α水晶體蛋白在水晶體中扮演很重要的角色，屬於小熱休克蛋白家族，具有類伴護活性用來維持水晶體透明，先前已在患白內障的水晶體中發現大量的銅離子及鋅離子，但目前對鋅、銅離子如何影響αA水晶體蛋白尚不是很清楚，且尚未有人研究銅、鋅離子存在下對於αA水晶體蛋白熱穩定性的影響。
因此本實驗利用圓二色光譜測量50、100、200μM銅、鋅離子對於αA/突變株F71W水晶體蛋白二級、三級結構的影響，另外藉由Trp螢光光譜及ANS螢光光譜了解蛋白質微環境及疏水性區暴露程度的影響；類伴護活性則是利用DTT誘導胰島素集結來測量；銅、鋅離子對αA水晶體蛋白多聚體分子量的影響則利用膠體過濾管柱層析來分析。此外，αA水晶體蛋白和銅、鋅離子於65℃預熱後，藉由Trp及ANS螢光光譜了解結構的變化及DTT誘導胰島素集結來測量類伴護活性的改變。
正常水晶體中存在大約3-10μM銅離子，本實驗的銅離子雖超過正常濃度，但隨銅離子濃度的增加，對αA/突變株F71W水晶體蛋白二級結構及伴護活性沒有太大影響，而三級結構與Trp殘基微環境則有明顯變化，且當銅離子濃度增加，會發現高分子量集結體的比例會隨之增加。鋅離子在水晶體中濃度大約為60-650μM，本實驗濃度在正常範圍中，隨濃度增加，對三級結構及Trp殘基微環境則沒有太大改變，但疏水性暴露與伴護活性隨鋅離子濃度增加而增加，且添加鋅離子會使多聚體平均分子量有些微增加。將含有100μM銅、鋅離子的αA/F71W水晶體蛋白在65℃下預熱半小時，發現銅離子之存在不影響伴護活性、鋅離子之存在會導致伴護活性喪失。
由實驗的結果推測：(1)αA/突變株F71W水晶體蛋白可能同時和銅離子及胰島素結合，且正常濃度範圍的銅離子具有穩定單體的功能、(2)鋅離子作用於αA/突變株F71W水晶體蛋白單體之間使多聚體結構穩定而使伴護活性增加、(3)在65℃預熱後，鋅離子反而使αA水晶體蛋白結構不穩定，造成伴護活性下降；銅離子存在則不影響伴護活性、(4)雖然Trp和Phe都屬於疏水性殘基，但對於αA水晶體蛋白和銅、鋅離子作用仍有影響，尤其在溫度65℃時影響更加明顯。

α- crystallin is very important protein in eye lens and it is a member of small heat shock protein family (sHsp). α-Crystallin has chaperone-like activity which can protect other lens proteins to maintain lens transparency. It has been found that there are high [Cu2+] and [Zn2+] in cataract eye lens. In order to explore the interactions between αA- /mutant F71W αA-crystallins and different concentrations of copper/zinc ions, we used circular dichroism (CD), tryptophen fluorescence and ANS fluorescence to study αA-/mutant F71W αA-crystallins’ structure change. Chaperone-like activity was studied by employing DTT induced insulin aggregation assay.
Concentrations of copper ion didn’t cause significant change for secondary structure, but for tertiary structure and tryptophen fluorescence did show big changes. Chaperone-like activity is independent to [Cu2+]. Zn2+ did not affect the secondary and tertiary structures, but increased the surface hydrophobicity and chaperone-like activity. Incubate αA-crystallin with Cu2+ or Zn2+ respectively at 65℃, Cu2+ didn’t reduce chaperon-like activity, whileas Zn2+ caused the decrease of chaperon-like activity.
Based on these studies, we suggest that (1) αA-crystallin may bind Cu2+ and insulin simultaneously. (2) Zn2+ stabilizes the structure of αA-crystallin by bridging monomers and increase interaction with insulin. (3) For Zn2+, may due to being in weak interactions, it dissociate as to cause chaperone-like activity decrease and insulin aggregation. (4) Both Phe and Trp, hydrophobic residues, may have influence on interaction between αA-crystallin and Cu2+/ Zn2+, especially at 65℃.

1.Berman, E.R., Biochemistry of the eye. 1991, New York and London: Plenum Press.
2.Newell, F.W., 眼科學精義. 6 ed, ed. 林和鳴. 1988, 台北市: 環球書社.
3.Harding, J.J. and K.J. Dilley, Structural proteins of the mammalian lens: a review with emphasis on changes in development, aging and cataract. Exp. Eye Res., 1976. 22: p. 1-73.
