LB기법을 이용한 cytochrome c 박막 제조

박 세정, 남 윤석, 오 병근, 최 정우, 이 원홍

서강대학교 화학공학과

Fabrication of cytochrome c LB Films

Sei-Jeong Park, Yun-Suk Nam, Byung-Keun Oh, 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 is one of the most widely studied proteins due to its stability and solubility in water as well as an electron transport property. The key feature of cytochrome c, the capability of electron transfer, is driven from the redox state change and conformational change of heme groups bound covalently via two thioether linkages formed by two cysteine side chains and two axial ligands, histidine and methionine. Since cytochrome c is acting as an electron acceptor in the bacterial photosynthetic reaction center, it is very reasonable approach to use cytochrome c as an electron acceptor in the development of molecular electronic device by mimicking biological photosynthesis mechanism. For these characteristics of cytochrome c, the fabrication of cytochrome c films is considered as the most important process in the development of bioelectronic device[1]. But, because cytochrome c is a water-soluble protein, it is difficult to directly fabricate the molecular multi-layers onto substrate[2].

The objective of this research is to fabricate cytochrome c multilayers onto substrate by the Schaefer technique[3] (a horizontal lifting technique), comparing with LB technique (a vertical lifting technique). It has not yet been reported that cytochrome c multilayers onto substrate by the Schaefer technique can be fabricated.

Experimental

1. Materials

Cytochrome c (cyt c) is a small single-domain protein with a well-defined three-dimensional structure as illustrated in Figure 1. The prosthetic heme group, located in the center of the protein and colored red, is covalently bounded to the polypeptide backbone by Cys14 and Cys17, also colored red. The heme iron is coordinated by His18 and Met26 or His33 may coordinated to the heme in place of Met80. The single tryptophan at position 59is colored purple.

Figure 1. Structure of cytochrome c

2. Methods

Cytochrome c from horse heart (type IV, Sigma Chemical Co.) was used. Cytochrome c was mixed with phosphate buffer solution with pH 8. Its concentration is 4.0M. For spreading cytochrome c solution on the subphase, cytochrome c was mixed with ethanol diluted with deionized distillrd water (DDW). The volume ratio of ethanol, cytochrome c and DDW is 2:2:1. To fabricate cytochrome c multilayers onto the substrate, an LB trough (Series 2022, NIMA) was used. DDW with a specific resistance of 18M after distillation was used as a subphase.

Result and Discussion

Fig. 1 shows the surface pressure-area isotherm of cytochrome c spread on the subphase. From the surface pressure-area isotherm, information on the stability of monolayer at the water-air interface, phase transitions and conformational transformation are obtained. As shown in Fig. 1, a phase transition is shown at 18 mN/m. Thus cytochrome c multilayer fabrication onto the substrate was carried out at 20mN/m.

Figure 2. □-A Isotherm of cytochrome c

It was investigated by UV-VIS spectroscopy that cytochrome c multilayers deposited onto substrate were formed well.

As shown in Fig. 2, the maximum absorbance peak of cytochrome c was about 410 nm and the absorbance intensity of cytochrome c thin films deposited onto substrate increased approximately proportional to its stroke number by Schaefer technique and LB technique. Also, the absorbance intensity (i.e. molecular density of cytochrome c) of the films by Schaefer technique is higher than that by LB technique. Based on UV-vis absorbance, it could be suggested that to form the cyt c films the Schaefer technique was more suitable than the LB technique. This may occur because the cytochrome c was removed by subphase at down stroke in the LB technique.

(a)                                    (b)

Figure 3. UV-vis absorbance as cytochrome c layer (a) by Schaefer

technique and (b) by LB technique

The morphology of cytochrome c multi-layers onto ITO-coated glass by Schaefer technique was observed using the AFM. As shown in Fig. 3, the height of cytochrome c films is about 150□ and the size of protein aggregates is about 0.5m. Based on these results, cytochrome c multi-layers were well formed onto substrate.

Figure 4. AFM Morphology of cytochrome c

It could be observed that cytochrome c multi-layers deposited onto substrate were well formed by Schaefer technique based on UV-VIS absorbance and AFM image. Thus Schaefer technique seems feasible for fabricating multilayers of cytochrome c, which opens the way to fabricate molecular bioelectronic device consisting of cytochrome c.

References

[1] J. W. Choi, H.G. Choi, J. Min and W.H. Lee, Proc. of the 5th World

Congress on Biosensors, 1, 129, (1998)

[2] T. Nakagawa, T. Nakagawa, M. Kakimoto, T. Miwa and M. Aizawa,

Solid Films, 202, 151-156 (1991)

[3] A. Ulman, An Introduction to Ultrathin Organic Films from

Langmuir-Blodgett to Self-Assembly, Academic Press, 127 (1991)