α-水晶體蛋白是脊椎動物水晶體蛋白中含量最多的蛋白質，由兩個同源性很高的單體αA和αB所組成。在囓齒類動物的α-水晶體蛋白主要以αA和αB莫耳數三比一的形式存在和一少量比例的αAins-水晶體蛋白。αB水晶體蛋白屬於熱休克性蛋白質家族的一員，且具有分子伴護功能，為了解αB水晶體蛋白之功能和結構特性，我們利用大腸桿菌大量表現大白鼠αB水晶體蛋白，並使用陰離子交換管柱和分子篩管柱進行純化。經由螢光光譜、圓二色光譜和類伴護功能分析，得知重組的αB水晶體蛋白是具有活性的。而在αA和αB水晶體蛋白類似伴護活性和熱穩定分析結果，知道αB水晶體蛋白分子伴護活性比αA水晶體蛋白好，但其熱穩定性αA水晶體蛋白差。以莫耳數三比一的比例混合的αA和αB水晶體蛋白，其類伴護活性比αA或αB水晶體單獨存在的類伴護活性更好。當再加入αAins水晶體蛋白於αA和αB混合之中，發現其並不會增加類伴護活性。本研究發現αB水晶體蛋白表面疏水性之存在並非伴護活性之唯一要素。

Alpha-crystallin, composed of two highly homologous subunits alpha A- and alpha B-crystallins, is a major protein component of the mammalian eye lens. In the rat lens, alpha-crystallin is a protein complex with a 3:1 molar ratio of alpha A- to alpha B-crystallin and an additional minor alpha Ains-crystallin. Alpha B-crystallin is a member of the small heat-shock protein family and has chaperone-like function. In order to understand the characteristics of Rat alpha B-crystallin, we constructed the recombinant proteins expressed in Escherichia coli BL21(DE3) cells and they were purified by an ion exchange column followed by a size exclusion HPLC.
In Tris-HCl buffer, the tryptophan fluorescence intensity of alpha B-crystallin was higher than that of alpha A-crystallin. The fluorescence intensity of ANS bound to alpha A-crystallin is higher than that bound to alpha B-crystallin, indicating a less extent of hydrophobicity of alpha B-crystallin. Far-UV CD spectroscopic study showed that the mainly secondary structure of alpha B-crystallin is beta-sheet. Near-UV CD spectroscopic study showed that the tertiary structure is similar to that of natural alpha B-crystallin.
The chaperone-like function and thermal aggregation analyses of alpha A and alpha B-crystallin showed that alpha B-crystallin has better chaperone-like activity better than alpha A-crystallin; whereas has less thermal stability than alpha A-crystallin. The chaperone-like activity of protein complex with a 3:1molar ratio of alpha A- to alpha B-crystallin showed greater thermal stability and chaperone-like activity than either alpha A- or alpha B-crystallin alone. Protein complex of alpha A- : alpha B- : alpha Ains -crystalline in the ratio of 3:1:0.3 (natural composition of rat lens alpha-crystallin) showed that alpha Ains-crystallin does not elevate the chaperone-like activity. The biological significance of alpha Ains-crystallin is being investigated.

1.Widmaier, E. P.; Raff, H.; Strang, K. T., Vander, Sherman, Luciano's Human Physiology - the Mechanisms of Body Function ninth edition, 2004.
2.Newell, F. W.; 林和鳴, 眼科學精義第六版, 環球書社, 1988.
3.Delaye, M.; Tardieu, A., Short-range order of crystallin proteins accounts for eye lens transparency. Nature, 302, (5907), 415-417, 1983.
4.Spector, A.; Roy, D.; Stauffer, J., Isolation and characterization of an age-dependent polypeptide from human lens with non-tryptophan fluorescence. Experimental Eye Research, 21, (1), 9-24, 1975.
