Raman and Surface Enhanced Raman Spectroscopy of2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamideLabeled Proteins: Bovine Serum Albumin and Cytochrome c

S. CAVALU,1 S. CINTA-PINZARU,2 N. LEOPOLD,2 W. KIEFER3

1 Biophysics Department, University of Oradea, 1 Dec. Square, Number 10, RO-3700 Oradea, Romania

2 Physics Department, Babes-Bolyai University, Kogalniceanu 1, RO-3400 Cluj-Napoca, Romania

3 Institut fur Physikalische-Chemie, Universitat Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

Received 1 March 2001; revised 19 March 2001; accepted 17 April 2001Published online 19 October 2001; DOI 10.1002/bip.10002

ABSTRACT: 2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bo-vine serum albumin and cytochrome c at different pH values were prepared andinvestigated using Raman–resonance Raman (RR) spectroscopy and surface enhancedRaman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteinsin the pH 6.7–11 range were compared to those of the corresponding free species. TheSERS spectra were interpreted in terms of the structural changes of the tempyo labeledproteins adsorbed on the silver colloidal surface. The tempyo spin label was found to beinactive in the Raman–RR and SERS spectra of the proteins. The a-helix conformationwas concluded to be more favorable as the SERS binding site of bovine serum albumin.In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocyclestretching mode allowed the supposition of the N-adsorption onto the colloidal surface.© 2001 John Wiley & Sons, Inc. Biopolymers (Biospectroscopy) 62: 341–348, 2001

Keywords: bovine serum albumin; cytochrome c; 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide spin label; Raman; surface enhanced Raman scattering

INTRODUCTION

In spite of the existence of a large amount of datain the biological applications fields of Ramanspectroscopy and particularly resonance Raman(RR) spectroscopy applied to heme proteins, it isvery difficult to outline a complete vibrationalcharacterization of them.1 The current traditionalmethods of analysis of proteins are time consum-ing and expensive, and therefore more efficienttechniques are desirable. Raman spectroscopy isan important tool for the determination of the

structure of biomolecules, and extensive researchhas been undertaken.2–4

The lower sensitivity and effects of fluores-cence on the normal Raman spectra of these mol-ecules led to the extension of research in the di-rection of surface enhanced Raman scattering(SERS).5 The latest consensus is that, as a resultof electromagnetic enhancement, the SERS spec-trum of molecules is virtually an enhanced ver-sion of the compounds of the normal Raman spec-trum. However, during charge transfer somevibrations will be altered because of the mole-cule’s interaction with the nanometric surface,resulting in the corresponding spectrum of themolecule–surface complex.6

Albumin is the most abundant protein in thecirculatory system and contributes 80% to colloid

Correspondence to: S. Cavalu (meda@medanet.ro).Contract grant sponsor: World Bank; contract grant num-

ber: T 131.Biopolymers (Biospectroscopy), Vol. 62, 341–348 (2001)© 2001 John Wiley & Sons, Inc.

341

osmotic blood pressure.7 It has now been deter-mined that serum albumin is chiefly responsiblefor the maintenance of blood pH.

The substantial information on bovine serumalbumin (BSA) has led to some contradictory re-sults and discussions. The previously reportedRaman spectra8 showed an intensity increase at1235 cm21 and a decrease at 940 cm21 for theBSA gel, indicating a drop in the a-helix contentaccompanied by b-sheet formation. An investiga-tion of thermal, acid, and alkali denaturation ofBSA by Raman spectroscopy reported that heat-ing to 70°C or a change in pH below 5 or above 10caused an increase of the 1246 cm21 band and adecrease of the 938 cm21 band. The interpretationwas that there is a decrease in the a-helix contentaccompanied by an increase in b sheets.9

In another reported Raman spectrum of BSA10

the interpretation was made with the aid of pre-vious amino acid data, highlighting a relationshipbetween the intensity of a given amide I band andthe amount of structural conformation to which itis assigned. Further assignments10 were found tobe not relevant for the conformation of the pro-tein.

