Raman and surface-enhanced Raman spectroscopy of tempyo spinlabelled ovalbumin

S. CõÃntaÄ-PõÃnzarua,*, S. Cavalub, N. Leopolda, R. Petryc, W. Kieferc

aPhysics Department, Babes-Bolyai University, Kogalniceanu 1, RO-3400 Cluj-Napoca, Romania;bBiophysics Department, University of Oradea, 1 Dec. Square, No. 1, RO-3700 Oradea, Romania

cInstitut fuÈr Physikalische-Chemie, UniversitaÈt WuÈrzburg, Am Hubland, D97074 WuÈrzburg, Germany

Received 31 August 2000; revised 17 November 2000; accepted 17 November 2000

Abstract

Tempyo labelled ovalbumin at different pH values was prepared and investigated using Raman and SERS spectroscopy.

Raman spectra of tempyo labelled ovalbumin in the pH range from 6.7 to 11 were compared to those of the corresponding free

ovalbumin. In the basic pH range from 6.7 to 11 the molecular conformation was found to be unaffected by the tempyo

presence. Adsorption versatility to the colloidal Ag particles of pure- and tempyo labelled ovalbumin was also found to be

unchanged in this basic pH range. As the SERS binding site of protein the a-helix conformation is favourable. q 2001 Elsevier

Science B.V. All rights reserved.

Keywords: Ovalbumin; Raman; SERS; Tempyo

1. Introduction

The biophysical activity of ovalbumin (protein of

385 amino acids) and its effect in reacting with other

enzymes provides contradicting results to the hypoth-

esis of the experiment. Ovalbumin is a good model for

the serpin family and a unique feature is that upon

proteolysis with enzymes, very little conformational

change or inhibitory functions can be observed [1,2].

The current traditional methods of investigation of

proteins turn more and more to the ef®cient techni-

ques, like Raman and its derivatives. Raman and

SERS spectroscopy plays a key role in exploring the

behaviour of biological samples. In particular, due to

its high sensitivity and selectivity, surface-enhanced

Raman technique can be successfully used to investi-

gate the versatility to adsorption, conformational

changes of proteins, which occur as a consequence

of proteolysis, lyophilization or dehydration [3]. A

typical stable nitroxyl free radical, widely used as

ESR spin label, tempyo (2,2,5,5-tetramethyl-3

pyrrolin-1-yloxy-3 carboxamide) was applied to

label the solutions of ovalbumin at various pH (6.7,

8.1, 9.5 and 11). In order to study the magnetic inter-

action between the spin label and the functional group

of this protein (spin±spin and exchange phenomena)

and also the motional effect in spin label spectra,

samples for EPR spectroscopy [4,5] were prepared

and parallel investigated. The EPR results were uncer-

tain in answering to the question if the ovalbumin

suffers conformational changes in the labelling

process at different pH. Therefore, in this paper we

present vibrational Raman and SERS investigations

on the tempyo labelled ovalbumin in the basic pH

Journal of Molecular Structure 565±566 (2001) 225±229

0022-2860/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.

PII: S0022-2860(00)00930-3

www.elsevier.nl/locate/molstruc

* Corresponding author. Tel.: 140-64-405-300; fax: 140-64-

191-906.

E-mail address: scinta@phys.ubbcluj.ro (S. CõÃntaÄ-PõÃnzaru).

range between 6.7 and 11 where the possible modi®-

cations in the mobility of tempyo into the protein were

suspected. The expansion of the study to the surface

enhanced Raman spectroscopy was performed in

order to check the in¯uence of tempyo label to oval-

bumin adsorption and to study binding sites and

binding mechanisms of tempyo in the tempyo-protein

complex, if any exists.

2. Experimental

2.1. Chemicals

The ovalbumin powder was received from Sigma

and used without further puri®cation. The protein was

rehydrated in phosphate buffer physiological saline at

a ®nal concentration of 1023 mol/l and than the corre-

sponding tempyo buffer solution was added to each

protein solution in a ®nal concentration of 1023 mol/l.

Protein/spin label molar ratio was 1:1. The pH range

was adjusted between 6.7 and 11. A small amount of

5 ml from each sample was lyophilised [6] for 30 h at

2508C and than used as Raman sample.