5.Brown, N. A.; Sparrow, J. M.; Bron, A. J., Central compaction in the process of lens growth as indicated by lamellar cataract. British Journal of Ophthalmology, Vol. 72, pp 538-544, 1988.
6.van Kamp, G. J.; Hoenders, H. J., The distribution of the soluble proteins in the calf lens. Experimental Eye Research, 17, (5), 417-426, 1973.
7.Fagerholm, P. P.; Philipson, B. T.; Lindstrom, B., Normal human lens--the distribution of protein. Experimental Eye Research, 33, (6), 615-620, 1981.
8.Cohen, L. H.; Westerhuis, L. W.; Jong, W. W.; Bloemendal, H., Rat alpha-Crystallin A Chain with an Insertion of 22 Residues. European journal of biochemistry, Vol. 89, pp 259-266, 1978.
9.Hawkins, J. W.; Vankeuren, M. L.; Piatigorsky, J.; Law, M. L.; Patterson, D.; Kao, F. T., Confirmation of Assignment of the Human Alpha-1-Crystallin Gene(Crya1) to Chromosome 21 with Regional Localization to Q22.3. Human Genetics, 76, (4), 375-380, 1987.
10.Ngo, J. T.; Klisak, I.; Dubin, R. A.; Piatigorsky, J.; Mohandas, T.; Sparkes, R. S.; Bateman, J. B., Assignment of the Alpha-B-Crystallin Gene to Human Chromosome-11. Genomics, 5, (4), 665-669, 1989.
11.Kato, K.; Shinohara, H.; Kurobe, N.; Goto, S.; Inaguma, Y.; Ohshima, K., Immunoreactive alpha A-crystallin in rat non-lenticular tissues detected with a sensitive immunoassay method. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1080, (2), 173-180, 1991.
12.Bhat, S. P.; Nagineni, C. N., Alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochemical and Biophysical Research Communications, 158, (1), 319-325, 1989.
13.Narberhaus, F., alpha-crystallin-type heat shock proteins: Socializing minichaperones in the context of a multichaperone network. Microbiology and Molecular Biology Reviews, 66, (1), 64-+, 2002.
14.Cohen, L. H.; Smits, D. P. E. M.; Bloemendal, H., Isolation and Characterization of Rat-Lens Messenger RNAs. Comparison of Lens Proteins, Synthesized in Lens Culture and in Homologous and Heterologous Cell-Free Systems. European journal of biochemistry, Vol. 67, pp 563-572, 1976.
15.Cohen, L. H.; Westerhuis, L. W.; Smits, D. P.; Bloemendal, H., Two Structurally Closely Related Polypeptides Encoded by 14-S mRNA Isolated from Rat Lens. European journal of biochemistry, Vol. 89, pp 251-258, 1978.
16.Andley, U. P., Crystallins in the eye: Function and pathology. Progress in Retinal and Eye Research, 26, (1), 78-98, 2007.
17.Ingolia, T. D.; Craig, E. A., Four Small Drosophila Heat Shock Proteins are Related to Each Other and to Mammalian alpha -crystallin. Proceedings of the National Academy of Sciences, Vol. 79, pp 2360-2364, 1982.
18.Klemenz, R.; Frohli, E.; Steiger, R. H.; Schafer, R.; Aoyama, A., Alpha-B-Crystallin Is a Small Heat-Shock Protein. Proceedings of the National Academy of Sciences of the United States of America, 88, (9), 3652-3656, 1991.
19.Horwitz, J., Alpha-Crystallin Can Function as a Molecular Chaperone. Proceedings of the National Academy of Sciences of the United States of America, 89, (21), 10449-10453, 1992.
20.Nelson, D. L.; Cox, M. M., Lehninger Principles of Biochemistry third edition. 2000.
21.Derham, B. K.; Harding, J. J., Alpha-Crystallin as a molecular chaperone. Progress in Retinal and Eye Research, 18, (4), 463-509, 1999.