Conformation-sensitive Raman bands of glob-ular proteins were also reported11 in which theamide I, amide III, and some skeletal modes areassigned and used to compare and differentiateamong the a-helix, b-sheet, and random coil con-tent. The Raman active amide I mode for thea-helix conformation appeared at the same fre-quency as in the IR spectra (1650 to 1657 cm21).Strong splitting of the amide I mode was observedfor the antiparallel b-sheet conformation.11 Anintense amide III band at 1235 cm21 could beassigned to the antiparallel b sheet, and the dis-ordered conformation appeared at about 1245cm21 in the amide III region of the Raman spec-trum.11 The lack of any strong Raman bands inthe range from 1200 to 1300 cm21 was reported asevidence for the a-helix conformation.

In the present Raman and SER spectroscopicstudies the conformational change of BSA wasinvestigated in a pH range of 6.7–11. All these pHvalues are greater than the isoelectric pH value,at which point the ONH2 groups are recoveredandOCOOH groups become anionic (OCOO2).11,12

Cytochrome c is an essential component of themitochondrial respiratory electron transportchain.13 The structure of cytochrome c is verysimilar to hemoglobin in regard to the hemegroup, the active site of this protein. This group isnot attached to the protein by only one axial li-gand (e-imidazole nitrogen of histidine) of the iron

atom as in hemoglobin. In both oxidation statesthere is a second axial ligand, the sulfur atom ofmethionine 80, which is part of the protein. Ad-ditionally, the heme is linked to cysteine 14 andcysteine 17 by two thioether bonds.13,14 The iso-electric pH value of this protein is 10.6.

A typical stable, nitroxyl-free radical widelyused as an electron spin resonance spin label,2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide(tempyo), was applied as a label on the solutionsof proteins at pH 6.7, 8.1, 9.5, and 11. In order tostudy the magnetic interaction between the spinlabel and the functional group of these proteins(spin–spin and exchange phenomena) and themotional effect in spin label spectra, samples forelectron paramagnetic resonance (EPR) spectros-copy were prepared and subjected to a parallelinvestigation (data not shown). The EPR investi-gations followed the pH influence on the rota-tional correlation time of tempyo with respect tothese proteins in the framework of the “moderatejump diffusion” model for rotational diffusion.15,16

The localization of the tempyo label in the pro-tein–tempyo complex structure could influencethe vibrational structure and, consequently, theadsorption behavior of the proteins.

Therefore, in this article we present vibra-tional Raman and SERS investigations on thetempyo labeled BSA and ferrocytochrome c in apH range between 6.7 and 11, which is where themobility of tempyo is assumed to be sensitive withrespect to these proteins. The extension of thestudy to SER spectroscopy was performed tocheck the influence of the tempyo label on BSA orcytochrome c adsorption and to study bindingsites and binding mechanisms of tempyo in thetempyo–protein complex, if any exist.

MATERIALS AND METHODS

Chemicals

The BSA, cytochrome c, and tempyo spin labelpowder were purchased from Sigma and usedwithout further purification. The cytochrome cwas reduced with a small excess of sodium dithio-nite. Samples were prepared in phosphate bufferphysiological saline at a final concentration of1023 mol/L. Noncovalent labeling of each samplewas made in a 1:1 protein/tempyo spin label mo-lar ratio. The pH range was adjusted between 6.7and 11. A small amount (5 mL) of each samplewas lyophilized for 30 h at 250°C and than usedas the powder Raman sample.

342 CAVALU ET AL.

Colloidal silver substrate was prepared accord-ing to the Lee–Meisel procedure.17 The maximumabsorption of the freshly prepared colloid was cen-tered at 423 nm. Proteins were rehydratated inbuffer solutions for each pH value. A smallamount (10 mL) of 1022 mol/L protein solutionwas added to 2 mL of colloidal silver, resulting ina final sample concentration of 5 3 1025 mol/L.

Apparatus

A micro-Raman setup was employed to record theRaman spectra of the lyophilized powder samples.The 514.5-nm line of an argon ion laser (model166, Spectra Physics) was applied for excitation.The scattered light was collected in backscatter-ing geometry by means of a 503 objective (Olym-pus ULWD MSPlan50). A LabRam Dilor with1800 grooves/mm diffractive grating was used fordispersing the scattered light. The detection sys-tem consisted of a charge-coupled multichanneldetector.

A 103 objective, a laser power of 1 mW, and anexposure time of 1000 s with four overlaps wereused for the SERS spectra of BSA. When usinghigh power in the excitation of the SERS spectra,the SERS signal was untrustworthy. When keep-ing the low incident power (up to 1 mW), theywere reproducible. (Three or four experimentswere reproduced.)

