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Surface enhanced Raman scattering investigation of protein-bound flavin adenine dinucleotide structure
Journal of MOLECULAR STRUCTURE Journal
of Molecular
Structure
349 (1995)
Surface enhanced Raman scattering investigation flavin adenine dinucleotide structure
5-8
of protein-bound
S.A. Maskevich”, N.D. Strekal”, I.M. Artsukevichb, L.N. Kivacha and 1.P Chernikevich ‘Yanka Kupala State University of Grodno, 22 Ozheshko Str., Grodno, 230023, Belarus bInstitute of Biochemistry, Academy of Sciences of Belarus, 50 BLK, Grodno, 230009, Belarus ABSTRACT
The SERS spectra of alcohol oxidase from Pichia pastoris adsorbed on a silver electrode were obtained. The similarities and differences of these spectra with the SERS spectrum of free flavin adenine dinucleiotide were considered. The dependence of relative intensity of 1258 cm-’ band from the electrode potential in the protein SERS spectra differed from that of free flavin. From the data on this band being sensitive to the protein-flavin interaction a suggestion was made about incomplete dissociation of flavin from the protein. This conclusion is confirmed both by the fluorescence data and the SERS data on alcohol oxidase purified from Carrdida boidinii. The results of the SERS investigation of the interaction between the substrate, ethanol and the cofactor,FAD, as well as between proteinbound cofactor with the substrate are presented. The problem of retaining the protein enzyme activity is discussed. 1. INTRODUCTION
At present the great potential of SERS spectroscopy is undoubtful. Reviews [l-3] have demonstrated the applications of SERS to investigations of the structure of biologically important molecules of different types. Among them flavins and flavin-containing proteins occupy a particular position The interest to such objects is related to their oxidation-reduction properties, The interfacial behavior of free and protein-bound flavins in an electrochemical cell allows to study the functional state and capability to participate directly in oxidation-reduction reactions. In this connection of a paramount importance is the problem of retaining the protein catalytic properties in the process of adsorption on a silver surface. The present work is concerned with SERS study of the functional state of alcohol oxidase (AO). 2. EXPERIMENTAL
Flavin adenine dinucleotide (FAD) was obtained from Sigma Chemical Co. The electrophoretically homogenous preparation of alcohol oxidase (AO) was obtained by the methods developed in our laboratory [4] using supplementary gel chromatography and 0022-2860/95/$09.50 0 1995 Elsevier SSDI 0022-2860(95)08695-l
Science
B.L?
All rights
reserved
6
hydrophobic chromatography. FAD and A0 were used at 10m7M and in the concentration range from 4.1 0e6 to 8.1 O-” M, correspondingly. The SERS spectra were recorded with a DFS-52M model laser Raman spectrometer (LOMO, Russia). The exciting light source was a model ILA-120 argon ion laser (Karl Zeiss, Germany). The 5 14.5 nm line was employed for the excitation, The silver working electrode was polished with aluminum powder, then boiled in 1 M NaOH solution during 5 min and washed with distilled water. The electrode was mounted in the electrochemical cell described earlier [3], 0.1 M NaCl being used as the supporting electrolyte. The oxidation reduction cycle (ORC) consisted of triangular sweeps from -0.65V to +0.25V and back to -0.65 V at the scan rate of 5 mV/s. FAD was placed into the ectrochemical cell before ORC. A0 was adsorbed onto the roughened electrode by placing the electrode in the buffer solution of AO. The SERS spectra were obtained after transferring electrode with adsorbed A0 to the electrochemical cell containing pure electrolyte. 3. RESULTS
AND DISCUSSION
Figure 1 shows the SERS spectra of free FAD absorbed on silver surface at different electrode potentials. In Fig. 1A one cannot see any features at the electrode potential of -0.65V in this spectral region. However, at the electrode potential of -0.5V intensive bands are developed in the spectrum which are assigned to vibrations of the FAD isoalloxazine moiety (Fig. IB). At the potential of -0.5V the FAD adsorbed on the anodized electrode represents the oxidized form. In Fig. lB, the spectrum demonstrates all lines typical of the oxidized FAD SERS spectrum [3]. At the electrode potential of -0.3V (Fig. 1C) certain redistribution of relative intensities of the 1258 and 13411 cm-1 bands occurs. , / The absorption spectrum of FAD (Fig. 2A) 600 950 1300 G< obtained in the electrochemical cell before ORC Raman shift, cm“ procedure is typical for spectra of this compound Fig. 1. The SERS spectra of FAD solutions [5]. The isoalloxazine moiety of this silver electrode at adsorbed on molecule is responsible for the fluorescence. After different electrode potentials:(A) -0.65 ORC treatment a bleaching of the solution is v; (B) -0.50 v; (C) -0.30 v observed. Changes in absorption spectrum (Fig. 2B) testi@ the appearance of the FAD reduced forms in cell, or in other words reduced FAD is produced in the solution. The reduction of FAD molecules takes place at the electrode potential
of q<-O.5V.