22.Siezen, R. J.; Bindels, J. G.; Hoenders, H. J., The quaternary structure of bovine alpha-crystallin. Effects of variation in alkaline pH, ionic strength, temperature and calcium ion concentration. European journal of biochemistry, Vol. 111, pp 435-444, 1980.
23.Wistow, G., Domain structure and evolution in alpha-crystallins and small heat-shock proteins. FEBS Letters, 181, (1), 1-6, 1985.
24.Dejong, W. W.; Leunissen, J. A. M.; Leenen, P. J. M.; Zweers, A.; Versteeg, M., Dogfish Alpha-Crystallin Sequences - Comparison with Small Heat-Shock Proteins and Schistosoma Egg Antigen. Journal of Biological Chemistry, 263, (11), 5141-5149, 1988.
25.Siezen, R. J.; Argos, P., Structural Homology of lens crystallins : III. Secondary structure estimation from circular dichroism and prediction from amino acid sequences. Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 748, (1), 56-67, 1983.
26.Liang, J. N.; Chakrabarti, B., Spectroscopic investigations of bovine lens crystallins. 1. Circular dichroism and intrinsic fluorescence. Biochemistry, Vol. 21, pp 1847-1852, 1982.
27.Li, L.-K.; Spector, A., Circular dichroism and optical rotatory dispersion of the aggregates of purified polypeptides of alpha-crystallin. Experimental Eye Research, 19, (1), 49-56, 1974.
28.Lamba, O. P.; Borchman, D.; Sinha, S. K.; Shah, J.; Renugopalakrishnan, V.; Yappert, M. C., Estimation of the Secondary Structure and Conformation of Bovine Lens Crystallins by Infrared-Spectroscopy - Quantitative-Analysis and Resolution by Fourier Self-Deconvolution and Curve Fit. Biochimica et Biophysica Acta, 1163, (2), 113-123, 1993.
29.Merck, K. B.; Groenen, P. J.; Voorter, C. E.; de Haard-Hoekman, W. A.; Horwitz, J.; Bloemendal, H.; de Jong, W. W., Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones. Journal of Biological Chemistry, Vol. 268, pp 1046-1052, 1993.
30.Horwitz, J.; Huang, Q. L.; Ding, L. L.; Bova, M. P., Lens alpha-crystallin: Chaperone-like properties. Molecular Chaperones, Vol. 290, pp 365-383, 1998.
31.Sreelakshmi, Y.; Sharma, K. K., The interaction between alpha A- and alpha B-crystallin is sequence-specific. Molecular Vision, 12, (64-65), 581-587, 2006.
32.Kelley, P. B.; Abraham, E. C., Thermally induced disintegration of the oligomeric structure of alpha B-crystallin mutant F28S is associated with diminished chaperone activity. Molecular and Cellular Biochemistry, 252, (1-2), 273-278, 2003.
33.Carver, J. A.; Aquilina, J. A.; Truscott, R. J. W., A Possible Chaperone-like Quaternary Structure for alpha-Crystallin. Experimental Eye Research, 59, (2), 231-234, 1994.
34.Groth-Vasselli, B.; Kumosinski, T. F.; Farnsworth, P. N., Computer-generated model of the quaternary structure of alpha crystallin in the lens. Experimental Eye Research, 61, (2), 249-253, 1995.
35.Haley, D. A.; Horwitz, J.; Stewart, P. L., The small heat-shock protein, alpha B-crystallin, has a variable quaternary structure. Journal of Molecular Biology, 277, (1), 27-35, 1998.
36.Takemoto, L.; Boyle, D., Molecular Chaperone Properties of the High-Molecular-Weight Aggregate from Aged Lens. Current Eye Research, 13, (1), 35-44, 1994.
37.Rao, P. V.; Huang, Q. L.; Horwitz, J.; Zigler, J. S., Evidence that alpha-crystallin prevents non-specific protein aggregation in the intact eye lens. Biochimica et Biophysica Acta-General Subjects, 1245, (3), 439-447, 1995.
