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High resolution imaging of native hemoglobin by scanning tunneling microscopy
ELSEVIER
Journal of Electroanalytical Chemistry 379 (1994) 535-539
Preliminary note
High resolution imaging of native hemoglobin by scanning tunneling microscopy J.-D. Zhang, Q. Chi, S.-J. Dong *, E. Wang * Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China
Received 29 June 1994
Keywords:
Native hemoglobin; Scanning tunneling microscope
1. Introduction
Hemoglobin (Hb) is essential to the life of all vertebrates, and also exists in some invertebrates and in the root nodules of leguminous plants. Hb is known to be a tetrameric protein with a molecular weight of ca 64250 Da and consists of two (Yand two p subunits. The Hb tetramer is formed by association of the alp, dimer with CQ& forming contacts between (or and & and symmetrical contacts between p1 and (Y*.Each subunit carries a same prosthetic heme group, iron011 protoporphyrin IX, associated with a polypeptide chain between 136 and 153 amino residues. In the Hb molecule the ferrous iron of the heme is linked to N, of a histidine, the porphyrin is wedged into its pocket by a phenylalanine, and about 35 other specific sites along the polypeptide chain are occupied by nonpolar residues [l-3]. According to the crystallographic studies, the unit cell dimensions of Hb in a native state are 6.4 x 5.5 X 5.0 nm3 [4,5]. In summary, Hb is one of the most important proteins for various living things, and its structure has been well-characterized mainly based on X-ray diffraction analysis. However, to our knowledge, direct observation of its structural features has not yet been performed. STM has been demonstrated to be a powerful tool in imaging many biological molecules such as DNA [6-81, enzymes [9,103, virus particles, polypeptides, amino acids, polysaccharides and many other proteins with varying degrees of resolution [ill. These successful examples encourage us to observe the structure of an individual Hb molecule directly using such a technique in this work, in order to provide the direct evidence of this protein structure.
l
Corresponding
author.
0022-0728/94/$07.00 0 1994 Elsevier Science S.A. All rights reserved SSDI 0022-0728(94)03661-6
Although the tunneling mechanism through biological macromolecules is not fully understood until now, the major obstacle to imaging biological specimens is not tunneling through the sample but rather fixation of the sample to the substrate [123. Several methods have been developed to overcome the fixation problem, which include covalent-bonding method and electrochemical deposition [9,13]. In this paper, electrochemical pretreatment was used to improve fixation of Hb molecules on the substrate.
2. Experimental Hb (from horse heart, H-4632) was obtained from Sigma, other reagents used were of analytical reagent grade. All solutions were prepared with double distilled water. Hb was dissolved in double-distilled water. Electra chemical experiments were carried out on an FDH 3204 potentiostat (Shanghai). A three-electrode system was employed with a Ag lAgC1 (saturated KCl) reference electrode, a platinum plate auxiliary electrode and an HOPG working electrode. A ESCALAB-MKII (VG Scientific Lt. Co.) was used in XPS assays. STM imagings were performed under a normal atmosphere at room temperature (15 &-2°C) with a TMX 2000 series (TopoMetrix. Co., USA), this STM instrument contains a monitor which was used in observing distance between the sample and the tip. The air humidity was kept at 40%-50%. The Pt/ Ir(80/ 20) tips were mechanically formed by cutting. Scanning was in a constant-current mode at a bias voltage of 0.1-0.4 V and a current of 0.6-1.2 nA. The treatment of all images involved only low pass filtering to remove high frequency noise without two- dimensional Fourier transformation.
536
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistv 379 (1994) 535-539
The freshly cleaved HOPG was firstly anodized at 1.95 V for 2 min in 0.1 M phosphate buffer (PB) solution and then washed thoroughly with double-distilled water. A 10 ~1 droplet of 4 mg ml-’ freshly-prepared Hb solution was syringed onto anodized substrate surface. The sample was then dried at room temperature (drying time 4-5 h) in the dark and finally washed carefully with double distilled water. The resulting samples were investigated by STM and XPS within a day.
