The Journal of
Physical Chemistcy
0 Copyright, 1987, b y the American Chemical Society
VOLUME 91, NUMBER 7 MARCH 26, 1987
LETTERS Surface-Enhanced Resonance Raman Spectroscopy of Oxyhemoglobin Adsorbed onto Colloidal Silver J. de Groot and R. E. Hester* Department of Chemistry, University of York. Heslington, York YO1 5DD. England (Received: September 16, 1986;In Final Form: January 29, 1987)
Surface-enhanced resonance Raman spectra of oxyhemoglobin on colloidal silver dispersions -.3ve been obtained with no observable denaturation resulting from adsorption at the silver surface. Excitation profiles of the SERR bands of oxyhemoglobin indicate that perturbations of the electronic states of the oxyhemoglobin occur upon adsorption, although the vibrational mode frequencies are unperturbed. An unusual enhancement of the bands associated with symmetric vibrational modes of the porphyrin macrocycle is reported for excitation of SERR spectra in the wavelength region of the p band. This is interpreted in terms of the lowered symmetry of the adsorbed species.
Introduction The discovery of the surface-enhanced Raman (SER) effect from roughened electrodes' opened up a new field of research. This was extended when it was realized that the S E R effect operated in a similar way with colloidal dispersions of metals such as silver, gold, and copper. This field of surface spectroscopy developed further when it was realized that resonance Raman (RR) and SER effects could combine to give the surface-enhanced reonance Raman (SERR) effect.2 This new effect has recently been used in the study of biological systems with chromophoric molecules such as hemoproteins being of particular i n t e r e ~ t . ~ - ~ ( 1 ) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett.
1974, 26, 163.
(2) Jeanmaire, D. L.;Van Duyne, R. P. J. Electroanal. Chem. 1977.84,
The results of these investigations are very controversial and even contradictory. A particular point at issue is whether molecular structure or conformational changes occur when a biological molecule is adsorbed at a SERR-active surface. A recent reportS concludes that adsorption of hemoproteins onto a silver colloid can facilitate the separation of the porphyrin macrocycle (hemolysis) from the protein. These may be either covalently (e.g. cytochrome c) or noncovalently (e.g., cytochrome b and oxyhemoglobin) bound to the protein moiety. The p-oxobridged iron porphyrin dimers were inferred as decomposition products. However, other work7 suggests that no hemolysis m u r s when cytochrome c is adsorbed on a silver colloid. We report here some new results on the SERR spectroscopy of hemoglobin and the suggested p o x 0 dimer (Fe"'PP),O decomposition product. These extend the previous studies and lead us to new conclusions.
1.
(3) Cotton, T.M.; Schultz, S . G.; Van Duyne, R. P. J. A m . Chem. SOC. 1980,. 102, 7960. (4) Sanchez, L. A.; Spiro, T. G. J. Phys. Chem. 1985,89, 763. ( 5 ) Smulevich, G.;Spiro, T. G . J . Phys. Chem. 1985, 89, 5168. (6) McMahon, J. J.; Baer, S.; Melendres, C. A. J. Phys. Chem. 1986,90, 1572. (7) Hildebrandt, P.;Stockburger, M. J . Phys. Chem. 1986, 90, 6017.
