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Raman and surface-enhanced Raman spectroscopy of tempyo spin
labelled ovalbumin
S. CõÃntaÄ-PõÃnzarua,*, S. Cavalub
, N. Leopolda
, R. Petryc
, W. Kieferc
a
Physics Department, Babes-Bolyai University, Kogalniceanu 1, RO-3400 Cluj-Napoca, Romania;
b
Biophysics Department, University of Oradea, 1 Dec. Square, No. 1, RO-3700 Oradea, Romania
c
Institut 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
RamanIntensity/a.u.
Wavenumbers/cm
-1
1040
615
638
754
822
849
896
954
998
1027
1076
1123
1151
1167
1204
1329
1314
1337
1393
1446
1461
1553
1583
1602
1665
1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600
RamanIntensity/a.u.
Wavenumbers/cm
-1
e
d
c
b
a
tempyo
1446
1665
1028
998
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
RamanIntensity/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
1569
1644
RamanIntensity/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.
References
[1] C.R. Cantor, P.R. Schimmel (Eds.), Biophysical Chemistry,
Part III, The Behavior of Biological Macromolecules
Freeman, San Francisco, 1980.
[2] P.C. Painter, J.L. Koenig (Eds.), Handbook of Biochemistry
and Molecular Biology, Proteins, Vol. III, Raman Spectro-
scopy of Polypeptides and Proteins, 1976.
[3] T.M. Herne, A.M. Ahern, R.L. Garell, J. Am. Chem. Soc. 113
(1991) 846±854.
[4] V. Budker, L.L. Du, M. Seiter, G.R. Eaton, S.S. Eaton,
Biophys. J. 68 (1995) 2531±2542.
[5] J.-L. Du, G.R. Eaton, S.S. Eaton, J. Magn. Reson. 115 (1995)
313±321.
[6] D. Marsch, L.I. Horvath (Eds.), Advanced EPR, Application
In Biology and Biochemistry, E.J. Hoff, Elsevier, Amsterdam,
1990.
[7] P.C. Lee, D. Meisel, J. Phys. Chem. 84 (1982) 339.
[8] A.M. Bellocq, R.C. Lord, R. Mendelson, Biochem. Biophys.
Acta 257 (1972) 280.
[9] S. Stewart, P.M. Fredericks, Spectrochim. Acta, Part A 55
(1999) 1615.
[10] I. Nabiev, M. Manfait, Rev. Ins. Fr. Pel. 48 (1993) 261.
[11] S. CIÃntaÄ, E. Vogel, D. Maniu, M. Aluas, T. Iliescu, O. Cozar,
W. Kiefer, J. Mol. Struct. 482-483 (1999) 679.
[12] B.G. Frushour, J.L. Koening, in: R.J. Clarck (Eds.), Advances
in Infrared and Raman Spectroscopy, Vol. I, England, 1975.
S. CõÃntaÄ-PõÃnzaru et al. / Journal of Molecular Structure 565±566 (2001) 225±229 229

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SIMONA CAVALU_Raman and surface-enhanced Raman spectroscopy of tempyo spin labelled ovalbumin

  • 1. Raman and surface-enhanced Raman spectroscopy of tempyo spin labelled ovalbumin S. CõÃntaÄ-PõÃnzarua,*, S. Cavalub , N. Leopolda , R. Petryc , W. Kieferc a Physics Department, Babes-Bolyai University, Kogalniceanu 1, RO-3400 Cluj-Napoca, Romania; b Biophysics Department, University of Oradea, 1 Dec. Square, No. 1, RO-3700 Oradea, Romania c Institut 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).
  • 2. 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
  • 3. 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 RamanIntensity/a.u. Wavenumbers/cm -1 1040 615 638 754 822 849 896 954 998 1027 1076 1123 1151 1167 1204 1329 1314 1337 1393 1446 1461 1553 1583 1602 1665 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 RamanIntensity/a.u. Wavenumbers/cm -1 e d c b a tempyo 1446 1665 1028 998 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.
  • 4. 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 RamanIntensity/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 1569 1644 RamanIntensity/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.
  • 5. 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. References [1] C.R. Cantor, P.R. Schimmel (Eds.), Biophysical Chemistry, Part III, The Behavior of Biological Macromolecules Freeman, San Francisco, 1980. [2] P.C. Painter, J.L. Koenig (Eds.), Handbook of Biochemistry and Molecular Biology, Proteins, Vol. III, Raman Spectro- scopy of Polypeptides and Proteins, 1976. [3] T.M. Herne, A.M. Ahern, R.L. Garell, J. Am. Chem. Soc. 113 (1991) 846±854. [4] V. Budker, L.L. Du, M. Seiter, G.R. Eaton, S.S. Eaton, Biophys. J. 68 (1995) 2531±2542. [5] J.-L. Du, G.R. Eaton, S.S. Eaton, J. Magn. Reson. 115 (1995) 313±321. [6] D. Marsch, L.I. Horvath (Eds.), Advanced EPR, Application In Biology and Biochemistry, E.J. Hoff, Elsevier, Amsterdam, 1990. [7] P.C. Lee, D. Meisel, J. Phys. Chem. 84 (1982) 339. [8] A.M. Bellocq, R.C. Lord, R. Mendelson, Biochem. Biophys. Acta 257 (1972) 280. [9] S. Stewart, P.M. Fredericks, Spectrochim. Acta, Part A 55 (1999) 1615. [10] I. Nabiev, M. Manfait, Rev. Ins. Fr. Pel. 48 (1993) 261. [11] S. CIÃntaÄ, E. Vogel, D. Maniu, M. Aluas, T. Iliescu, O. Cozar, W. Kiefer, J. Mol. Struct. 482-483 (1999) 679. [12] B.G. Frushour, J.L. Koening, in: R.J. Clarck (Eds.), Advances in Infrared and Raman Spectroscopy, Vol. I, England, 1975. S. CõÃntaÄ-PõÃnzaru et al. / Journal of Molecular Structure 565±566 (2001) 225±229 229