3. Results and discussion Having an easily-cleaved structure and atomically flat surface, HOPG has been widely used as a substrate supporting various isolated organic and biological molecules or films derived from these for STM imaging [14]. However, in practice the cleaved HOPG surface is markedly hydrophobic and lacks high affinity for the hydrophilic organic and biological molecules commonly encountered [14]. This results in the fixation of the sample becoming difficult. For this reason, many chemical and physical methods, such as chemical oxidation, sputtering ion irradiation, laser activation, electrochemical oxidation, etc. have been used in an attempt to reduce the intrinsic undesirable hydrophobic properties of the HOPG surface or to create surface binding sites for organic and biological molecules 115-171. In our preliminary experiments, Hb was directly de-
5 3
530
posited onto a freshly cleaved HOPG surface. Under this situation, Hb molecules usually aggregate together and the fixation is not stable enough, so that high resolution STM images could not be obtained. This is because Hb being a water soluble heme protein, protein molecules cannot be dispersed uniformly on a high hydrophobic surface. In our further studies, therefore, electrochemical pretreatment at a high positive voltage was used to produce active sites on the HOPG surface and reduce the hydrophobic property. STM images of bare anodized HOPG show clearly the nanometer-sized pits formed on the substrate plane, as described in our previous reports [lS]. These etching pits provide effective active sites for the adsorption of Hb and improve obviously the hydrophilic ability of the HOPG surface. The Hb molecules can be adsorbed onto the anodized HOPG to yield a stable and uniformly-distributed sample. XPS assays show that N and Fe can be found in an Hb-adsorbed anodized HOPG surface, but not in a bare anodized HOPG surface, as shown in Fig. 1. This result confirms that Hb molecules were adsorbed on the substrate surface. STM images with different scan ranges, as shown in Figs. 2 and 3, exhibit a uniform monolayer composed of Hb molecules. Fig. 2 shows lots of individual Hb molecules and only a few aggregated cells compared with the STM image of Hb adsorbed on an untreated HOPG surface. Many scans were performed in other regions of the sample, the pattern shown in Fig. 2 was found to be reproducible. The scanning range was then limited to 28 X 28 nm2 or
532
534
Fe2
280
282
284
286
288
290
I 702 Eb
Fig. 1. XPS spectra
704
706
708
L
396
6
1
398
\
400
402
Y
710
712
714
716
/eV
of bare (solid lines) and Hb-deposited
(broken
lines) HOPG
surfaces.
718
404
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistry 379 (1994) 535-539
537
smaller, which allows detailed observation of a few Hb molecules, as shown in Fig. 3. As visualized from Fig. 3, an individual Hb molecule exhibits an oval-shaped structure containing a depressed space. Quantitative size determinations were made by the TopoMetrix Instrument software. Fig. 4 shows the typical line profiles obtained through the directions noted in Fig. 3. The line profiles indicate clearly that the depressed space exists. The dimension of each individual Hb molecule is almost the same allowing for experimental error. For example, the average values of major axis and minor axis for globe A are 6.40 and 5.38 nm (n = 151, for globe B are 6.34 and 5.40 nm (n = 12), for globe C are 6.54 and 5.18 nm, respectively. The aver, age height of the molecule was measured as 6.8 A (n = 20) on the basis of the calculated average displacement of the probe tip from the substrate while the molecule was scanned. As a different electronic work function exists between the substrate (HOPG) and the adsorbed Hb, the measured vertical distance will not be consistent with the physical height. In other words,
Fig. 3. STM images of Hb adsorbed on the anodized HOPG, scan area: 28x28 nm’, I = 0.84 nA, V, = 0.24 V.
the absolute thickness of the molecule cannot be obtained accurately from these STM measurements. However, the relative height must be proportional to the absolute height. Based on these measurements, the size of a Hb molecule can be approximately described as 6.4nm X 5.3nm X 0.7nm. When scan range was further limited to 6 X 6 nm2 or smaller, the structural feature of Hb molecule could be observed more clearly. As shown in Fig. 5, a Hb molecule is composed of two parts and possesses a space in its center, each part represents a dimer. Obviously the structural feature ol Hb observed here is in accordance with that previously obtained from X-ray analysis shown in Fig. 6.