In cytochrome c the acquisition of a single spec-trum typically takes about 100 s, and four repeatson each sample were done. Each Raman spectrumis the result of four accumulations with 100-sexposure time using a laser power of about 12mW. The spectral resolution was 3 cm21.

RESULTS AND DISCUSSION

BSA Analysis

The Raman spectra of pure and tempyo labeledBSA at different pH values and in the spectralrange of 500–1800 cm21 are presented in Figure 1.

In comparison with previous Raman reports onBSA,11 the spectral region between 1200 and1300 cm21 is poorly represented, indicating thedominant presence of a-helix content. The second-ary structure (a helix, b-pleated sheet, and ran-dom coil)10–12,18 is observed in the Raman spectrathrough the contribution of the strong amide Iband (1653 cm21) and weak amide III band (1270cm21). The phenyl stretching bands at 998, 1027,and 1603 cm21 are present in the Raman spectra,

indicating the aromatic amino acid residues. TheCH2 and CH3 scissoring modes are located at1444 and 1461 cm21, respectively, similar to thatof the ovalbumin Raman spectrum.19 In addition,bands from the side chains of some of the aminoacids residues (tyrosine, tryptophan, phenylala-nine, etc.) are present.

Besides the common amide bands, the supple-mentary17 disulfide bridges are characteristic forBSA. The characteristic frequencies of COS andSOS groups are present as weak–medium bandsin the 500–750 cm21 spectral region. Comparingthe spectra from Figure 1 (spectra a–e), the onlymajor difference observed is the presence of a newband at 1042 cm21 for the pH 6.7 and 8.1 values.This band was uncertain for any assignments;moreover, the tempyo contribution to this wasexcluded after studying the Raman behavior ofpure tempyo.

The label does not bring any contribution to thevibrational Raman structure of the protein, inspite of its large Raman cross section. This is

Figure 1. Raman spectra of pure (spectrum a) andtempyo labeled BSA at pH 6.7 (spectrum b), 8.1 (spec-trum c), 9.5 (spectrum d), and 11 (spectrum e). A514.5-nm laser line at 100-mW power was used.

RAMAN AND SERS OF BSA AND CYTOCHROME c 343

probably due to the folding complexity of the pro-tein, which limits the scattering effect of thesmall tempyo label.

The SERS spectra of tempyo labeled BSA atdifferent pH values (Fig. 2) strongly differ fromtheir corresponding Raman spectra in both theband positions and relative intensities. These dif-ferences could have at least two possible explana-tions: SERS and normal Raman probably span adifferent portion of the protein structure, andthus the bands of different wavenumbers and rel-ative intensities are observed; further, based onSERS theory,12,20 the molecules can be phy-sisorbed or chemisorbed, the last case being char-acterized through the drastic spectral change onpassing from Raman to SERS. When the chemi-sorption takes place, the molecule of interest to-gether with the nanometric surface builds a so-called “metal–molecule SERS complex” where acharge transfer contribution to the total enhance-ment mechanism20 is present. According to theliterature,21,22 the amide I bands correspondingto the a-helix, b-sheet, and random coil conforma-tions occur in the 1658–1640, 1680–1665, and1666–1660 cm21 ranges, respectively. On theother hand, the amide III band positions of these

conformations were localized in the 1310–1260,1242–1235, and 1250–1240 cm21 regions, respec-tively. In our SERS spectra of tempyo labeledBSA the amide I band can be observed at 1645cm21, which could be supposed as a Raman shifttoward a lower wavenumber, and the amide IIIband is localized at 1304 cm21. A very strongSERS band was observed at 1357 cm21. At thisposition there is no correspondence in the Ramanspectra (Fig. 1). It is difficult to believe that theweak band at 1400 cm21 from the Raman isshifted to such lower wavenumbers in SERS. Itwould be more convincing if there was a SERScontribution derived from the large Raman bandat 1334 cm21.

On comparing the SERS spectrum of pure BSAwith the SERS spectra at various pH values, onlyone notable difference could be observed (Fig. 2):the presence of the weak and medium phenylbands at 1615 and 1024 cm21, respectively. In theprevious published SERS spectrum of BSA23 theauthors reported that only those vibrations corre-sponding to aromatic side chains are visible, inaddition to the amide I and III major bands. Onthis basis they suggested a random coil conforma-tion on the surface. However, it is not appropriateto compare our SERS spectra with those obtainedin completely different conditions of surface, sur-face plasmon resonance, or sample drying.