The FAD reduced
form probably
has lesser adsorption
capability
than
the oxidized form because of the loss of the x-conjugated system of ring II and ring III. We obtained a fluorescence spectrum of the adsorbed FAD which was somewhat shifted towards the long wavelength-range and which was wider than that of FAD dissolved in
7
the cell. The fluorescence intensity of the adsorbed FAD depended on the electrode potential. This dependence was the same as for the SERS signal. 0.8 f3 The maximum intensity was observed at q=-0.5 V. ‘Zp 0.6 This potential obviously corresponds to the optimum s: 0.4 adsorption of the molecule oxidized form In fi contrast to the free FAD, the protein-bound flavin in 0.2 A0 had very weak fluorescence and its spectrum 0.0 ’ was shifted to the shorter wavelength-range. This 500 300 400 may indicate incomplete dissociation of FAD from Wavelength, nm A0 under these experimental conditions. Figure 3 shows the SERS spectra of A0 at Fig. 2. Spectra of FAD absorption different electrode potentials. No spectral features cell (A) - in electrochemical related to absorbed molecules are manifested at the containing 0.1 M ofNaC1 at pII 7.8; electrode potential of -0.65 V (Fig. 38 and Fig. RA). (I3) -the same as in item A, but after At the electrode potential of -0.5 V (Fig. 3B) a ORC spectrum emerges which is very similar to that shown in Fig. IB. That is why an appropriate question arises as to the spectrum shown in Fig. 3B belonging to either dissociated FAD or protein-bound FAD. It is known [I] that SERS spectra of flavin proteins are similar to coenzyme spectra. It could provide evidence for protein dissociation. A0 may be an exception from this rule. The comparison of the SERS spectra of free and protein-bound FAD (Fig. 1 and Fig.3) indicates differences mainly in changing the relative intensities of the band at I2S8 cm-’ in changing the electrode potential from -0.5V to -0.3V. In addition individual bands at 1258, 153 1 and 1625 cm-i of SERS spectra of the free FAD SERS spectra are shifted in protein-bound FAD spectra to the alues of 1255, 1525 and 1622 cm-l correspondingly. On the basis of the RR study of riboflavin bound to egg-white flavoprotein Kitagava [6] indicated a particular importance of the band at 1258 cm-’ since it characterizes the N3-II’“protein interaction From this point of view the different microsorroundings of FAD possibly exist when FAD is adsorbed in free and in protein-bound state. Considering our results described above, including the fluorescence data and also our data [7] on SERS investigation of A0 from various sources (Pichiapastoris and Candida boidinii), we suggest the following. There is incomplete dissociation of FAD from A0 under our experimental conditions. The SERS spectra shown in Fig. 3 can be interpreted as proteinbound FAD spectra. Earlier T. Cotton and coworkers [8] studied the SERS spectra of enzyme glucose oxidase adsorbed on a silver electrode. They reported that the anodization procedure disrupted the native FAD-enzyme interaction, allowing the FAD to contact the silver surface directly. They noticed that the free flavin was present in commercial preparations of the enzyme and was also released from the protein solution upon standing at room temperature. The authors [8] also conclude that free FAD obscures the spectrum of enzyme-bound FAD, which was extremely weak. In the purification procedure of our A0 enzyme (see above) the presence of free FAD in protein solution was essentially excluded. To elucidate the question about the functional state of adsorbed A0 we carried outa series of experiments on the influence of ethanol (which is a substrate of AO) on adsorption of
8
c
J
600
950 Raman
1300
1650
shift, cm-’
Fig. 3. The SERS spectra of AO from PP adsorbed on silver electrode at different electrode potentials: (A) - 0.65 V; (B) - 0.50 V; (C) -0.30 V.