38.Datta, S. A.; Rao, C. M., Differential temperature-dependent chaperone-like activity of alpha A- and alpha B-crystallin homoaggregates. Journal of Biological Chemistry, 274, (49), 34773-34778, 1999.
39.Sun, T. X.; Liang, J. J. N., Intermolecular exchange and stabilization of recombinant human alpha A- and alpha B-crystallin. Journal of Biological Chemistry, 273, (1), 286-290, 1998.
40.Jakob, U.; Lilie, H.; Meyer, I.; Buchner, J., Transient Interaction of Hsp90 with Early Unfolding Intermediates of Citrate Synthase - Implications for Heat-Shock in-Vivo. Journal of Biological Chemistry, 270, (13), 7288-7294, 1995.
41.Kantorow, M.; Piatigorsky, J., alpha-Crystallin/Small Heat Shock Protein has Autokinase Activity. In Proceedings of the National Academy of Sciences of the United States of America, Vol. 91, pp 3112-3116, 1994.
42.Stevens, A.; Augusteyn, R. C., Binding of 1-anilinonaphthalene-8-sulfonic acid to alpha crystallin. European Journal of Biochemistry, 243, (3), 792-797, 1997.
43.Smulders, R.; deJong, W. W., Hydrophobic probe 4,4'-bis(1-anilino-8-naphthalene sulfonic acid) is specifically photoincorporated into the N-terminal domain of alpha B-crystallin. FEBS Letters, 409, (1), 101-104, 1997.
44.Jobling, A. I.; Stevens, A.; Augusteyn, R. C., Binding of dexamethasone by alpha-crystallin. Investigative Ophthalmology & Visual Science, 42, (8), 1829-1832, 2001.
45.Doss-Pepe, E. W.; Carew, E. L.; Koretz, J. F., Studies of the denaturation patterns of bovine alpha-crystallin using an ionic denaturant, guanidine hydrochloride and a non-ionic denaturant, urea. Experimental Eye Research, 67, (6), 657-679, 1998.
46.Carver, J. A.; Nicholls, K. A.; Aquilina, J. A.; Truscott, R. J. W., Age-related changes in bovine alpha-crystallin and high-molecular-weight protein. Experimental Eye Research, 63, (6), 639-647, 1996.
47.Das, K. P.; Surewicz, W. K., Temperature-Induced Exposure of Hydrophobic Surfaces and Its Effect on the Chaperone Activity of Alpha-Crystallin. FEBS Letters, 369, (2-3), 321-325, 1995.
48.Mandal, K.; Dillon, J.; Gaillard, E. R., Heat and concentration effects on the small heat shock protein, alpha-crystallin. Photochemistry and Photobiology, 71, (4), 470-475, 2000.
49.Vanhoudt, J.; Abgar, S.; Aerts, T.; Clauwaert, J., The native structure of bovine alpha-crystallin: Temperature and concentration dependency. Biophysical Journal, 76, (1), A118-A118, 1999.
50.Marini, I.; Bucchioni, L.; Voltarelli, M.; Delcorso, A.; Mura, U., Alpha-Crystallin-Like Molecular Chaperone against the Thermal-Denaturation of Lens Aldose Reductase - the Effect of Divalent Metal-Ions. Biochemical and Biophysical Research Communications, 212, (2), 413-420, 1995.
51.Koretz, J. F.; Doss, E. W.; LaButti, J. N., Environmental factors influencing the chaperone-like activity of alpha-crystallin. International Journal of Biological Macromolecules, 22, (3-4), 283-294, 1998.
52.del Valle, L. J.; Escribano, C.; Perez, J. J.; Garriga, P., Calcium-induced decrease of the thermal stability and chaperone activity of alpha-crystallin. Biochimica et Biophysica Acta-Proteins and Proteomics, 1601, (1), 100-109, 2002.