In our study we suggest a carboxylate groupinteraction with the colloidal surface through thepresence of the enhanced amide I band and lessthrough the aromatic side chains for all the pHvalues investigated. The secondary structure ofthe title compound is obviously modified by theadsorption induced structural changes.

These remarks suggest that the a-helix confor-mation is probably the SERS binding site of BSA.Moreover, in the pH range that was studied thetempyo labeled BSA was found to be stable whenadsorbed onto the colloidal silver particles (withinsignificant differences from the pure BSA). Thepure tempyo was found to be SERS inactive, in-dependent of the concentration in the colloidalfinal sample or laser power.

Cytochrome c

Excitation with the 514.5-nm line falls into one ofthe absorption bands (408, 520, and 544 nm) ofcytochrome c, leading to the RR spectra of thesample. Figure 3 presents the RR spectrum ofpure cytochrome c in the solid state in comparisonwith the spectra of cytochrome c at pH 6.7, 8.1,9.5, and 11.

Figure 2. SERS spectra of pure (spectrum a) andtempyo labeled BSA at pH 6.7 (spectrum b), 8.1 (spec-trum c), 9.5 (spectrum d), and 11 (spectrum e). A514.5-nm laser line at 100-mW power was used.

344 CAVALU ET AL.

The very strong at 1580 n(CAN) and 1308 cm21

(porphyrin stretching); the strong bands at 1530n(CAC), 1393, and 1356 cm21 n(ACON); and themedium bands at 1225 d(COH), 1166, and 1124cm21 are observed in concordance with the previ-ously reported results on cytochrome c.24–26,29

The band at 1225 cm21 is believed to arise froman in-plane bending vibration of the methine hy-drogens. In our RR spectra this vibration seems tonot be affected by the pH variation, even if H-NMR and visible absorption studies30 showedthat pH, temperature, or ionic strength perturba-tions readily displaced the methionine ligand. Itwas suggested that the loss of the methionineligand results in the loss of the electron transportcapability of cytochrome c. When comparing theRR spectra of cytochrome c in both oxidationstates, ferricytochrome c exhibited a very differ-ent Raman spectrum from ferrocytochrome c,which is much more sensitive to RR.26

For the ferricytochrome (oxidized form, Fe31)three bands of medium intensity at 1560, 1585,and 1638 cm21 are reported26 and interpreted asCAC and CAN stretching vibrations of the por-phyrin macrocycle. Other RR spectra27 of oxidizedand reduced cytochrome c in solution correlatedthe spectral band at 1584 cm21 with the existence

of low spin iron in both oxidation states and theposition of the spectral band at 1375 or 1360 cm21

with oxidized or reduced cytochrome c, respec-tively. A general shift toward a lower frequencywas concluded upon reduction of Fe31 to Fe21. Onthe other hand, chemical modification of the hemeprotein resulting from the conversion of iron froma low to a high spin state was correlated with a1584–1566 cm21 shift, which is ascribed to themovement of the iron atom out of the heme planeupon the increasing of its spin state.29,31 Based onthe previous observations, we conclude that thestrong band located at 1580 cm21 (Fig. 1, spec-trum a) indicates the presence of low spin Fe21.

The tempyo spin label was expected to reveal ascattering contribution in the cytochrome c–tem-pyo complex, based on its Raman spectrum (Fig.4, spectrum a). In Figure 4 one can observe thatthe label does not make any contribution to thevibrational structure of this protein, in spite of itslarge Raman cross section. This is probably due tothe folding complexity of the protein, as in theBSA, which limits the scattering effect of thesmall tempyo label. We suppose that the label isbound within a certain distance from the hemegroup in the basic pH range of 6.7–11.

A comparison of Figures 3 and 4 reveals the

Figure 3. Resonance Raman spectra of pure (spectrum a) and lyophilized cytochromec at pH 6.7 (spectrum b), 8.3 (spectrum c), 9.5 (spectrum d), and 11 (spectrum e). Theexcitation was at 514.5 nm, and there was 12-mW power on the powder sample.

RAMAN AND SERS OF BSA AND CYTOCHROME c 345

very stable conformation of the cytochrome cheme group in the pH range from 6.7 to 11, inspite of the fact that the amino acid residues arevery sensitive to the pH variation. Furthermore,the RR spectra do not reflect changes in the pro-tein content around the porphyrin ring.