the enzyme. The influence of ethanol on the Raman spectra of absorbed free FAD was studied. In this experiment the working electrode with absorbed FAD was replaced in the electrolyte solution, containing O.lM of ethanol and SERS spectra were recorded. ORC was additionally done and the SERS spectra were recorded again. As a result we came to a conclusion that the SERS spectra of FAD in the electrolyte+ethanol system did not distinguish from those in pure electrolyte. Finally the attempt to carry out a reaction of A0 with ethanol directly on the charge Ag surface was undertaken. The electrode with adsorbed protein was placed in the electrolyte solution containing 0.1 M ethanol. As a result of this experiment the SERS signal irreversibly vanished. The reanodization of electrode did not result in appearance of a signal at any
potential either. We suggest the following possible explanation of later result. A0 interacts with substrate near the surface. This interaction may be accompanied by two processes: either the protein desorption from the electrode or irreversible reduction of FAD. Considering the results of the above experiments one may suggest that FAD does not dissociate or partially dissociate from the protein. Moreover it is possibly that adsorbed AO retains its enzymatic activity. Further work is needed to detect the product of enzymatic reaction in bulk solution The potential of SERS technique for probing the structure and functional state of flavoproteins is undoubtfirly very high. REFEBENCES 1. T Cotton, Spectroscopy of Surfaces, Eds. by R.J.H. Clark and R.E. Hester. (1988) 91. 2. E Moglin and J.-M Sequaris, Topics in Current Chemistry, 134 (1986). 3. I.R. Nabiev and R.G. Efremov, UFN (Russia), 154 (1988) 459 (in Russian). 4. I. Artsukevich, I. Chernikevich, Yu. Ostrovsky, Method of Yeast Alkohol Qxidase Production, SU patent No. 488 00 40/13 (1991). 5. A.J.W.G. Visser and F. Muller, Helv. Chim. Acta, 62 (1979) 593. 6. T. Kitagava, Y. Nishina, T. Kyogoku, et al.,Biochemistry, 18 (1979) 1804. 7. S.A. Maskevich, ND. Strekal, et al.,Proc. XIY Int. Conf. Raman Spectr.,Nai Teng Yu (eds.), John Wiley & Sons, New York (1994) A-199. 8. R. Holt and T. Cotton, J. Am. Chem. Sot., 109 (1987) 1841.
of Molecular
Structure
349 (1995)
Surface enhanced Raman scattering investigation flavin adenine dinucleotide structure
5-8
of protein-bound
S.A. Maskevich”, N.D. Strekal”, I.M. Artsukevichb, L.N. Kivacha and 1.P Chernikevich ‘Yanka Kupala State University of Grodno, 22 Ozheshko Str., Grodno, 230023, Belarus bInstitute of Biochemistry, Academy of Sciences of Belarus, 50 BLK, Grodno, 230009, Belarus ABSTRACT
The SERS spectra of alcohol oxidase from Pichia pastoris adsorbed on a silver electrode were obtained. The similarities and differences of these spectra with the SERS spectrum of free flavin adenine dinucleiotide were considered. The dependence of relative intensity of 1258 cm-’ band from the electrode potential in the protein SERS spectra differed from that of free flavin. From the data on this band being sensitive to the protein-flavin interaction a suggestion was made about incomplete dissociation of flavin from the protein. This conclusion is confirmed both by the fluorescence data and the SERS data on alcohol oxidase purified from Carrdida boidinii. The results of the SERS investigation of the interaction between the substrate, ethanol and the cofactor,FAD, as well as between proteinbound cofactor with the substrate are presented. The problem of retaining the protein enzyme activity is discussed. 1. INTRODUCTION
At present the great potential of SERS spectroscopy is undoubtful. Reviews [l-3] have demonstrated the applications of SERS to investigations of the structure of biologically important molecules of different types. Among them flavins and flavin-containing proteins occupy a particular position The interest to such objects is related to their oxidation-reduction properties, The interfacial behavior of free and protein-bound flavins in an electrochemical cell allows to study the functional state and capability to participate directly in oxidation-reduction reactions. In this connection of a paramount importance is the problem of retaining the protein catalytic properties in the process of adsorption on a silver surface. The present work is concerned with SERS study of the functional state of alcohol oxidase (AO). 2. EXPERIMENTAL
Flavin adenine dinucleotide (FAD) was obtained from Sigma Chemical Co. The electrophoretically homogenous preparation of alcohol oxidase (AO) was obtained by the methods developed in our laboratory [4] using supplementary gel chromatography and 0022-2860/95/$09.50 0 1995 Elsevier SSDI 0022-2860(95)08695-l
Science
B.L?
