STUDIA UNIVERSITATIS BABES-BOLYAI, PHYSICA, SPECIAL ISSUE, 2003

ADVANTAGES AND SHORTCOMINGS IN FT-RAMAN AND SURFACE ENHANCED RAMAN SCATTERING OF TWO DIFFERENT ENZYMES S. Cavalu 1, S.Canta-Panzaru2, W.Kiefer3 University of Oradea, Faculty of Medicine and Pharmacy, Biophysics dept., P-ta 1 Decembrie nr.10, Oradea, 3700,Romania, scavalu@rdslink.ro 2 Babes-Bolyai University,Physics Faculty, Optics and Spectroscopy Dept, Kogalniceanu 1, RO 3400 ClujNapoca, Romania 3 Institut fr Physikalische Chemie, Universitt Wrzburg, Am Hubland, 97074 Wrzburg, Germany
Abstract. Mammalian peroxidases are fundamentally distinct from plant peroxidases. The resonant Raman spectrum, in the high frequencies region, is dominated by modes other then the heme oxidation state marker band (4) which is the most intense of the porphyrin modes under Soret excitation. However, on passing from RR to SERRS, this marker band is upshifft and strongly enhanced. As shown in the RR spectrum of lactoperoxidase (powder sample), this enzime exhibit only a fluorescent features upon excitation with the green light, but the SERRS spectra were successfully obtain upon excitation both with the green and the red laser line .
1

INTRODUCTION An important field of application of lasers in medicine is related to laser spectroscopic characterization of biomedically relevant molecules and processes in order to study structural-functional properties. Laser spectroscopic methods can be applied non-invasively under ambient conditions in a biological environment. This work summarize some interesting results concerning resonant Raman and SERRS investigations on silver nanoparticles of enzymes from two different classes: lactoperoxidase and horseradish peroxidase. SERS techniques, due to the high sensitivity and selectivity, can be successfully used to investigate the versatility to adsorption and conformational changes of proteins which occur as a consequence of proteolysis, lyophilization or dehydration.[1]. In the case of hemic proteins, it is possible to investigate by means of SERRS exclusively the vibrations of the chromophore, without interference by scattering of the huge surrounding protein. As a further advantage, the fluorescence background, which can make normal Raman spectroscopy extremely difficult, has been quenched in many SERRS experiments by new nonradiative decay channels provided by the SERSactive metal.

S. CAVALU, S.CANTA-PANZARU, W.KIEFER

EXPERIMENTAL Chemicals. Lactoperoxidase and horseradish peroxidase (type VI) were received from Sigma and used without further purification. Samples were prepared in phosphate buffer physiological saline at a final concentration of 10 -3 mol/l. A small amount of 5 ml from each sample was lyophilized for 30 hours at 50 0 C and than used as powder Raman sample. Colloidal silver substrate was prepared according to the Lee-Meisel procedure [4]. The absorption maximum of the freshly prepared colloid was centered at 423 nm.. For the SERRS measurements, a small amount of about 10 l 10-2 mol/l protein solution was added to 2 ml colloidal silver, resulting a final sample concentration of 5 x 10-5 mol/l. Apparatus. A micro-Raman setup was employed in order to record the Raman spectra of lyophilized powder samples. The 514.5 nm and 630 cm line of an argon ion laser (Spectra Physics, Model 166) was applied for excitation. The scattered light was collected in backscattering geometry by focusing a x50 objective (Olympus ULWD MSPlan50) on the entrance slit of a spectrometer LabRam, Dilor with 1800 grooves/mm diffractive grating. The detection system consisted of a chargecoupled multichannel detector (CCD, SDS 9000 Photometrics). For the SERS spectra we used an x10 objective, a laser power of 1 mW and an exposure time of 1000 s with 4 overlaps. Each Raman spectrum is the result of 4 accumulations with 100 sec. exposure time using a laser power of about 12 mW. The spectral resolution was 3 cm-1. RESULTS AND DISCUSSION The structure of the prosthetic group in lactoperoxidase and horseradish peroxidase is presented in fig.1. Native form of lactoperoxidase is six coordinated, high spin and the sixth ligand is suggested to be a water molecule or an alcoholic hydroxyl group[3]. The first coordination shell structure is different from that of horseradish peroxidase which is a high spin five coordinate heme system [4,5].

Fig.1. Structure of the prosthetic group in lactoperoxidase and horseradish peroxidase .

FT-RAMAN AND SURFACE ENHANCED RAMAN SCATTERING OF TWO DIFFERENT ENZYMES

1623

1527

1378 1346

5000000

1592

Raman Intensity / arb. units

1484 1428

4000000
1569

1372

1251 1222

1019

951

798 753

651

c)
492

1171

3000000

961

673

b)

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1000000

a)

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Wavenumber / cm

-1

Fig.2 RR spectrum of lactoperoxidase powder, the native protein (a), and SERRS spectra of lactoperoxidase at 630 nm (c) and 514 nm (b) excitation line.