4.Mörner, C.T., Untersuchungen der Protein-substanzen in den lichtbrechenden Medien des Auges. Hoppe-Seyl. Zeit Physiol. Chem., 1894. 18(1): p. 61-106.
5.M, D. and T. A, Short-range order of crystallin proteins accounts for eye lens transparency. Nature, 1983. 302: p. 415–417.
6.Augusteyn, R.C., Growth of the lens: in vitro observations. Clin Exp Optom, 2008. 91(3): p. 226–239.
7.Bloemendal, H., The verterbrate eye lens. Science, 1977. 197: p. 127–138.
8.Sharma, K.K. and P. Santhoshkumar, Lens aging: Effects of Crystallins. Biochimica et Biophysica Acta., 2009. 1790: p. 1095-1108.
9.Bassnett, S. and D. Mataic, Chromatin degradation in differentiating fiber cells of the eye lens. J Cell Biol, 1997. 137: p. 37-49.
10.Y, O., The oxidative stress in the cataract formation. Nippon Ganka Gakkai Zasshi, 1995. 99(12): p. 1303-1341.
11.Andley, U.P., Effects of α-Crystallin on Lens Cell Function and Cataract Pathology. Curr Mol Med, 2009. 9: p. 887-892.
12.Horwitz, J., Alpha-crystallin. Exp. Eye Res., 2003. 76: p. 145-153.
13.Michael, R. and A.J. Bron, The aging lens and cataract: a model of normal and pathological aging. PHILOS T R SOC B, 2011. 366 (1568): p. 1278-1292.
14.Andley, U.P., Effects of α-Crystallin on Lens Cell Function and Cataract Pathology. Curr Mol Med, 2009. 9(7): p. 887-892.
15.Dubin, R.A., E.F. Wawrousek, and J. Piatigorsky, Expression of the murine alpha B-crystallin gene is not restricted to the lens. Mol.Cell Biol, 1989. 9: p. 1083-1091.
16.Bhat, S.P. and C.N. Nagineni, alpha B subunit of lens-specific protein alpha-crystallin is present in other ocular and non-ocular tissues. Biochem.Biophys.Res.Commun, 1989. 158: p. 319-325.
17.Jong, W.W.d., G.-J. Caspers, and J.A.M. Leunissen, Genealogy of the α-crystallin—small heat-shock protein superfamily. Int J Biol Macromol., 1998. 22(3-4): p. 151-162.
18.S, R., et al., Role of arginine-163 and the 163REEK166 motif in the oligomerization of truncated alpha A-crystallins. Biochemistry, 2006. 45(51): p. 15684-91.
19.Arthur Laganowsky, et al., Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function. Protein Sci., 2010. 19: p. 1031-1043.
20.Siezen, R.J. and P. Argos, Structural Homology of lens crystallins: III. Secondary structure estimation from circular dichroism and prediction from amino acid sequences. Biochim.biophys.acta, 1983. 748: p. 56-67.
21.TOMPA, P. and P. CSERMELY, The role of structural disorder in the function of RNA and protein chaperones. FASEB J, 2004. 18: p. 1169-1175.
22.Tardieu, A., et al., Calf lens α-crystallin quaternarystructure:a three-layer tetrahedral model. J.Mol.Biol, 1986. 192: p. 711-724.
23.Augustcyn, R.C. and J.F. Koretz, A possible structure for α-crystallin. FEBS Lctt, 1987. 223: p. l-5.
24.Walsh, M.T., A.C. Sen, and B. Chakrsbarti, Micellar subunit assembly in a three-layer model of oligomeric α-crystallin. J. Biol. Chcm, 1991. 266: p. 20079-20084.
25.G, W., Possible tetramer-based quaternary sructures for α-crystallin and small heat shock proteins. Exp Eye Res, 1993. 56: p. 729-732.
26.JA, C., A. JA, and T. RJW, A possible chaperone-like quaternary structure for α-crystallin. Exp. Eye Res., 1994. 59: p. 231-234.
27.J, V., et al., Quaternary structure of bovine alpha-crystallin: influence of temperature. Int J Biol Macromol., 1998. 22(3-4): p. 229-37.
28.RJ, S., B. JG, and H. HJ, The quaternary structure of bovine alpha-crystallin. Effects of variation in alkaline pH, ionic strength, temperature and calcium ion concentration. Eur J Biochem. Oct, 1980. 111(2): p. 435-44.
