Protein Structure in Computer Aided Molecular Design; Proceedings of the 1st European Seminar on Computer-Aided Molecular Design, London, Oct 1984; Oyez Scientific: London, 1985; pp 99-106. (31) Creighton, J. A.; Alvarez, M. S.; Weitz, D. A,; Garoff, S.; Kim, M. W. J . Phys. Chem. 1983, 87, 4793.
The Journal of Physical Chemistry, Vol. 92, No. 7, 1988 2045
1650
Wavenumberlcm-l
1000
Figure 1. SERR spectra of (a) 1.4 X M HbCO (Aex = 514.5 nm) M deoxyHb (& = 441.6 nm) adsorbed at a colloidal and (b) 1.4 X silver surface. RR spectrum (Aex = 441.6 nm) of 1 X M deoxyHb pH 6.8 buffer) is shown for comparison (c). in solution (0.05 M Pod3-,
Citrate-prepared sols also could be stored indefinitely without any appreciable aggregation occurring. The SERR band wavenumbers in the HbCO spectrum shown in Figure 1 were found to be virtually identical with those in the corresponding R R spectrum. However, in addition to the expected overall intensity enhancement of the SERR spectrum, the v4 band was found to be anomalously enhanced with 514.5-nm excitation (ca. 25 mW, defocused). This wavelength lies within the /3 absorption bands and, under these conditions, is known not to photolyze the Fe-CO bond in HbC0.32 A similar effect was reported previously for the SERR effect of HbO2I1and also for cyt d o on colloidal silver. Photolysis of this surface-adsorbed HbCO with 441.6-nm laser radiation, in an evacuated cell, resulted in the production of deoxyHb. The SERR spectrum of this deoxyHb product is shown in Figure 1 for comparison with the normal R R spectrum of deoxyHb in solution (aqueous phosphate buffer, pH 6.8). The SERR spectrum of adsorbed deoxyHb shows marker bands characteristic of five-coordinate, Fez+ high-spin, viz. v4, v3, and vl0 at 1358, 1473, and 1605 cm-I, respectively (see Figure 1 and Table I). It can be seen that the relative intensities of the v4, v3, and vl0 marker bands are closely similar to those of the corresponding bands in the R R spectrum of deoxyHb in solution, although minor differences are apparent. This indicates that the primary mechanism for the surface enhancement is electromagnetic rather than chemical; significant spectral perturbations normally are characteristic of the chemical or charge-transfer mechanism. This point could be important in the evident retention of biological activity of the adsorbed hemoglobin. It is interesting to note that Smulevich and Spirog observed a Raman band at 1500 cm-' in their SERR spectrum of deoxyHb, which they assigned to the v3 Fe3+ low-spin state marker band. They also observed a high-wavenumber shoulder on the 1358-cm-' band and attributed this to the v4 band of the Fe3+state, thus (32) Armstrong, R. S.; Irwin, M. J.; Wright, P. E. J . A m Chem. SOC. 1982, 104, 626.
de Groot et al.
The Journal of Physical Chemistry, Vol. 92, No. 7, 1988
2046
TABLE I: Wavenumbers (cm-I) and Mode Assignments for Heme Complexes in Solution and Adsorbed at a Silver Surface
assigntab VI0 bl, W==C y37
ell
v i 9 a2g y2
YlI u38
a,, bl,
u3 a,, u28
6,(=CH2) ( I ) y29 b2g y4 alg 6,(=CH2) (2)
deoxyhemoglobin RR SERR
oxyhemoglobin RRC SERR
carbonylhemoglobin RR SERR
1607 1622 1590
1609 1622 1590
d
d
1567 1550 1529
1567 1550 1525 1500 1473 1455 1430 1395 1358 1340 1310 1304 1285 1225 1176 1133 1124
1640 1620 1606 1586 1583 1564 1552
d
1642 1625 1605 1588 1583 1564 1552
1506
1501‘
1508
1500‘
1431 1400 1375 1342
1432 1399 1370 1342 1308f 1308f
1432 1400 1374 1345 1308
1432 1397 1375 1340 1308 d
1473 1450 1430 1400 1358 1340
u21 a2g
a(CH2) us
+ u9 a,,
y13
y30 b2g
u6
+ v8 a,,
y22 a2g
1305 1284 1215 1174 1135 1120
1639 1620 1607 1586 1583 1565
i
1638 1622 1602 1585
d 1564 1554
>I
L
1305 1225 1173 1133
1225 1167 1147 1125
1172
1225 1167
1137
1128
LI)
aEl
L
a Mode designations follow Kitagawa et a1.23,59nd ref 60. bFrom ref 61 and 62.-CFromref 14. dNot resolved. eSee text. /Accidental degeneracies.