Colloidal silver substrate was prepared according to

the Lee±Meisel procedure [7]. The absorption

maximum of the freshly prepared colloid was centred

at 423 nm. Buffer solutions were prepared for each

corresponding pH value of the ovalbumin. A small

amount of about 10 ml 1022 mol/l ovalbumin aqueous

solution at each pH was added to 2 ml colloidal silver,

resulting a ®nal SERS sample concentration of

5 £ 1025 mol/l.

2.2. Apparatus

For the recording of the Raman spectra from

lyophilised powdered sample a micro-Raman setup

was employed. The 514.5 nm line of an argon ion

laser (Spectra Physics, Model 166) was applied for

excitation. The scattered light was collected in back-

scattering geometry by focusing a £ 50 objective

(Olympus ULWD MSPlan50) on the entrance slit of

a spectrometer LabRam, Dilor with 1800 grooves/mm

diffractive grating. The laser power at the focus spot

with a beam diameter of about 0.1 mm on the sample

was kept below ca. 160 mW. The spectral resolution

was 3 cm21. The detection system consisted of a

charge-coupled multichannel detector (CCD, SDS

9000 Photometrics). The acquisition of a single spec-

trum typically takes about 100 s and 4 repeats on each

sample were done. Each Raman spectrum is the result

of 4 accumulations with 100 s exposure time. For the

SERS spectra we used a £ 10 objective, a laser power

of 1 mW and an exposure time of 1000 s with 4 over-

laps. Using high power in excitation of the SERS

spectra, the SERS signal was untrustworthy. Keeping

the low incident power (up to 1 mW) they were found

reproducible (3±4 experiments were reproduced).

3. Results and discussions

Raman spectra of solid pure ovalbumin at different

pH values (lyophilised powder sample) in the spectral

range 550±1700 cm21 are presented in Fig. 1A

whereas Fig. 1B presents the Raman spectra of the

corresponding tempyo labelled ovalbumin. Raman

spectrum of solid tempyo (Fig. 1B, a) is presented

as a proof that the label does not bring any contribu-

tion in the vibrational Raman structure of the protein

in spite of its large Raman cross-section. This is prob-

ably due to the complex folded macrostructure of

protein, which limits the scattering effect of the

small tempyo label. The spectra are interpreted with

the aid of the amino acid and proteins studied earlier

[8,9]. Several vibrational modes in this region are

sensitive to protein conformation, e.g. amide I and

amide III. The spectra of pure ovalbumin at different

pH were compared with the corresponding spectra of

the various ovalbumin-tempyo complexes. The pH

dependence of ovalbumin Raman spectra (Fig. 1A)

suggests a stabile structure of ovalbumin molecule

between pH 6.7 and 11. The only one observed differ-

ence was the presence of a new band at 1040 cm21,

which is decreased for pH values over 9.5. This band

was uncertain for any assignments, anyway the

tempyo contribution to this was excluded (Fig. 1B, a).

The amino acid polymer structure of the ovalbumin

basically has the peptide bonds that regularly repeat

the conformation of the polypeptide backbone

forming the secondary structure, like the a-helix b-

pleated sheet and random coil. All these three contri-

bution are observed in the Raman spectra as a large

band centred at 1665 (Fig. 1A and B) through the

amide I and III contribution. The phenyl stretching

bands at 998, 1027 and 1602 cm21 are present in the

S. CõÃntaÄ-PõÃnzaru et al. / Journal of Molecular Structure 565±566 (2001) 225±229226

S. CõÃntaÄ-PõÃnzaru et al. / Journal of Molecular Structure 565±566 (2001) 225±229 227

1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600

e

d

c

b

a

Ram

anIn

tens

ity/a

.u.

Wavenumbers/cm-1

1040

615

638

754

822

849

896

954

998

1027

1076

1123

1151

116712

041329

1314

1337

1393

1446

1461

1553

158316

02

1665

1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600

Ram

anIn

tens

ity/a

.u.