4. Conclusions
Fig. 2. STM images of Hb adsorbed on the anodized HOPG, scan area: 112~ 112 nm2, I = 0.84 nA, V,, = 0.24 V.
Electrochemical pretreatment has been demonstrated to be an effective means in improving fixation of Hb molecules and reducing the hydrophobic prop. erty of HOPG surface. Hb can be adsorbed strongly onto the treated HOPG surface to form a very stable
J.-D. Zhang et al. /Journal
538
4 \
2
of Electroanalytical
Chemistry 379 (1994) 535-539
b
1.90 0.00
4.56
9.17
6.75
13.51
h
0.00
Distance
/
M
Fig. 4. Line profiles obtained from globe B along the direction and cd noted in Fig. 3(A), respectively.
of ab
sample for STM measurements. High resolution images of Hb have been obtained successfully. The structural feature and the dimensions of Hb from this study are well consistent with those from X-ray analysis.
Fig. 6. (A) Three-dimensional
structure
of the Hb molecule
obtained
Fig. 5. STM image of single Hb molecule adsorbed on the anodized HOPG, scan area: 9.12 x 9.12 nm’, I = 0.84 nA, Vt, = 0.24 V.
from X-ray analysis
and (B) external
shape of this molecule.
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistry 379 (1994) 535-539
Acknowledgement
This research was supported by the National Natural Science Foundation of China.
[9] 1101
References [l] G. Zubay, Biochemistry, Addison-Wesley Publishing Company (1984), Reprinted with Corrections, New York. [2] J. Baldwin and C. Chothia, J. Mol. Biol., 129 (1979) 175. [3] M.F. Peru@ Ann. Rev. Biochem., 48 (1979) 327. [4] G. Fermi, J. Mol. Biol., 97 (1975) 237. [5] R.C. Ladner, E.J. Heidner and M.F. Peru@ J. Mol. Biol., 114 (1977) 385. [6] D.D. Dunlap and C. Bustamante, Nature, 342 (1989) 204. [7] SM. Lindsay, T. Thundat, L. Nagahara, U. Knipping and R.L. Rill, Science, 244 (1989) 1063. [8] Jing-De Zhu, Min-Qian Li, Liu-Zhang Xiu, Jie-Qing Zhu, Ju
[ll] 1121 [13] [14] 1151 [161 1171 [18]
539
Hu, Min-Ning Gu, Yao-Liang Xu, Lan-Ping Zhang, Ze-Qi Huang, B.K. Chernov, Y.D. Nechipurenko and N.A. Tchurikov, Biochem. Biophys. Acta, 1115 (1992) 239. R.N. Keller, D. Bear and C. Bustamante, J. Vat. Sci. Technol., 39 (1991) 1291. R. Czajika, C.G.J. Koopal, M.C. Feiters, J.W. Gerritsen, R.J.M. Nolte and H. Van Kempen, Bioelectrochem. Bioenerg., 29 (1992) 47. S.R. Snyder and H.S. White, Anal. Chem., 64 (1992) 116R. M. Salmeron, T.P. Beebe, Jr., J. Odriozola, T. Wilson, D.F. Ogletree and W. Siekhaus, J. Vat. Sci. Technol., A8 (1990) 635. A. Cricenti, S. Selci, A.C. Felici, R. Generosi, E. Gori, W. Djaczenko and C. Bustamante, Nature, 342 (1989) 204. N.M.D. Brown and H.-X. You, J. Struct. Biol., 107 (1991) 250. Hong-Xing You, N.M.N. Brown and K.F. Al-Assadi, Surf. Sci., 279 (1992) 189. A.A. Gewirth and A.J. Bard, J. Phys. Chem., 92 (1988) 5563. C.A. Goss, J.C. Brumfield, E.A. Irene and R.W. Murray, Anal. Chem., 65 (1993) 1378. Qijin Chi, Jingdong Zhang, Shaojun Dong and Erkang Wang, J. Chem. Sot. Faraday Trans. 1, (19941, in press.