150
250 Wavenumber/cm
0
b M
-
h
Figure 2. Change in intensity of the u4 marker band with successive uptake of O2following laser photolysis of adsorbed HbCO in an open cell (Aex = 441.6 nm).
signaling the presence of Fe3+ low-spin complexes at the silver surface. In our SERR spectrum of deoxyHb no high-wavenumber shoulder is observed on the 1358-cm-I band, indicating that only Fe2+ hemes are present. Recombhation of the adsorbed deoxyHb with carbon monoxide was demonstrated by observation of the characteristic SERR spectrum of adsorbed HbCO, using nonphotolyzing 5 14.5-nm laser excitation. Laser photolysis of adsorbed HbCO in an open cell yields first adsorbed deoxyHb which then, over a period of ca. 3-4 min, binds dioxygen to form the photostable adsorbed HbOz. Figure 2 shows the increase in intensity of the 1370-cm-l band, u4. of adsorbed H b 0 2 and the corresponding decrease in the intensity of the 1358-cm-’ band, v4, of adsorbed deoxyHb. After ca. 3-4 min, subsequent to introducing air into the photolysis cell, the SERR spectrum of adsorbed deoxyHb was completely replaced by that of adsorbed HbO,. This was identical with that reported previously,” using Soret excitation (406.7 nm), showing the v4, u3, and vlo bands at 1370, 1501, and 1640 cm-I, respectively. Complete oxygenation of the adsorbed deoxyHb could be achieved virtually instantaneously by flushing the sol with air. Thus, the rate of oxygenation of the adsorbed hemoglobin in a static cell appears to be limited by the rate of diffusion of dioxygen through the sol. Atteppts to remove the dioxygen ligand from the adsorbed HbOz by addition of a slight excess of sodium dithionite were
Figure 3. Fe-NH, stretching band (Aex = 441.6 nm): (a) 1.4 X IO-’ M Hb adsorbed at a colloidal silver surface; (b) 1 X lo-) M Hb in solution (0.05 M PO,‘-,pH 6.8 buffer).
frustrated by rapid precipitation of the colloid and subsequent loss of all SERR signals. The dioxygen ligand of the adsorbed Hb0, could, however, be replaced by carbon monoxide by flushing the sol with carbon monoxide gas. The adsorbed HbCO so produced underwent laser photolysis (at 441.6 nm) to produce adsorbed deoxyHb. Hence, the adsorbed hemoglobin will reversibly bind both dioxygen and carbon monoxide. It may be argued that the adsorbed hemoglobin exists in a dynamic equilibrium with unadsorbed hemoglobin and that reversible ligand binding only takes place to those hemoglobin molecules in solution. The fact that the SERRS and RRS spectra-in particular of the Fe-NHls vibration-are virtually identical (see Figure 3) strongly suggests, however, that the observed effects are truly properties of the adsorbed species. Since denaturation always is associated with drastic changes in the spectroscopic properties of the heme group and to a loss of the O2 binding capacity,*’ we can conclude that the forces involved in adsorption of the hemoglobin to the silver surface do not cause major changes in the hemoglobin structure or conformation. The low-wavenumber region of the RR spectrum of hemoglobin contains useful information about the protein structure. Although much of this information has not yet been elucidated, some assignments have been made.33-36 The 21 5-cm-’ band in native human T-state deoxyHb has been assigned to the stretching vibration of the bond between the heme iron atom and the nitrogen atom of the imidazole ring of the protein-bound proximal histidine residue (Fe-NH,,). The Fe-NH,, bond provides a link between the heme group and the protein moiety and has been shown to be extremely sensitive to the protein quaternary s t r ~ c t u r e . ~It~ ~ ~ * ~
~
~~
~~
~~
~
~
~~
~
(33) Kitagawa, T.; Nagai, K.; Tsubaki, M. FEBS Len. 1982, 140, 626. (34) Nagai, K.; Kitagawa, T.; Morimoto, H. J . Mol. Biol. 1980,136, 271. (35) Hori, H.; Kitagawa, T. J . A m . Chem. SOC.1980, 102, 3608. (36) Kincaid, J.; Stein, P.; Spiro, T. G. Proc. N a f l .Acad. Sci. U.S.A.1979, 76, 4156. (37) Nagai, K.; Kitagawa, T. Proc. N ~ t lAcad. . Sci. U.S.A.1980, 77,2033. (38) Friedman, J. M.; Rousseau, D. L.; Ondrias, M . R. Annu. Reo. Phys. Chem. 1982, 33, 471.