As shown in fig.2., the RR spectrum of lactoperoxidase (powder sample) exhibit only a fluorescent features upon excitation with the green light, but the SERRS spectra were successfully obtain upon excitation both with the green and the red laser line .On can observe that upon the red light excitation, the resonant response offer much more details then the other one (fig.2). In the high frequencies region, the common features of the skeletal stretching modes of metalloporphyrin are present (table 1) and assigned to in plane vibrational modes 10 ,2 ,11 ,19 , 3 ,4. Those frequencies,which are enhanced under the red light excitation in our SERRS spectrum, have been generally used to characterize the oxidation and spin state of the heme iron [6,4]. There are also contributions from the peripheral vinyl stretching vibrations, that might be overlapped the 10 band, and the scissor modes of the two vinyls, (=CH2).The assignments in Table 1 are based on the comparison with the solution spectra obtained under different excitation line [23,24]. In the low frequencies region we can notice an enhancement of the modes assigned to the phenyl ring breathing, out of plane vibrations, pyrrole fold vibrations and especially the stretching vibration of the Fe-N histidine mode upon the red light excitation. Fig.3. presents the resonance Raman spectrum of horseradish peroxidase (native protein, powder sample) at 630 nm excitation together with the SERRS spectrum on silver sol at 514 nm excitation. As compared to the literature[7,9-11], RR spectrum of horseradish peroxidase upon red light excitation (630 nm) show a modified intensity pattern relative to that observed under Soret excitation (413 nm). This spectrum, in the high frequencies region, is dominated by modes other then

250

238

S. CAVALU, S.CANTA-PANZARU, W.KIEFER

the heme oxidation state marker band ( 4) which is the most intense of the porphyrin modes under Soret excitation. However, on passing from RR to SERRS, this marker band is upshifft and strongly enhanced (Table 2). The band at 1631 cm 1 , which has been proposed to be a vinyl stretching mode, appears almost invariably in Soret excited spectra [4,7], obscuring the 10 mode , a spin state marker frequency. This band is absent in our RR spectrum upon excitation with the green light. The 2, 10 and some other modes above 1550 cm -1 ( in the RR spectrum) involve expansion and contraction motion of the outer periphery of the porphyrin ring (C-C) to a greater extent then 4 which is a ring breathing mode involving motions of atoms (C-N) in the inner core. The selective enhancement of 2 and 10 modes might be therefore reflective of displacement of equilibrium nuclear positions in the excited state relative to the ground state of the heme periphery [5]. In the SERRS spectrum, only the 10,2 and 4 stretching modes are selectively enhanced.

Table 1. Assignment of the main frequencies in RR and SERRS spectra of lactoperoxidase (native protein) upon exitation with 630 nm and 514 nm (ref.7,8). Abreviations: w-weak, vw-very weak, s-strong, vs-very strong, m-medium, sh-sholder.

Assignments RR (solution, pH=7) 1622 1593 1560 1527 1484 1428 1373 1340 1212 10 ,(C=C) 2 11 19 (=CH2) (=CH2) 5 +8 4 3

SERRS 630 nm 1623 m 1592 vs --1527 m 1484 w ---1378 vs 1346 w 1222 s 1171 w 1019 vs 951 m 798 m 753 w 651 m 492 m 250 vs 238 sh

SERRS 514 nm 1569 vs -------1372 vs --1222 vw 1171 m ---961 w ----673 m ---240 sh 238 sh

1170 (C-C) 994 Phenyl breathing 752 out of 674 plane 496 Pyrrole fold 258 (Fe-Nhis) 228 (Fe-pyrrole)

FT-RAMAN AND SURFACE ENHANCED RAMAN SCATTERING OF TWO DIFFERENT ENZYMES

1632 1623 1579 1377

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1622 1569

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1606

1447 1428 1396 1370 1341 1308

1241

973

1001

755

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1167 1126

761

b)

a)

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800

700

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Fig.3. Resonance Raman spectrum of horseradish peroxidase (native protein, powder sample) at 630 nm excitation (a) and SERRS spectrum on silver sol at 514 nm excitation (b). Table 2. Assignment of the main frequencies in RR and SERRS spectra of horseradish peroxidase (native protein) upon exitation with 630 nm and 514 nm respectively (ref. 5,7,9,10). Abreviations: w-weak, s-strong, vs-very strong, m-medium

RR solution (pH=7) 163110 1620 (C=C) 1575 2 1430 (=CH2) 1374 4(=C-N) 1341 (=CH2) 1302 1238 (C-H) 1170 (C-C) 1127 (C-C) 1000 ph breathing 973 out of 755 plane

RR Powder 630 nm ---1622 vs 1569 m 1447 s 1370 m 1341 m 1308 m 1241 vs 1167 m 1126 vs 1001 vs 973 m 755 vs

SERRS 514 nm 1632 m 1623 m 1579 m 1430 w 1377 vs 1341 w 1310 w ---1170 m 1128 w ----761 m

S. CAVALU, S.CANTA-PANZARU, W.KIEFER

CONCLUSIONS. Normal resonance Raman and SERRS spectroscopy allowed an interesting comparison between the results obtained with these techniques. On passing from RR to SERRS large differences can be observed both in band positions and relative intensities. SERRS technique is a very important tool for measuring small amounts (nmol, pmol and even lower) of proteins due to its high selectivity and huge enhancement of the Raman signal combined with (pre-) resonance amplification contribution. SERRS spectra of peroxidases under red light excitation offer much more details then the green light excitation.
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