Figures 5 and 6 respectively present theSERRS spectra of pure cytochrome c at the corre-sponding pH values with or without tempyo spinlabel. The first SERRS of cytochrome c24 showedthat this spectrum appeared to be very similar toits solution RR spectrum. Other previous works,which were based on a comparison between theSERRS and RR spectra of the oxidized form, con-cluded that the heme was detached from the pro-tein at the level of the silver surface and formedoxo-dimers.25

When passing from RR to SERRS in thepresent work, large differences can be observed inthe band positions and relative intensities. Thedominant SERRS bands are observed at 1633(nCAN), 1581 (nCAN), 1562, 1399, 1369, 1166, and1127 cm21. In contrast to the corresponding RRspectra, the in-plane bending vibration of the me-thine hydrogens (1227 cm21) is poorly repre-sented but it seems to be pH dependent. Theblueshift from 1356 (RR) to 1369 cm21 (SERRS)

and the enhancement indicate an interaction ofthe N atoms from n(ACON) bonds of the porphyrinring through the bonds with the metal surface.32

According to the literature,33 porphyrin macro-cycles in cytochromes and other porphyrin com-pounds bind edge-on to silver surfaces via propi-onate functional groups. Other reports1 showedthat the protein envelope prevents the hemegroup from coming in direct contact with the sil-ver electrode surface. If the heme group werelocated far from the surface, the local electromag-netic enhancement of the heme modes would beless representative. Because the resonance contri-bution in the signal intensity is the only one re-maining, this would be similar to the RR signal,which is not observed in our SERRS spectra.

The SERRS spectral features of cytochrome care certainly attributable to the adsorption in-duced structural changes in the heme pocket, re-sulting in the corresponding enhancement of theporphyrin cytochrome c–Ag complex modes, inde-pendent of the presence of the tempyo label. Thepure tempyo was found to be SERS inactive oncemore and independent of the protein concentra-tion in the colloidal final sample or the laserpower.

Figure 4. The Raman spectrum of tempyo (spectrum a) and resonance Raman spectraof the lyophilized tempyo labeled cytochrome c at pH 6.7 (spectrum b), 8.3 (spectrum c),9.5 (spectrum d), and 11 (spectrum e). The excitation was at 514.5 nm, and there was12-mW power on the sample.

346 CAVALU ET AL.

CONCLUSIONS

The suspected tempyo induced motional effects orconformational changes of tempyo labeled BSA in

the basic pH region between 6.7 and 11 wereconcluded to be absent from the Raman spectra.Pure or tempyo labeled BSA in the basic pH range(6.7–11) was found to be adsorbed on the silver

Figure 5. SERRS spectra of pure (spectrum a) and lyophilized cytochrome c at pH 6.7(spectrum b), 8.3 (spectrum c), 9.5 (spectrum d), and 11 (spectrum e). The excitation wasat 514.5 nm, and there was 12-mW power on the sample.

Figure 6. SERRS spectra of tempyo labeled cytochrome c at pH 6.7 (spectrum a), 8.3(spectrum b), 9.5 (spectrum c), and 11 (spectrum d). The excitation was at 514.5 nm, andthere was 12-mW power on the sample.

RAMAN AND SERS OF BSA AND CYTOCHROME c 347

colloidal particles, and there was a chemical con-tribution to the total enhancement mechanism.The amide I and III band contributions in theSERS spectra suggest that the a-helix domain ofthe free or labeled protein is closer and interactswith the colloidal surface.

In the studied pH range, the RR spectra ofcytochrome c (pure or labeled) indicate a stableconformation of the heme group. The tempyo la-bel was supposed to be bound within a certaindistance from the heme group in the basic pH6.7–11 range. From the SERRS study of the tem-pyo labeled cytochrome c on the silver surface, achemical contribution to the total enhancementwas concluded. The enhancement of the bandsassigned to the porphyrin macrocycle stretchingmodes allowed the supposition of the N-adsorp-tion from the porphyrin ring to the colloidal sur-face. The adsorption of the cytochrome c on the Agsurface under resonance conditions is indepen-dent of the pH in the range from 6.7 to 11 or thepresence of the tempyo spin label.

The financial support from the World Bank is grate-fully acknowledged.

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