Wavenumbers/ cm-1

e

d

c

b

atempyo

1446

1665 10

2899

8

Fig. 1. A (top). Raman spectra of pure ovalbumin (a) and at various pH: (b) 6.7; (c) 8.3; (d) 9.5; (e) 11; B (bottom). Raman spectra of tempyo (a)

and of the lyophilized tempyo labelled ovalbumin, at various pH: (b) 6.7; (c) 8.3; (d) 9.5; (e) 11. Excitation: 514.5 nm, 160 mW on the powder

sample.

S. CõÃntaÄ-PõÃnzaru et al. / Journal of Molecular Structure 565±566 (2001) 225±229228

1600 1500 1400 1300 1200 1100 1000 900 800 700 600

d

c

b

a

Wavenumbers/cm-1

Ram

anIn

tens

ity/a

.u.

1600 1500 1400 1300 1200 1100 1000 900 800 700 600

e

d

c

b

a

1025

605

770

1123

1174

1303

1357

1506

156916

44

Ram

anIn

tens

ity/a

.u.

Wavenumbers/cm-1

Fig. 2. A (top). SERS spectra of pure (a) and lyophilized ovalbumin at various pH: (b) 6.7; (c) 8.3; (d) 9.5; (e) 11. B (bottom). SERS spectra of

tempyo labelled ovalbumin, at different pH: (a) 6.7; (b) 8.3; (c) 9.5; (d) 11. Excitation: 514.5 nm, 1 mW on the sample.

Raman spectra indicating the aromatic amino acids

residues. The CH2 and CH3 scissoring modes are

located at 1461 and 1446 cm21, respectively, similar

to that of the bovine serum albumin Raman spectrum

[8]. In addition, bands from the side chains of some of

the amino acids residues (Tyr, Trp, Phe, etc.) are

present.

SERS spectra of pure ovalbumin and at different pH

values are presented in Fig. 2A and the corresponding

SERS spectra of the tempyo labelled samples in Fig.

2B, respectively. The most signi®cant information in

the SERS spectra are contained in the region

550±1700 cm21 and for this reason the spectra are

displayed in this spectral range only. They strongly

differ from their corresponding Raman spectra, in

band positions and relative intensities. These differ-

ences could have at least two possible explanations:

SERS and normal Raman probably span a different

portion of the protein structure, and hence bands of

different frequencies and relative intensities are

observed; further, based on SERS theory [10], the

molecules can be physisorbed or chemisorbed, the

last case being characterised through the drastic spec-

tral change on passing from Raman to SERS. When

the chemisorption takes place, the molecule of interest

together with the nanometric surface builts the so-

called ªmetal-molecule SERS complexº [11] where

a charge transfer contribution to the total enhance-

ment mechanism [10] is present. According to the

literature [12], the amide I band for the a-helix b-

sheet and random coil conformation occurs in the

region 1658±1640, 1680±1665 and 1666±

1660 cm21, respectively. For the amide III band

position of these conformations were reported the

regions 1310±1260, 1242±1235 and 1250±

1240 cm21, respectively. In our SERS spectra of

ovalbumin with or without tempyo label the amide I

band can be observed at 1644 cm21 while the amide

III band is located at 1303 cm21, both of them

indicating the a-helix conformation as the SERS

binding site of protein. The phenyl stretching modes

that are the most intense in the Raman spectra, are

completely absent in SERS, suggesting that the

aromatic residues are not involved in adsorption or

far from the colloidal surface. Moreover, in the

basic pH region studied the ovalbumin was found to

be stable in adsorption to the colloidal silver particles.

The pure tempyo was found to be SERS inactive,

independent on the concentration in the colloidal ®nal

sample or laser power.

4. Conclusions

The suspected tempyo induced motional effects or

conformational changes of tempyo labelled oval-

bumin in the basic pH region between 6.7 and 11

were concluded to be absent from the Raman spectra.

Pure or tempyo labelled ovalbumin in the basic pH

region between 6.7 and 11 was found to adsorb on the

silver colloidal particles, with a chemical contribution

to the total enhancement mechanism. The amide I and

III band contribution in the SERS spectra suggests

that the a-helix domain of the protein is closer to

interact with the colloidal surface.

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