작용기가 변형된 cytochrome c의 특성

박 세정, 최 정우, 이 원홍

서강대학교 화학공학과

Characteristics of modified functional group on the Cytochrome c surface

Sei-Jeong Park, Jeong-Woo Choi, and Won Hong Lee

Department of Chemical Engineering, Sogang University

C.P.O. BOX 1142, Seoul 100-611, Korea

Introduction

Cytochrome c takes part in the process of electron transfer in biological system and plays a role as an electron acceptor in bioelectronic device developed by mimicking biological system[1]. To make a molecular assembly onto gold substrate, cytochrome c was cross-linked with SPDP for bioelectronic device. The cross-linking reagent, SPDP, was frequently applied for the synthesis of protein-protein conjugates as well as for the synthesis of polymer-protein conjugates[2]. It is known that the NH2 group in proteins, which binds with these agents to form conjugates, may form disulfide bridges.  When the SPDP with a disulfide bonds is exposed to the outer surface of cytochrome c, the succinimide group in SPDP is removed. So the amino group on the cytochrome c is cross-linked with 2-pyridyldithio-propionamide moieties in the SPDP. And the disulfide bond of 2-pyridyldithio-propionamide moieties in the SPDP can be cleaved with dithiothreitol(DTT). DTT is also a famous reagent for cleavage of the disulfide bond. The amino groups of cytochrome c are translated into thiol group, using the SPDP method as first described by Carlsson et al.[3], so cytochrome c modified using the SPDP method can be adsorbed onto the Au substrate selectively. In this paper, It is reported a demonstration of the modification of cytochrome c using the SPDP method and suggest the feasibility for fabrication of biomolecular electronic device.

Experimental

1. Materials

Cytochrome c (type IV, M.W.=12,384) and GFP (rEGFP) were purchased from Sigma Chemical Company (St. Louis, USA) and CLONTECH CO. (Palo Alto, California, USA) respectively. N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP; M.W.=312.4) and dithiothreitol (DTT; 99%), also purchased from Sigma Chemical Company, were used as a cross-linker and a cleavage reagent, respectively, of disulfide bonding reagent for modification of functional group on the cytochrome c surface. Figure 1 shows the structure of SPDP with a disulfide bond(a) and dithiothreitol (DTT)(b). The gold was thermally evaporated on cleaned glass substrate.

(a)                                  (b)

Figure.1. The structure of SPDP(a) and the structure of DTT(b)

2. Methods

Cytochrome c (type IV, Sigma Chemical Co.; 3.2mM, M.W.=12,384) was mixed with phosphate buffer solution (pH:8, 10ml). SPDP (3.2mM, M.W.=312.4) and DTT (25mM) were mixed with deionized distilled water (10ml) repectively. For synthesis of cytochrome c was synthesizes with SPDP for 30minites at room temperature, and excess reagent and N-hydroxysuccinimide were removed by gel filtration. The gold was thermally evaporated on cleaned glass substrate. The reaction scheme of synthesis of cytochrome c and SPDP is shown in Fig. 2.

Figure 2. Reaction scheme of synthesis of cytochrome c and SPDP

Result and Discussion

For introduction of 2-pyridyldithio-propionamide moieties in the cytochrome c by reaction with SPDP and DTT, UV-visible spectra and FT-IR spectra were measured. As shown in Fig. 3, the maximum absorbance peak of SPDP was 280nm(a) and cytochrome c was about 410 nm(c). The maximum absorbance peak of 2-pyridyldithio-propionamide moieties in the cytochrome c was 340nm(b). Also, Fig. 4 shows the FT-IR spectra of N-H group in cytochrome c(a) and cytochrome c having a 2-pyridyldithio-propionamide(b). The precipitate showed FT-IR bands at 1550.4 and 1639.2 cm-1 typical for amides. But FT-IR band at 1639.2 is not found in Fig. 4(a). It might indicate that the amount of NH2 groups on the cytochrome c is relatively reduced as the coupling reaction is preceded, and the amount of NH group is increased relatively. So the synthesis of cytochrome c and SPDP are fulfilled by the SPDP method.

FIGURE 3.   UV-visible spectra       FIGURE 4.   FT-IR spectra of

of (a) SPDP, (b) cytochrome c having   (a) cytochrome c and (b) cy

a 2-pyridyldithio-propionamide, and      tocrome c having a 2-pyrid-

(c) cytochrome c                      yldithio-propionamide

The cytochrome c having a 2-pyridyldithio-propionamide is constructed with 2-pyridyldithio-propionamide and cytochrome c. And 2-pyridyldithio-propionamide and cytochrome c are linked with disulfide bridge. To cleave this disulfide bridge, DTT was used. Then cytochrome c having a 2-pyridyldithio-propionamide has a thiol group. Because thiol group is adsorbed onto the gold substrate, so this modified cytochrome c is adsorbed onto the gold substrate. Fig. 5 shows surface morphology of modified cytochrome c onto the gold substrate.

FIGURE 5. AFM image of modified cytochrome c onto the gold substrate

( a: 5□m, b: 3□m, c: 1□m )

This SPDP method has simple but powerful tool for protein synthesis, because of the easy handling, high resolution and no wholly heating. In this study, the authors mainly focused on the modification of functional group on the cytochrome c surface and its potential application to the field of bioelectronics.

Acknowledgement

This work was supported by the grants from the Korea Science and Engineering Foundation (KOSEF), the ADvanced Environmental Monitoring Research Center at Kwangju Institute of Science and Technology, and the Korea Ministry of Science and Technology (98-NF-02-07-A-01).

References

[1] J.W. Choi, B.S. Seok, J. Min, W.H. Lee and D.M. Shin, Molecular Crystals and Liquid Crystals, 316, 393-396 (1998)

[2] W. N. E. van Dijk-Wolthuis, P. van de Wetering, W. L. J. Hinrichs, L. J. F. Hofmeyer, R. M. J. Liskamp, D. J. A. Crommelin, and W. E. Hennink, Bioconjugate Chem., 10, 687-692 (1999)

[3] Carlsson, J., Drevin, H., and Axen, R., Biochem. J., 173, 723-737 (1978)