The Journal of Physical Chemistry, Vol. 92, No. 7, 1988 2047
Hemoglobins Adsorbed on Colloidal Silver la)
(bl
I
p H 5.8
(TI
I
r”
Figure 4. Fe-N,,, stretching band (Aex = 441.6 nm) of deoxyHb,, (a) in solution (1.4 X lo4 M) and (b) adsorbed at a colloidal silver surface (1.4 X lo-’ M ) at both acidic and basic pH’s; (T) = T state, (R) = R state.
has been suggested that the F-NH~, bond plays an essential role in controlling the oxygen affinity of the heme by relaying structural information from the protein moiety to the heme iron.I3J9 Thus, overall, the Fe-NHi, stretching vibration provides a sensitive probe which can be used to monitor structural changes occurring in the protein moiety of deoxyHb as a result of adsorption at the silver surface. Unfortunately, this mode evidently is inactive in the R R spectra of liganded (e.g., oxy) hemoglobin^.^,^^ In Figure 3 the Fe-NHls stretching vibration in the SERR and R R spectra of deoxyHb are compared. The Fe-NHi, band in the SERR spectrum of adsorbed deoxyHb was found to be located at 215 cm-I, which is identical with its value in the corresponding solution R R spectrum. This indicates that the adsorbed deoxyHb is in the native T-state quaternary structure, with no additional strain imposed on the Fe-NHi, bond due to adsorption at the silver surface. The relative intensity and shape of the Fe-NHi, band in the SERR spectrum of deoxyHb are virtually identical with those in the R R spectrum of deoxyHb in solution, indicating little or no change to the heme environment. Hemoglobins in submillimolar concentrations are usually dissociated into Lyp dimers or monomer^.^' If this occurred, however, the Fe-NHi, stretching mode should be shifted to higher wavenumber (ca. 222 ~ m - l ) .The ~ ~ invariance of the wavenumber position of this stretching mode between the solution and colloidal silver experiments suggests that the tetrameric structure is preserved after absorption at the silver sols, partly due to locally higher concentrations on the colloidal surface. To examine whether the adsorbed hemoglobins remain functionally active in switching from one quaternary structure to the other, we investigated the SERRS of carp hemoglobin (Hbcarp), DeoxyHb,,,, is known to switch quaternary structure upon a change of pH.28*29942’44 A recent report by Coppey et aL4 suggests that a small fraction of ligated hemoglobin must be present for this quaternary structure change to occur. In Figure 4 the Fe-NHi, band of deoxyHb,,,, in solution is compared with that from deoxyHb,, adsorbed at a colloidal silver surface, at both acidic and basic pH. At pH 5.8 it is in the T state, as shown by the Fe-NH, stretching band located at ca. 212 cm-’. Upon changing to pH (39) Matsukawa, S.; Mawatari, K.; Yoneyama, Y.; Kitagawa, T. J . A m . Chem. SOC.1985, 107, 1108. (40) Tsubaki, M.; Srivastava, R. B.; Yu, N.-T.Biochemistry 1982,21, 842. (41) Tsubaki, M.; Yu, N.-T. Biochemistry 1982, 21, 842. (42) Pennelly, R. R.; Tan-Wilson, A. L.; Noble. R. W. J . Biol. Chem. 1975, 250, 7 2 3 9. (43) Tan-Wilson, A. L.;Noble, R. W.; Gibson, Q.H. J . Biol. Chem. 1973, 248, 2880. (44) Coppey, M.; Dasgupta, S.; Spiro, T. G. Biochemistry 1986, 25, 1940.