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Experimental Section Silver colloids were prepared according to the method of Lee and Meise18 by dropwise addition of a 1% solution of trisodium ~
~~~~~
~
(8) Lee, P. C.; Meisel, D. J. Phys. Chem. 1982, 86, 3391
0 1987 American Chemical Society
1694 The Journal of Physical Chemistry, Vol, 91, No. 7, 1987
citrate (10 cm3) to a boiling solution of AgN0, (90 mg) in triply distilled water (500 cm3) under an atmosphere of nitrogen. The resulting solution was kept boiling for approximately 1 h. A small concentration of NaNO, (5 X loW3M) was added to the silver sol to increase the S E R signal^.^ Human oxyhemoglobin was extracted from whole blood by the method of Rossi et a1.I0 and was stored at 4 "C in a 0.05 M phosphate buffer (pH 6.8) at ca. 8 X lo-, M concentration. Methemoglobin was prepared by the method of Antonini and Brunori" by addition of a slight excess of potassium ferricyanide to the oxyhemoglobin solution followed immediately by Sephadex G-25 gel chromatography. The oxidation of oxyhemoglobin was monitored to completion by UV-vis spectrophotometry. (Fe111PP)20was prepared by dissolving hemin chloride (Sigma) in aqueous base (pH 10.5).4 The (Fe"'PP),O species was characterized by UV-vis spectrophotometry. SERR spectra of oxyhemoglobin and (Fe111PP)20were obtained with 3 X lo-? and 6 X lo-? M solutions, respectively. Both 25% and 50% v/v glycerol were used as intensity standards for Raman excitation profile measurements of hemoglobin and (Fe"'PP),O, respectively, it having been found that the glycerol had no observable effect on the Raman spectra. The SERR spectra of hemin chloride were obtained as suggested by Stockburger and Hildebrandt,' by the addition of a dilute solution of hemin chloride, dissolved in ethanol, to the silver sol, so that the final concentrations were 3 X lo-? M hemin chloride and 1% ethanol. Raman excitation profile measurements were obtained with 90" illumination using a Jobin-Yvon Ramanor HG2 double monochromator, equipped with an RCA 31304A02 cooled photomultiplier tube and photon-counting electronics, controlled by a Nicolet 1074 data processor. SERR spectra excited at 406.7 nm were obtained with 90" illumination using a Spex 1403 double monochromator, equipped with an RCA 3 1304A02 cooled photomultiplier tube and photon-counting electronics, controlled by a Spex SCAMP data station. SERR spectra excited at 441.6 nm were obtained with 90" illumination using a JEOL-400D Raman spectrometer, equipped with an RCA 31034a cooled photomultiplier tube. Spectra recorded at 406.7- and 441.6-nm excitation have not been included in the Raman excitation profiles. SERR spectra were recorded from a rotating cell using a spectral band-pass of 6 cm-' or less. 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 (Kimmon Electronics Model CDRIOMGE) lasers.
Results and Discussion The surface potential of the silver colloid with adsorbed oxyhemoglobin was found to be approximately -0.3 V (relative to the SCE) by the method of Wetzel et a1.I2 This is much less negative than the potential reported to cause unfolding of the protein moiety.I3 Indeed, since the potential of zero charge for silver is ca. -0.9 V (SCE), the colloidal particle surfaces are positive in absolute terms. Since borohydride reduction was used by Smulevich and S p i r ~it, ~is possible that their colloid carried a different surface charge. Bands observed in the SERR spectra of hemin chloride, (Fel''PP),O, and oxyhemoglobin are readily identifiable with RR-active vibrational modes (see Table I). This coincidence suggests that no drastic changes occur in the bonding in oxyhemoglobin upon addition to the silver colloid. The SERR spectra of oxyhemoglobin indicate the presence of low-spin Fe2+only. The absence of the characteristic Fe3+heme marker band at 1488 cm-l in the SERR spectra of oxyhemoglobin, even with 406.7-nm excitation which causes this to become one (9) Wetzel, H.; Gerischer, H. Chem. Phys. Lett. 1980, 76, 460. (10) Rossi, F. A.; Antonini, E.; Caputo, A. J. Biol. Chem. 1961, 236, 391. (1 1) Antonini, E.; Brunori, M.In Frontiers of Biology: Haemoglobin and Myoglobin in Their Reactions with Ligands; Neuberger, A,, Takum, E. L., Eds.; Elsevier: Amsterdam, 197 1. (12) Wetzel, H.; Gerischer, H.; Pettinger, B. Chem. Phys. Lett. 1982, 85, 187. (13) Hildebrandt, P. Ph D. Thesis, Universitat Gottingen, 1985
Letters TABLE I: Mode Assignment and Wavenumbers (em-') for Heme Complexes in Solution and Adsorbed at a Silver Surface hemin oxy-
VI0
chloride
(Fe11'PP)20
assignto,*
RRb SERR
RR" SERR
RR'
SERR
blg
1626 1626 1591 1571 1570 1553 1533 1491 1453 1435 1403 1403 1373 1340 1309 1308 1260 1228 1170 1130 1127
1624
1624
1591 1569 1569
1592 1570d 1570d
1532 1491
1534 1488
1640 1620 1606 1586 1583 1564 1552 1506
1639 1620 1607 1586 1583 1565 e 1501
1429
1431 1398
1431 1400
1432 1399
1375 1339
1370 1342 1305d 1305d
1375 1342
1370 1342 1 308d 130@
UC==C u37
e,
u19 a2g
u2 "I1
alg blg
u3a
alg "28 b2g u3
6,(=CH2) (1) u29 b2g
azg alg 6, (=CH2) (2) u21 a2g u20 V4
6(CHJ u5 u13
+ u9 a l g
u30 b2g
u6+ u8alg y22
a2g
1625d 1625d 1592 1571d 157Id 1554 1534 1492 1452 1431 1398d 1398d 1373 1342 1307d 1307d 1228 1167 1150 1127
1307 1225 1170 1127
1225 1167 1147 1125
hemoglobin
1305 1225 1173 1133
1225 1167 1147 1125
From ref 4. bFrom ref 17. From ref 18. dAccidental degeneracies. e Not resolved from the broad 1564-cm-' band
1000
Wavenumber (cm-0
1700
Figure 1. SERR spectra of (A) 6
X M (Fe"'PP),O, (B) 3 X M oxyhemoglobin (partially denatured; see text), and (C) 3 X IO-' M oxyhemoglobin, recorded between 1000 and 1700 cm-' and excited at 406.7 nm.