All rights
reserved
6
hydrophobic chromatography. FAD and A0 were used at 10m7M and in the concentration range from 4.1 0e6 to 8.1 O-” M, correspondingly. The SERS spectra were recorded with a DFS-52M model laser Raman spectrometer (LOMO, Russia). The exciting light source was a model ILA-120 argon ion laser (Karl Zeiss, Germany). The 5 14.5 nm line was employed for the excitation, The silver working electrode was polished with aluminum powder, then boiled in 1 M NaOH solution during 5 min and washed with distilled water. The electrode was mounted in the electrochemical cell described earlier [3], 0.1 M NaCl being used as the supporting electrolyte. The oxidation reduction cycle (ORC) consisted of triangular sweeps from -0.65V to +0.25V and back to -0.65 V at the scan rate of 5 mV/s. FAD was placed into the ectrochemical cell before ORC. A0 was adsorbed onto the roughened electrode by placing the electrode in the buffer solution of AO. The SERS spectra were obtained after transferring electrode with adsorbed A0 to the electrochemical cell containing pure electrolyte. 3. RESULTS
AND DISCUSSION
Figure 1 shows the SERS spectra of free FAD absorbed on silver surface at different electrode potentials. In Fig. 1A one cannot see any features at the electrode potential of -0.65V in this spectral region. However, at the electrode potential of -0.5V intensive bands are developed in the spectrum which are assigned to vibrations of the FAD isoalloxazine moiety (Fig. IB). At the potential of -0.5V the FAD adsorbed on the anodized electrode represents the oxidized form. In Fig. lB, the spectrum demonstrates all lines typical of the oxidized FAD SERS spectrum [3]. At the electrode potential of -0.3V (Fig. 1C) certain redistribution of relative intensities of the 1258 and 13411 cm-1 bands occurs. , / The absorption spectrum of FAD (Fig. 2A) 600 950 1300 G< obtained in the electrochemical cell before ORC Raman shift, cm“ procedure is typical for spectra of this compound Fig. 1. The SERS spectra of FAD solutions [5]. The isoalloxazine moiety of this silver electrode at adsorbed on molecule is responsible for the fluorescence. After different electrode potentials:(A) -0.65 ORC treatment a bleaching of the solution is v; (B) -0.50 v; (C) -0.30 v observed. Changes in absorption spectrum (Fig. 2B) testi@ the appearance of the FAD reduced forms in cell, or in other words reduced FAD is produced in the solution. The reduction of FAD molecules takes place at the electrode potential
of q<-O.5V.
The FAD reduced
form probably
has lesser adsorption
capability
than
the oxidized form because of the loss of the x-conjugated system of ring II and ring III. We obtained a fluorescence spectrum of the adsorbed FAD which was somewhat shifted towards the long wavelength-range and which was wider than that of FAD dissolved in
7
the cell. The fluorescence intensity of the adsorbed FAD depended on the electrode potential. This dependence was the same as for the SERS signal. 0.8 f3 The maximum intensity was observed at q=-0.5 V. ‘Zp 0.6 This potential obviously corresponds to the optimum s: 0.4 adsorption of the molecule oxidized form In fi contrast to the free FAD, the protein-bound flavin in 0.2 A0 had very weak fluorescence and its spectrum 0.0 ’ was shifted to the shorter wavelength-range. This 500 300 400 may indicate incomplete dissociation of FAD from Wavelength, nm A0 under these experimental conditions. Figure 3 shows the SERS spectra of A0 at Fig. 2. Spectra of FAD absorption different electrode potentials. No spectral features cell (A) - in electrochemical related to absorbed molecules are manifested at the containing 0.1 M ofNaC1 at pII 7.8; electrode potential of -0.65 V (Fig. 38 and Fig. RA). (I3) -the same as in item A, but after At the electrode potential of -0.5 V (Fig. 3B) a ORC spectrum emerges which is very similar to that shown in Fig. IB. That is why an appropriate question arises as to the spectrum shown in Fig. 3B belonging to either dissociated FAD or protein-bound FAD. It is known [I] that SERS spectra of flavin proteins are similar to coenzyme spectra. It could provide evidence for protein dissociation. A0 may be an exception from this rule. The comparison of the SERS spectra of free and protein-bound FAD (Fig. 1 and Fig.3) indicates differences