Figure 5. Molecular graphics representation of R-state H b 0 2 , showing the propionate groups attached to the porphyrin macrocycle protruding out through the protein envelope.
8.5 the quaternary structure switches to that of the R state, the
Fe-NHi, band now being located at ca. 216 cm-I. These wavenumber values are consistent with those reported previo~sly.~~ The Fe-NHi, band of the adsorbed species reversibly shifts by ca. 4 cm-’ upon change of pH, showing that the quaternary structure is reversibly switching between the T and R states. It has been suggested that selective ligand binding to a chain could provide an alternative interpretation of the observed shift in the Fe-NHi, band,44but the important point is that this shift is seen in our work to be reversible. It is widely accepted that the SER effects operate from a direct interaction of the substrate with the SERS-active surface (charge-transfer model)46-48and/or from the interaction of the substrate lying close to the metal surface with the large electric field produced when the incident photon frequency is in resonance with a normal mode of the conduction electrons in the metal (plasmon resonance m ~ d e l ) . ~Our ~ - ~work ~ with the hemoglobin apoprotein has shown that this possesses no suitable sites on the exterior of the globular molecule for generating SERS effects on adsorption to the silver surface. A similar result has been reported for the cyt c apoprotein.1° It should be noted, however, that SER spectra are readily observable from denatured hemoglobin apoprotein, indicating that adsorption is taking place. Thus, the protein conformation of the intact native protein appears not to favor adsorption with SER activity at the silver surface. Evidently, it is the aromatic amino acid residues that dominate the SER spectra of protein^.^ This leads us to speculate that not only are the aromatic residues of native hemoglobin unable to contact a silver surface at which the heme propionate groups provide the primary binding mechanism, but even in the absence of the heme groups (apoprotein) the SERS-active aromatic residues are “buried”. In our previous report on the SERRS of H b 0 2 ” w’e first suggested that hemoglobin binds to a silver surface via the carboxylate functions of the propionate groups attached to the porphyrin macrocycle. Molecular graphics representations of R-state H b 0 2 using the coordinates obtained from X-ray diffractions3 (2.1-8, resolution) clearly show that the propionate (45) Dalvit, C.; Cerdonio, M.; Fontana, A.; Mariotto, G.; Vitale, S.; De Young, A.; Noble, R. W. FEBS Left. 1982, 140, 303. (46) Otto, A. Appl. Surf. Sci. 1980, 6, 309. (47) Furtak, T. E.; Trott, G.; Loo, B.-H. Surf. Sci. 1980, 101, 374. (48) Seki, H. J . Chem. Phys. 1982, 76, 4412. (49) Gersten, J.; Nitzan, A. J . Chem. Phys. 1980, 73, 3023. (50) Kerker, M.; Wang, D. S.; Chew, H. Appl. Opt. 1980, 19, 4159. (51) Gersten, J.; Nitzan, A. J . Chem. Phys. 1981, 75, 1139. (52) Jha, S. S.; Kirtley, J. R.; Tsang, T. C. Phys. Rev. E: Condens. M a t e r 1980, 22, 3973.
2048
The Journal of Physical Chemistry, Vol. 92, No. 7, 1988
I
I
,c"-o
?,+O
0 Ag
S u r f ace
(11 (11) Figure 6. Possible binding geometries of carboxylate groups a t a silver surface.