Journal of Electroanalytical Chemistry 379 (1994) 535-539
Preliminary note
High resolution imaging of native hemoglobin by scanning tunneling microscopy J.-D. Zhang, Q. Chi, S.-J. Dong *, E. Wang * Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China
Received 29 June 1994
Keywords:
Native hemoglobin; Scanning tunneling microscope
1. Introduction
Hemoglobin (Hb) is essential to the life of all vertebrates, and also exists in some invertebrates and in the root nodules of leguminous plants. Hb is known to be a tetrameric protein with a molecular weight of ca 64250 Da and consists of two (Yand two p subunits. The Hb tetramer is formed by association of the alp, dimer with CQ& forming contacts between (or and & and symmetrical contacts between p1 and (Y*.Each subunit carries a same prosthetic heme group, iron011 protoporphyrin IX, associated with a polypeptide chain between 136 and 153 amino residues. In the Hb molecule the ferrous iron of the heme is linked to N, of a histidine, the porphyrin is wedged into its pocket by a phenylalanine, and about 35 other specific sites along the polypeptide chain are occupied by nonpolar residues [l-3]. According to the crystallographic studies, the unit cell dimensions of Hb in a native state are 6.4 x 5.5 X 5.0 nm3 [4,5]. In summary, Hb is one of the most important proteins for various living things, and its structure has been well-characterized mainly based on X-ray diffraction analysis. However, to our knowledge, direct observation of its structural features has not yet been performed. STM has been demonstrated to be a powerful tool in imaging many biological molecules such as DNA [6-81, enzymes [9,103, virus particles, polypeptides, amino acids, polysaccharides and many other proteins with varying degrees of resolution [ill. These successful examples encourage us to observe the structure of an individual Hb molecule directly using such a technique in this work, in order to provide the direct evidence of this protein structure.
l
Corresponding
author.
0022-0728/94/$07.00 0 1994 Elsevier Science S.A. All rights reserved SSDI 0022-0728(94)03661-6
Although the tunneling mechanism through biological macromolecules is not fully understood until now, the major obstacle to imaging biological specimens is not tunneling through the sample but rather fixation of the sample to the substrate [123. Several methods have been developed to overcome the fixation problem, which include covalent-bonding method and electrochemical deposition [9,13]. In this paper, electrochemical pretreatment was used to improve fixation of Hb molecules on the substrate.
2. Experimental Hb (from horse heart, H-4632) was obtained from Sigma, other reagents used were of analytical reagent grade. All solutions were prepared with double distilled water. Hb was dissolved in double-distilled water. Electra chemical experiments were carried out on an FDH 3204 potentiostat (Shanghai). A three-electrode system was employed with a Ag lAgC1 (saturated KCl) reference electrode, a platinum plate auxiliary electrode and an HOPG working electrode. A ESCALAB-MKII (VG Scientific Lt. Co.) was used in XPS assays. STM imagings were performed under a normal atmosphere at room temperature (15 &-2°C) with a TMX 2000 series (TopoMetrix. Co., USA), this STM instrument contains a monitor which was used in observing distance between the sample and the tip. The air humidity was kept at 40%-50%. The Pt/ Ir(80/ 20) tips were mechanically formed by cutting. Scanning was in a constant-current mode at a bias voltage of 0.1-0.4 V and a current of 0.6-1.2 nA. The treatment of all images involved only low pass filtering to remove high frequency noise without two- dimensional Fourier transformation.
536
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistv 379 (1994) 535-539
The freshly cleaved HOPG was firstly anodized at 1.95 V for 2 min in 0.1 M phosphate buffer (PB) solution and then washed thoroughly with double-distilled water. A 10 ~1 droplet of 4 mg ml-’ freshly-prepared Hb solution was syringed onto anodized substrate surface. The sample was then dried at room temperature (drying time 4-5 h) in the dark and finally washed carefully with double distilled water. The resulting samples were investigated by STM and XPS within a day.