mainly in changing the relative intensities of the band at I2S8 cm-’ in changing the electrode potential from -0.5V to -0.3V. In addition individual bands at 1258, 153 1 and 1625 cm-i of SERS spectra of the free FAD SERS spectra are shifted in protein-bound FAD spectra to the alues of 1255, 1525 and 1622 cm-l correspondingly. On the basis of the RR study of riboflavin bound to egg-white flavoprotein Kitagava [6] indicated a particular importance of the band at 1258 cm-’ since it characterizes the N3-II’“protein interaction From this point of view the different microsorroundings of FAD possibly exist when FAD is adsorbed in free and in protein-bound state. Considering our results described above, including the fluorescence data and also our data [7] on SERS investigation of A0 from various sources (Pichiapastoris and Candida boidinii), we suggest the following. There is incomplete dissociation of FAD from A0 under our experimental conditions. The SERS spectra shown in Fig. 3 can be interpreted as proteinbound FAD spectra. Earlier T. Cotton and coworkers [8] studied the SERS spectra of enzyme glucose oxidase adsorbed on a silver electrode. They reported that the anodization procedure disrupted the native FAD-enzyme interaction, allowing the FAD to contact the silver surface directly. They noticed that the free flavin was present in commercial preparations of the enzyme and was also released from the protein solution upon standing at room temperature. The authors [8] also conclude that free FAD obscures the spectrum of enzyme-bound FAD, which was extremely weak. In the purification procedure of our A0 enzyme (see above) the presence of free FAD in protein solution was essentially excluded. To elucidate the question about the functional state of adsorbed A0 we carried outa series of experiments on the influence of ethanol (which is a substrate of AO) on adsorption of
8
c
J
600
950 Raman
1300
1650
shift, cm-’
Fig. 3. The SERS spectra of AO from PP adsorbed on silver electrode at different electrode potentials: (A) - 0.65 V; (B) - 0.50 V; (C) -0.30 V.
the enzyme. The influence of ethanol on the Raman spectra of absorbed free FAD was studied. In this experiment the working electrode with absorbed FAD was replaced in the electrolyte solution, containing O.lM of ethanol and SERS spectra were recorded. ORC was additionally done and the SERS spectra were recorded again. As a result we came to a conclusion that the SERS spectra of FAD in the electrolyte+ethanol system did not distinguish from those in pure electrolyte. Finally the attempt to carry out a reaction of A0 with ethanol directly on the charge Ag surface was undertaken. The electrode with adsorbed protein was placed in the electrolyte solution containing 0.1 M ethanol. As a result of this experiment the SERS signal irreversibly vanished. The reanodization of electrode did not result in appearance of a signal at any
potential either. We suggest the following possible explanation of later result. A0 interacts with substrate near the surface. This interaction may be accompanied by two processes: either the protein desorption from the electrode or irreversible reduction of FAD. Considering the results of the above experiments one may suggest that FAD does not dissociate or partially dissociate from the protein. Moreover it is possibly that adsorbed AO retains its enzymatic activity. Further work is needed to detect the product of enzymatic reaction in bulk solution The potential of SERS technique for probing the structure and functional state of flavoproteins is undoubtfirly very high. REFEBENCES 1. T Cotton, Spectroscopy of Surfaces, Eds. by R.J.H. Clark and R.E. Hester. (1988) 91. 2. E Moglin and J.-M Sequaris, Topics in Current Chemistry, 134 (1986). 3. I.R. Nabiev and R.G. Efremov, UFN (Russia), 154 (1988) 459 (in Russian). 4. I. Artsukevich, I. Chernikevich, Yu. Ostrovsky, Method of Yeast Alkohol Qxidase Production, SU patent No. 488 00 40/13 (1991). 5. A.J.W.G. Visser and F. Muller, Helv. Chim. Acta, 62 (1979) 593. 6. T. Kitagava, Y. Nishina, T. Kyogoku, et al.,Biochemistry, 18 (1979) 1804. 7. S.A. Maskevich, ND. Strekal, et al.,Proc. XIY Int. Conf. Raman Spectr.,Nai Teng Yu (eds.), John Wiley & Sons, New York (1994) A-199. 8. R. Holt and T. Cotton, J. Am. Chem. Sot., 109 (1987) 1841.