of the most prominent bands in the R R spectrum, indicates that oxyhemoglobin does not denature to form the Fe3+ heme species upon adsorption on the silver colloid (see Figure 1). This is in agreement with Stockburger and Hildebrandt,? who observed no bands from Fe3+decomposition products in their SERR spectra of cytochromes, but contradicts the finding of Smulevich and Spiro: who reported that oxyhemoglobin denatured in the presence of colloidal silver to form a (Fe"'PP),O decomposition product. The SERR spectra of oxyhemoglobin and (Fe"'PP),O are clearly different (see Figures 1 and 2), indicating that in our experiments the oxyhemoglobin has not denatured to form a (Fe"'PP),O species upon addition to the silver colloid. However, we found that oxyhemoglobin which had been set aside at room temperature for several hours did denature to form a (Fe'"PP),O species upon addition to the silver colloid (see Figure 1). Thus,
The Journal of Physical Chemistry, Vol. 91, No. 7, 1987
Letters
1695
Y/cm-l x 1125 A 1167
+ 1370 1359
- Visible absorption
t
1000
Wavenumber (cm-1)
1700
Figure 2. SERR spectra of (A) 3 X lo-' M oxyhemoglobin and (B) 6 X lO-' M (Fe**'PP)20, recorded between loo0 and 1700 cm-I and excited at 514.5 nm.
the possibility existed that (Fe1T1PP),Ospecies forms, upon addition to the silver colloid, from either thermally denatured or autoxidized hemoglobin, as suggested by Smulevich and Spiro.s Investigations using chemically oxidized oxyhemoglobin (methemoglobin) showed that no (Fei11PP)20species formed upon addition to the silver colloid; thus, its formation evidently arises from thermally denatured protein. Raman excitation profiles of SERR bands of oxyhemoglobin measured with respect to the 1466-cm-' band of glycerol are given in Figure 3, along with the electronic absorption spectrum of the unadsorbed oxyhemoglobin. The excitation profiles show that the SERR bands rise in the region of 540 nm but fall by 568 nm, which is highly indicative of the (3 band of the oxyhemoglobin. The excitation profiles also show a very small rise around 490 nm which could arise from perturbations of the electronic states upon adsorption to the silver colloid. We anticipate that the excitation profile would rise also in the region of the a band, but the limitations of our dye laser prevented Gonfirmation of this. This SERR band excitation profiles also show, unlike those for the RR bands, that excitation in the a- and @-bandregions gives rise to enhancement of totally symmetric as well as asymmetric vibrational modes of the porphyrin macrocycle (see Table I and Figure 3). Indeed, with 568.2-nm excitation the symmetric stretching mode u4 of oxyhemoglobin is the dominant band in the SERR spectrum, in contrast to the R R spectrum in which the v4 mode is totally absent at this excitation wavelength. In addition, excitation at 568.2 nm appears to be less in resonance with the a electronic absorption band. These results indicate a significant perturbation of the electronic states associated with the a and (3 absorption bands. Activation of the a l gvibrational modes with excitation in the wavelength region of the (3 band has also been observed for cytochrome c on colloidal silver,' and an explanation offered in terms of spin state changes. However, the lowering of the symmetry of the porphyrin moiety, brought about by adsorption to the silver surface, could itself be responsible for the activation of the alg modes in affecting mixing of the transition moments associated with the Soret and a bands. Unfortunately, due to the strong absorption of the silver sol itself, changes in the oxyhemoglobin absorption are not directly observable. The Raman excitation profiles of the (Fe"*PP),O species measured with respect to the 1466-cm-' band of glycerol are given
4 50
500
5 50
Wavelength lnm)
Figure 3. Visible absorption spectrum of unadsorbed oxyhemoglobin and excitation profiles of some prominent SERR bands normalized to the 1466-cm-I band of glycerol.