3. Results and discussion Having an easily-cleaved structure and atomically flat surface, HOPG has been widely used as a substrate supporting various isolated organic and biological molecules or films derived from these for STM imaging [14]. However, in practice the cleaved HOPG surface is markedly hydrophobic and lacks high affinity for the hydrophilic organic and biological molecules commonly encountered [14]. This results in the fixation of the sample becoming difficult. For this reason, many chemical and physical methods, such as chemical oxidation, sputtering ion irradiation, laser activation, electrochemical oxidation, etc. have been used in an attempt to reduce the intrinsic undesirable hydrophobic properties of the HOPG surface or to create surface binding sites for organic and biological molecules 115-171. In our preliminary experiments, Hb was directly de-
5 3
530
posited onto a freshly cleaved HOPG surface. Under this situation, Hb molecules usually aggregate together and the fixation is not stable enough, so that high resolution STM images could not be obtained. This is because Hb being a water soluble heme protein, protein molecules cannot be dispersed uniformly on a high hydrophobic surface. In our further studies, therefore, electrochemical pretreatment at a high positive voltage was used to produce active sites on the HOPG surface and reduce the hydrophobic property. STM images of bare anodized HOPG show clearly the nanometer-sized pits formed on the substrate plane, as described in our previous reports [lS]. These etching pits provide effective active sites for the adsorption of Hb and improve obviously the hydrophilic ability of the HOPG surface. The Hb molecules can be adsorbed onto the anodized HOPG to yield a stable and uniformly-distributed sample. XPS assays show that N and Fe can be found in an Hb-adsorbed anodized HOPG surface, but not in a bare anodized HOPG surface, as shown in Fig. 1. This result confirms that Hb molecules were adsorbed on the substrate surface. STM images with different scan ranges, as shown in Figs. 2 and 3, exhibit a uniform monolayer composed of Hb molecules. Fig. 2 shows lots of individual Hb molecules and only a few aggregated cells compared with the STM image of Hb adsorbed on an untreated HOPG surface. Many scans were performed in other regions of the sample, the pattern shown in Fig. 2 was found to be reproducible. The scanning range was then limited to 28 X 28 nm2 or
532
534
Fe2
280
282
284
286
288
290
I 702 Eb
Fig. 1. XPS spectra
704
706
708
L
396
6
1
398
\
400
402
Y
710
712
714
716
/eV
of bare (solid lines) and Hb-deposited
(broken
lines) HOPG
surfaces.
718
404
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistry 379 (1994) 535-539
537
smaller, which allows detailed observation of a few Hb molecules, as shown in Fig. 3. As visualized from Fig. 3, an individual Hb molecule exhibits an oval-shaped structure containing a depressed space. Quantitative size determinations were made by the TopoMetrix Instrument software. Fig. 4 shows the typical line profiles obtained through the directions noted in Fig. 3. The line profiles indicate clearly that the depressed space exists. The dimension of each individual Hb molecule is almost the same allowing for experimental error. For example, the average values of major axis and minor axis for globe A are 6.40 and 5.38 nm (n = 151, for globe B are 6.34 and 5.40 nm (n = 12), for globe C are 6.54 and 5.18 nm, respectively. The aver, age height of the molecule was measured as 6.8 A (n = 20) on the basis of the calculated average displacement of the probe tip from the substrate while the molecule was scanned. As a different electronic work function exists between the substrate (HOPG) and the adsorbed Hb, the measured vertical distance will not be consistent with the physical height. In other words,
Fig. 3. STM images of Hb adsorbed on the anodized HOPG, scan area: 28x28 nm’, I = 0.84 nA, V, = 0.24 V.
the absolute thickness of the molecule cannot be obtained accurately from these STM measurements. However, the relative height must be proportional to the absolute height. Based on these measurements, the size of a Hb molecule can be approximately described as 6.4nm X 5.3nm X 0.7nm. When scan range was further limited to 6 X 6 nm2 or smaller, the structural feature of Hb molecule could be observed more clearly. As shown in Fig. 5, a Hb molecule is composed of two parts and possesses a space in its center, each part represents a dimer. Obviously the structural feature ol Hb observed here is in accordance with that previously obtained from X-ray analysis shown in Fig. 6.