53.Ghosh, J. G.; Shenoy, A. K.; Clark, J. I., N- and C-terminal motifs in human alpha B crystallin play an important role in the recognition, selection, and solubilization of substrates. Biochemistry, 45, (46), 13847-13854, 2006.
54.Bova, M. P.; Yaron, O.; Huang, Q.; Ding, L.; Haley, D. A.; Stewart, P. L.; Horwitz, J., Mutation R120G in alpha B-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function. Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, pp 6137-6142, 1999.
55.Zobel, A. T. C.; Loranger, A.; Marceau, N.; Theriault, J. R.; Lambert, H.; Landry, J., Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alpha B-crystallin mutant. Human Molecular Genetics, 12, (13), 1609-1620, 2003.
56.van Noort, J. M., Multiple sclerosis: an altered immune response or an altered stress response? Journal of Molecular Medicine, 74, (6), 285-296, 1996.
57.Renkawek, K.; Voorter, C. E. M.; Bosman, G. J. C. G. M.; Workum, F. P. A.; Jong, W. W., Expression of αB-crystallin in Alzheimer's disease. Acta Neuropathologica, 87, (2), 155-160, 1994.
58.Renkawek, K.; Jong, W. W.; Merck, K. B.; Frenken, C. W. G. M.; Workum, F. P. A.; Bosman, G. J. C. G. M., αB-Crystallin is present in reactive glia in Creutzfeldt-Jakob disease. Acta Neuropathologica, 83, (3), 324-327, 1992.
59.Head, M. W.; Corbin, E.; Goldman, J. E., Overexpression and abnormal modification of the stress proteins alpha B- crystallin and HSP27 in Alexander disease. American Journal of Pathology, Vol. 143, pp 1743-1753, 1993.
60.傅麗君, The Chaperone-like activity and thermal aggregation studies of rat lens alpha A and alpha Ains-crytallins, 國立成功大學化學研究所碩士論文, 2005.
61.Smulders, R.; Carver, J. A.; Lindner, R. A.; vanBoekel, M. A. M.; Bloemendal, H.; deJong, W. W., Immobilization of the C-terminal extension of bovine alpha A-crystallin reduces chaperone-like activity. Journal of Biological Chemistry, 271, (46), 29060-29066, 1996.
62.Sun, T. X.; Das, B. K.; Liang, J. J. N., Conformational and functional differences between recombinant human lens alpha A- and alpha B-crystallin. Journal of Biological Chemistry, 272, (10), 6220-6225, 1997.
63.Shroff, N. P.; Cherian-Shaw, M.; Bera, S.; Abraham, E. C., Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function. Biochemistry, 39, (6), 1420-1426, 2000.
64.Gupta, R.; Srivastava, O. P., Deamidation Affects Structural and Functional Properties of Human alpha A-Crystallin and Its Oligomerization with alpha B-Crystallin. Journal of biological chemistry, Vol. 279, pp 44258-44269, 2004.
65.黃秀慧, Function and structural study of mutant rat lens F71W and F71R alpha A-crystallins, 國立成功大學化學研究所碩士論文, 2006.
66.Reddy, G. B.; Das, K. P.; Petrash, J. M.; Surewicz, W. K., Temperature-dependent Chaperone Activity and Structural Properties of Human alpha A- and alpha B-crystallins. In Journal of Biological Chemistry, Vol. 275, pp 4565-4570, 2000.
67.Raman, B.; Rao, C. M., Chaperone-like activity and temperature structural changes of alpha-crystallin. Journal of Biological Chemistry, 272, (38), 23559-23564, 1997.
68.Liao, J. H.; Lee, J. S.; Chiou, S. H., Distinct roles of alpha A- and alpha B-crystallins under thermal and UV stresses. Biochemical and Biophysical Research Communications, 295, (4), 854-861, 2002.