X
Y/cm-l 1370
A 1399
+ 1488 0
1570 1592
- Visible absorption
49
500
5 50
Wavelength (nm)
Figure 4. Visible absorption spectrum of unadsorbed (Fe"'PP),O and excitation profiles of some prominent SERR bands normalized to the 1466-cm-' band of glycerol.
in Figure 4 along with the electronic absorption spectrum of the unadsorbed (Fe111PP)20species. The excitation profiles of this species show no rise in the region of 540 nm and follow the electronic absorption of the (Feil'PP),O species, indicating little or no change to its electronic states upon adsorption. The only vibrational band wavenumber shifts observed upon adsorption of oxyhemoglobin to the silver colloid are in the v3 and u4 bands. v3, the algsymmetric stretching mode C,-C,, is shifted
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J . Phys. Chem. 1987, 91, 1696-1698
from 1506 to 1501 cm-I. However, other structure-sensitive bands above 1450 cm-’ remain unchanged upon adsorption to the silver colloid; the reason for this is at present unclear. v4, the al, symmetric stretching mode C,-N (the porphyrin “ring breathing” mode), is shifted from 1375 to 1370 cm-I. This is a marker band for the oxidation state of the central metal atom as well as for alteration in the ring a bonding.I4 The 5-cm-I decrease relative to the R R band of the solution species suggests that a partial transfer of charge from the silver metal to the heme complex is taking place. Overall, these data indicate that the vibrational mode wavenumbers are less sensitive to electronic state perturbations than are the band intensities and excitation profiles. Other evidence has pointed to an edge-on binding of the porphyrin macrocycle to the silver surface, probably through the propionate functional groups, as suggested by Stockburger and Hildebrandt’ for adsorption of cytochromes onto silver colloids and by McMahon6 and CottonIs for adsorption of porphyrin model compounds onto silver electrodes. Adsorption of oxyhemoglobin through the negatively charged proprionate groups, which extend out through the protein envelope, would not result in large perturbation of the porphyrin electronic states, since the propionate groups appear not to be involved in coupling the molecular ground state to the electronic excited state, as evidenced by their absence from the R R spectra.6 Adsorption of the porphyrin macrocycle to the silver surface via overlap of the A electrons of the pyrrole rings would result in the porphyrin plane lying parallel to the surface. This seems unlikely since it would be expected to lead to significant perturbation in the vibrational mode energies, but no such perturbations are observed. It would also require major conformational changes to the protein structure to enable the silver to come into contact with the porphyrin macrocycle (e.g., opening of the heme pocket). Moreover, we have shown that this binding geometry can be ruled out since we now have established that the adsorbed hemoglobin remains functionally active in reversible ligand binding of O2and CO. The detailed experimental evidence in support of this statement will be the subject of a separate publication. Hence, the sixth axial ligand position of the heme iron does remain available for reversible ligand binding. The (14) Spiro, T. G. In Iron Porphyrins, Part I I ; Physical Bioinorganic Chemistry Series; Leuer, A. B., Gray, H. B., Eds.; Addison-Wesley: Reading, MA, 1982. (15) Cotton, T. M.; Schultz, S. G.: Van Duyne, R . P. J . Am. Chem. Soc. 1982, 104, 6528.