groups do protrude out through the protein envelope (see Figure 5 ) . Computer simulation of a 20% randomly roughened (atomic scale) silver surface54suggests that the propionate groups can contact the metal surface, with the protein moiety remaining unattached. The pK, value of the carboxylate functions of these propionate groups is expected to be similar to that of free propionic acid (pK, = 4.92) since they are shielded from the tetrapyrrole ring by two saturated carbon atoms. Thus, over the pH range used in the SERR experiments (pH 5.8-9) the propionate groups of the porphyrin macrocycle will be largely deprotonated. The measured surface potential of the silver sol was -0.3 V (SCE);" the point of zero charge for silver under these conditions is ca. -0.9 V (SCE).55 Assuming that the colloidal particles are spheres of average diameter 35 nm, composed of hexagonal close-packed ,~~ silver atoms of radius 1.34 A, and capacitance 50 ~ F / c m *the calculated charge per surface atom is ca. +0.1 unit of elementary charge. Hence, the deprotonated carboxylate groups will interact strongly with the positively charged silver surface. In hemoglobin the propionate groups are not involved in coupling the molecular ground state to the electronically excited aa* state and therefore do not contribute to the R R ~pectrum.~'In the SERR spectrum of hemoglobins, however, the carboxylate groups would be expected to receive maximum surface enhancement since they evidently interact most strongly with the silver surface. The v,(COO) mode for ionized carboxylate groups normally occurs at ca. 1415 cm-' but is raised to higher wavenumber values on binding to metal ions.s8 Figure 6 shows possible (53) Shaanan, B. J . Mol. Biol. 1983, 171, 31. (54) Harris, M., personal communication. (55) Hampson, N. A,; Larkin, D.; Morely, J. R. J . Electrochem. SOC.1967, 114, 817. (56) Cooper,I. L.; Harrison, J . A.; Sandbach, D. R. Electrochim. Acta 1978, 23, 527. (57) McMahon, J. J.; Baer, S.; Melendres, C. A. J . Phys. Chem. 1986, 90, 1572. (58) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds; Wiley-Interscience: New York, 1978.
de Groot et al. binding geometries for the surface-bound carboxylate groups. A highly asymmetric binding geometry could be a necessary consequence of the conformational constraints in hemoglobin, and this would result in high-wavenumber v,(COO) (cf. ester CO). In the SERR spectrum of adsorbed deoxyHb (Figure 1) all vibrational bands observed, except that at 1500 cm-', can be matched with R R bands arising from vibrational modes of Fez+ high-spin deoxyHb in solution. We previously attributed the appearance of a band at 1501 cm-1 in the SERR spectrum of HbO, to a 5-cm-' shift from the R R value of the 1506-cm-I v 2 stretching mode, which has a large C,-C, character.23 However: we were unable to explain why the 1639-cm-' vl0 stretching mode, which also has significant C,-C, character,23 remained unchanged. Thus, it may well be that the 1500-cm-' band in the SERR spectrum arises from the symmetric stretching, v,(COO), of the adsorbed carboxylate groups. An accidental overlap of the ca. 1500-cm-' v,(COO) stretching mode of the adsorbed carboxylate groups with the v3 1506-cm-' low-spin state marker band in the SERR spectrum of adsorbed H b 0 2 could account for the apparent wavenumber lowering of the u3 marker band. The weakness of the 1500-cm-' band, by comparison with the bands due to the porphyrin ring modes in the SERR spectrum of deoxyHb, would suggest that this is a S E R and not a SERR band. The value of model compounds for checking this assignment is limited by the fact that, in the absence of protein, metalloporphyrins probably adsorb flat on a metal surface rather than in the edge-on arrangement found for hemoglobin.1° Thus, a much weaker surface enhancement effect can be expected for metalloporphyrin models. In conclusion, the similarities between the R R spectra of hemoglobins in solution and the SERR spectra of the adsorbed species indicate that denaturation does not occur upon adsorption at a silver surface. In addition, the functional activity exhibited by the adsorbed hemoglobins indicates little or no distortion of the protein moiety.
Acknowledgment. We thank the SERC for financial support, particularly in enabling this York-Okazaki collaboration to take place through a special grant to J.deG. We also thank Dr. R. E. Hubbard, Dr. M. Harris, and Miss P. Harris (York) for their help with the molecular graphics work. Registry No. Ag, 7440-22-4; 02, 7782-44-7; CO, 630-08-0. (59) Abe, M.; Kitagawa, T.; Ogoshi, H. J . Chem. Phys. 1978, 69, 4516. (60) Choi, S.;Spiro, T. G.; Langry, K. C.; Smith, K. M. J . Am. Chem. SOC.1982, 104, 4337. (61) Choi, S . ; Spiro, T. G.; Langry, K. C.; Smith, K. M.; Budd, D. L.; La Mar, G. N. J . Am. Chem. SOC.1982, 104, 4345. (62) Sanchez, L. A,; Spiro, T. G. J . Phys. Chem. 1985, 89, 763.