4. Conclusions
Fig. 2. STM images of Hb adsorbed on the anodized HOPG, scan area: 112~ 112 nm2, I = 0.84 nA, V,, = 0.24 V.
Electrochemical pretreatment has been demonstrated to be an effective means in improving fixation of Hb molecules and reducing the hydrophobic prop. erty of HOPG surface. Hb can be adsorbed strongly onto the treated HOPG surface to form a very stable
J.-D. Zhang et al. /Journal
538
4 \
2
of Electroanalytical
Chemistry 379 (1994) 535-539
b
1.90 0.00
4.56
9.17
6.75
13.51
h
0.00
Distance
/
M
Fig. 4. Line profiles obtained from globe B along the direction and cd noted in Fig. 3(A), respectively.
of ab
sample for STM measurements. High resolution images of Hb have been obtained successfully. The structural feature and the dimensions of Hb from this study are well consistent with those from X-ray analysis.
Fig. 6. (A) Three-dimensional
structure
of the Hb molecule
obtained
Fig. 5. STM image of single Hb molecule adsorbed on the anodized HOPG, scan area: 9.12 x 9.12 nm’, I = 0.84 nA, Vt, = 0.24 V.
from X-ray analysis
and (B) external
shape of this molecule.
J.-D. Zhang et al. /Journal
of Electroanalytical Chemistry 379 (1994) 535-539
Acknowledgement
This research was supported by the National Natural Science Foundation of China.
[9] 1101
References [l] G. Zubay, Biochemistry, Addison-Wesley Publishing Company (1984), Reprinted with Corrections, New York. [2] J. Baldwin and C. Chothia, J. Mol. Biol., 129 (1979) 175. [3] M.F. Peru@ Ann. Rev. Biochem., 48 (1979) 327. [4] G. Fermi, J. Mol. Biol., 97 (1975) 237. [5] R.C. Ladner, E.J. Heidner and M.F. Peru@ J. Mol. Biol., 114 (1977) 385. [6] D.D. Dunlap and C. Bustamante, Nature, 342 (1989) 204. [7] SM. Lindsay, T. Thundat, L. Nagahara, U. Knipping and R.L. Rill, Science, 244 (1989) 1063. [8] Jing-De Zhu, Min-Qian Li, Liu-Zhang Xiu, Jie-Qing Zhu, Ju
[ll] 1121 [13] [14] 1151 [161 1171 [18]
539
Hu, Min-Ning Gu, Yao-Liang Xu, Lan-Ping Zhang, Ze-Qi Huang, B.K. Chernov, Y.D. Nechipurenko and N.A. Tchurikov, Biochem. Biophys. Acta, 1115 (1992) 239. R.N. Keller, D. Bear and C. Bustamante, J. Vat. Sci. Technol., 39 (1991) 1291. R. Czajika, C.G.J. Koopal, M.C. Feiters, J.W. Gerritsen, R.J.M. Nolte and H. Van Kempen, Bioelectrochem. Bioenerg., 29 (1992) 47. S.R. Snyder and H.S. White, Anal. Chem., 64 (1992) 116R. M. Salmeron, T.P. Beebe, Jr., J. Odriozola, T. Wilson, D.F. Ogletree and W. Siekhaus, J. Vat. Sci. Technol., A8 (1990) 635. A. Cricenti, S. Selci, A.C. Felici, R. Generosi, E. Gori, W. Djaczenko and C. Bustamante, Nature, 342 (1989) 204. N.M.D. Brown and H.-X. You, J. Struct. Biol., 107 (1991) 250. Hong-Xing You, N.M.N. Brown and K.F. Al-Assadi, Surf. Sci., 279 (1992) 189. A.A. Gewirth and A.J. Bard, J. Phys. Chem., 92 (1988) 5563. C.A. Goss, J.C. Brumfield, E.A. Irene and R.W. Murray, Anal. Chem., 65 (1993) 1378. Qijin Chi, Jingdong Zhang, Shaojun Dong and Erkang Wang, J. Chem. Sot. Faraday Trans. 1, (19941, in press.