Fe-N,,, stretching band of adsorbed deoxyhemoglobin (obtained by 44 1.6-nm laser photolysis of adsorbed carbonylhemoglobin) is found at 215 cm-’. This indicates that the adsorbed deoxyhemoglobin is in the T-state quaternary structure and shows that the adsorption at the silver surface imposes no additional strain on the Fe-N,,, bond. The reversible ligand-binding property of the adsorbed hemoglobin is a clear indication that the hemoglobin does not denature to form (Fe111PP)20decomposition products. Assuming that oxyhemoglobin (- 6-nm molecular diameter) is bound to the silver colloid (- 35-nm average particle diameter) through the propionate groups, with the heme plane lying perpendicular to the silver surface, the iron atom will be approximately 1.5 nm from the silver surface. In this configuration the porphyrin macrocycle lies parallel to the electric field resulting from the net positive charge on the colloidal metal. Hence, the interaction of the porphyrin T electrons with the electric field will be favored, even though somewhat distant from the silver surface. Although the absorbances are similar at 457 nm, the SERR signals of oxyhemoglobin are approximately 500 times less intense than those of hemin chloride and (Fe”’PP),O. It has been shown16 that the intensity of SER signals is a function of the distance of the adsorbate from the metal surface. Since it is believed that these heme complexes all bind to the silver surface in the same manner, it seems likely that the increase in the SERR signals of the protein-free heme complexes is due to the fact that they are able to approach closer to the silver surface than the heme groups in the oxyhemoglobin. These are encased in a protein envelope that not only restricts the movement of the porphyrin macrocycle but also shields it from the electric field surrounding the silver particle. This interpretation is consistent with our overall conclusion that oxyhemoglobin is not denatured but remains intact when adsorbed on colloidal silver.
Acknowledgment. We are grateful to Mr. R. B. Girling for technical assistance, to Dr. T. Kitagawa for helpful discussions and for placing the facilities of his laboratory at the I.M.S., Okazaki, at our disposal, and to the SERC for financial support to J.deG. (16) Murray, C. A.; Allara, D. L. J . Chem. Phys. 1982, 76, 1290. (17) Choi, S.; Spiro, T. G.; Langry, K. C.; Smith, K. M.; Budd, D. L.; La Mar. G. N.J . A m . Chem. Soc. 1982, 104, 4345. (18) Spiro, T. G.; Strekas, T. C. J . Am. Chem. SOC.1974, 96, 338.
Transient Grating Method Applied to Electron-Transfer Dynamics at a Semiconductor/Liquid Interface Seiichiro Nakabayashi, Shuji Komuro, Yoshinobu Aoyagi, and Akira Kira* The Institute of Physical and Chemical Research, Wako-Shi, Saitama 351 -01, Japan (Received: September 18, 1986; In Final Form: December 2, 1986) The transient grating method utilizing a fringe pattern of excited states was first applied to a semiconductor/electrolyte interface. The dynamics of a photoelectrochemical oxidation of 2-propanol aqueous solution on a Ti02 electrode was observed by this method. The results were explained in terms of participation of additional electron injection to the conduction band from an oxidized intermediate species of 2-propanol, which has been proposed as a mechanism of the current-doubling effect. Introduction This letter reports the first application of a transient grating method to the measurement of electron-transfer dynamics at a semiconductor/electrolyte interface. The transient grating method has recently been employed for the study of the recombination and the diffusion of charge carriers in semiconductors,l-3and (1) Aoyagi, Y.; Segawa, Y.; Namba, S. Phys. Reo. B: Condenr. Matrer
1982, 25, 1453.
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studies refer to a surface or a solid/solid interface.* In this method, a holographic diffraction grating consisting of a fringe of photoinduced charge carriers is utilized for the monitoring of carrier (2) Komuro, S.; Aoyagi, Y.; Namba, S.; Masuyama, A,; Okamoto, H . ; Hamakawa, y. Phys. Lett. 1983, 43,968. Newell, v. J.; Rose, T.s.; Fayer, M. D. Phys. Rev. B Condens. Marrer 1985, 32, 8035. (3) Hoffman, C. A.; Jarasiunas, K.; Gerritsen, H. J.: Kurmikko, A. V. Appl. Phys. Lert. 1978, 33, 536. Salcedo, J. R.: Siegman, A. E.; Dlott, D. D.;Fayer, M . D. Phyr. Rei!. Lert. 1978, 4 1 , 131.
0 1987 American Chemical Society
