摘要

　 小牛血清蛋白(Bovine Serum Albumin, BSA)，是免疫學分析以及食品科學研究裡最重要的蛋白質。外界環境中熱能的輸入，會造成蛋白質的熱變性，產生物理、化學以及生物性的變化，導致其結構上的改變。如何即時觀察蛋白質之熱變性過程，並推測其結構的變化，例如展開與摺疊，是我們所感興趣的。目前許多量測蛋白質結構的方法如蛋白質電泳法、X-ray晶格繞射、圓的二色性光譜法，皆無法完整研究BSA快速的熱變性現象。
在本研究中，我們架設一套角度放大率為12.8倍的光學外差式偏光儀，設計各種不同的加熱流程，以量測旋光角度的即時變化及雷射穿透功率的改變，來探討BSA詳細的熱變性過程，並搭配蛋白質電泳法來佐證其結果。實驗結果顯示，以濃度為2.5% (g/ml) 之BSA水溶液為例，當溫度超過攝氏66度，BSA的結構開始發生熱變性反應，此為可逆的過程。當溫度超過攝氏68度，BSA的結構開始發生聚集反應，此為不可逆的過程。我們也發現在維持特定溫度一段時間後，BSA旋光角度有回復的情形，表示在BSA的熱變性過程中，尚存在另一未知的變性過程。

Abstract

Bovine serum albumin (BSA), a highly complex protein, plays important functions in immunology analysis and food science. In the denaturation study of proteins, heat treatments are frequently required for a variety of purposes and they can change the physical, chemical, biological and functional properties of proteins. Therefore, during thermal processing to understand, monitor, and control protein changes, particularly unfolding and refolding are important. There are many methods to detect the structures of protein, such as electrophoresis, X-ray diffraction crystallography, circular dichroism. However, these methods are not easy to monitor the denaturation process of BSA in real time.
In this study, we have built up an optical heterodyne polarimeter capable of 12.8-fold of amplification factor in measuring the optical rotation angle and then understand the structure changes of protein. BSA under heat treatment can proceed structure changes. We observed thermal denaturation of BSA by measuring the real-time changes of optical rotation angle and transmission power, and then used electrophoresis to verify the results. For 2.5% (g/ml) BSA solution, the results indicate that BSA begins to denature at 66℃ and then soon goes to an unknown state which can restore the optical rotation angle. The denatured state is a reversible process. When the temperature goes beyond 68℃, BSA solution begins an irreversible aggregation state.

參考文獻

[1] J. R. Macdonald, and H. P. Bachinger, “HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding,” J Biol Chem, vol. 276, no. 27, pp. 25399-403, 2001.
[2] J. N. de Wit, “Structure and functional behaviour of whey proteins,” Netherlands Milk and Dairy Journal, vol. 35 no. 1, pp. 47-64, 1981.
[3] P. Walstra, and R. Jenness, Dairy Chemistry and Physics, New York: John Wiley & Sons, 1984.
[4] M. Murata, F. Tani, T. Higasa et al., “Heat-induced transparent gel formation of bovine serum albumin,” Bioscience, biotechnology, and biochemistry, vol. 57, no. 1, pp. 43-46, 1993.
[5] P. Relkin, “Thermal unfolding of beta-lactoglobulin, alpha-lactalbumin, and bovine serum albumin. A thermodynamic approach,” Crit Rev Food Sci Nutr, vol. 36, no. 6, pp. 565-601, 1996.
[6] T. Peter, “Serum albumin,” Adv Protein Chem, vol. 37, no. 2,
pp. 161-245, 1985.
[7] D. M. Mulvihill, and M. Donovan, “Whey proteins and their thermal denaturation: A review.,” Irish Journal of Food Science and Technology, vol. 11, no. 1, pp. 43-75, 1987.
[8] http://www.ncbi.nlm.nih.gov.
[9] T. Kongraksawech, “Characterization by optical methods of the heat denaturation of bovine serum albumin (BSA) as affected by protein concentration, pH, ionic strength and sugar concentration,” Food Science and Technology, Oregon State University, 2006.
[10] J. I. Boye, I. Alli, and A. A. Ismail, “Interactions Involved in the Gelation of Bovine Serum Albumin,” Journal of Agricultural and Food Chemistry, vol. 44, no. 4, pp. 996-1004, 1996.
[11] J. Oakes, “Thermally denatured proteins: Nuclear magnetic resonance, binding isotherm, and chemical modification studies of thermally denatured bovine serum albumin.,” Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases, vol. 72, no. 1, pp. 228-237, 1976.
[12] R. Wetzel, M. Becker, J. Behlke et al., “Temperature behaviour of human serum albumin,” Eur J Biochem, vol. 104, no. 2, pp. 469-78, 1980.
[13] C. N. Pace, “The stability of globular proteins,” CRC Crit Rev Biochem, vol. 3, no. 1, pp. 1-43, 1975.
[14] J. P. Attfield, A. W. Sleight, and A. K. Cheetham, “Structure determination of α-CrPO4 from powder synchrotron X-ray data”, Nature, vol. 322, no. 1, pp. 620-622 , 1986.
[15] T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaki, H. Yamaguchi, K. I. Shinzawa, R. Nakashima, R. Yaono, and S. Yoshikawa, “Structures of metal sites of oxidized bovine heart cytochrome,” Science, vol. 269, no. 2, pp. 1069-1074, 1995.
[16] Y. A. Antonov and B. A. Wolf, “Calorimetric and Structural Investigation of the Interaction between Bovine Serum Albumin and High Molecular Weight Dextran in Water”, Biomacromolecules, vol. 6, no. 5, pp. 2980-2989, 2005.
[17] K. Yamada, J. Sato, H. Oku, and R. Katakai, “Conformation of transmembrane partial peptides of peripheral myelin protein,”
J. Peptide Res, vol. 62, no. 2, pp. 78-87, 2003.
[18] Mackenzie, R.C., “Nomenclature in thermal analysis, ” Thermochimica Acta, vol. 28, no. 1, pp. 1-6, 1979.
[19] Oldfield, D. J.; Singh, H.; Taylor, M. W. “Kinetics of
heatinduced whey protein denaturation and aggregation in
skim milks with adjusted whey protein concentration,”
J. Dairy Research, vol. 72, no. 3, pp. 369-378, 2005.
[20] W. B. Gratzer and D. A. Cowburn, “Optical Activity of Biopolymers,” Nature, vol. 222, no. 2, pp. 426-431, 1969.
[21] E. Hecht, Optics, 4ed ed., ch. 8, New York: Addison Wesley, 2002.
[22] D. J. Caldwell and H. Eyring, The Theory of Optical Activity, ch. 1, New York: Wiley-Interscience, 1971.
[23] T. W. King and G. L. Cote,“Closed loop polarimetric glucose sensing using the Pockels effect,” Ann. Inter. Conf. Of IEEE EMBS, vol. 1, no. 3, pp. 161-162, 1992.

[24] M. P. Silverman, N. Ritchie, G. M. Cushman, and B. Fisher,
“Experimental configurations using optical phase modulation to measure chiral asymmetries in light specularly reflected from a naturally gyrotropic medium,”J. Opt. Soc, vol. 5, no. 7,
pp. 1852-1862, 1988.
[25] A. J. Majewski, M. Sanzari, H. L. Cui et al., “Effects of ultraviolet radiation on the type-I collagen protein triple helical structure: a method for measuring structural changes through optical activity,” Phys Rev E Stat Nonlin Soft Matter Phys, vol. 65, no. 3, pp. 319-328, 2002.
[26] 陳欣樂，“小牛血清蛋白熱變性之旋光量測研究”，2009.