29.Horwitz, J., alpha crystallin:The quest for a homogeneous quaternary structure. Exp. Eye Res., 2009. 88: p. 190-194.
30.Ingolia, T.D. and E.A. Craig, For small heat-shock proteins are related to each other and to mammalian α-crystallin. Proc. Nat. Acad. Sci USA, 1982. 79: p. 2360-2364.
31.Klemenz, R., et al., Alpha B-crystallin is a small heat-shock protein. Proc. Nat. Acad. Sci USA 1991. 88: p. 3652-3656.
32.Horwitz, J., alpha-crystallin can function as a molecular Chaperone. Proc.Natl.Acad.Sci.USA, 1992. 89: p. 10449-10453.
33.Brady, J.P., et al., Targeted disruption of the mouse alpha A-crystallin gene induces cataract and cytoplasmic inclusion bodies containing the small heat shock protein alpha B-crystallin. Proc.Natl.Acad.Sci. USA, 1997. 94: p. 884-889.
34.Brady, J.P., et al., alpha B-crystallin in lens development and muscle integrity:a gene knockout approach. Invest. Ophthalmol. Vis. Sci, 2001. 42: p. 2924-2934.
35.S, A., et al., The structural differences between bovine lens alphaA- and alphaB-crystallin. Eur.J.Biochem, 2000. 267: p. 5916-5925.
36.K, W. and S. A, The chaperone activity of bovine alpha crystallin. Interaction with other lens crystallins in native and denatured states. J Biol Chem., 1994. 269(18): p. 13601-8.
37.Carver, J.A., et al., On the interaction of α-crystallin with unfolded proteins. Biochimica et Biophysica Acta., 1995. 1252: p. 251-260.
38.Smulders, R.H.P.H. and W.W. de Jong, The hydrophobic probe 4,4′-bis(1-anilino-8-naphthalenesulfonic acid) is specifically photoincorporated into the N-terminal domain of αB-crystallin. FEBS Lett, 1997. 409: p. 101-104.
39.Das, K.P. and W.K. Surwicz, Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of α-crystallin. FEBS Lett., 1995. 369: p. 321-325.
40.Bhagyalaxmi, S.G., et al., A novel mutation (F71L) in αA-Crystallin with defective chaperone-like function associated with age-related cataract. Biochimica et Biophysica Acta, 2009. 1792: p. 974–981.
41.Laha, B., Influence of Thermal Perturbation of the Oligomeric Size of α-Crystallin on its Chaperone Function – A Biophysical Study, National Instituteof Technology: Rourkela , Orissa, India.
42.DS, M., A. UP, and S. A, cell death triggered by a novel mutation in the alpha A-crystallin gene underlies autosomal dominant cataract linked to chromosome 21q. Eur J Hum Genet., 2003. 11: p. 784 –793.
43.M, L., et al., Autosomal dominant congenital cataract associated with a missense mutation in the human alpha crystallin gene CRYAA. Hum Mol Genet, 1998. 7: p. 471– 474.
44.J, G., L. J, and S. D, Characterization of a new, dominant V124E mutation in the mouse alpha A-crystallin-encoding gene. Invest Ophthalmol Vis Sci., 2001. 42: p. 2909 –2915.
45.L, H., Y. W, and E. H, Genetic heterogeneity in microcornea-cataract: five novel mutations in CRYAA, CRYGD, and GJA8. Invest Ophthalmol Vis Sci., 2007. 48: p. 3937–3944.
46.E, P., F. M, and L.-N. E, A nonsense mutation (W9X) in CRYAA causes autosomal recessive cataract in an inbred Jewish Persian family. Invest Ophthalmol Vis Sci., 2000. 41: p. 3511– 3515.
47.AO, K., A. MA, and M. B, Recessive congenital total cataract with microcornea and heterozygote carrier signs caused by a novel missense CRYAA mutation (R54C). Am J Ophthalmol., 2007. 144: p. 949 –952.
48.B, C., et al., Chromosomal localization of a new mouse lens opacity gene (lop18). Genomics., 1996. 36: p. 171–173.
49.CH, X., L. H, and C. B, Arginine 54 and Tyrosine 118 residues of alphaA-crystallin are crucial for lens formation and transparency. Invest Ophthalmol Vis Sci., 2006. 47: p. 3004 –3010.
50.P, V., C. A, and G. P, A missense mutation in the alpha B-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet., 1998. 20: p. 92–95.
51.MP, B., Y. O, and H. Q, Mutation R120G in alpha B-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function. Proc Natl Acad Sci USA., 1999. 96: p. 6137– 6142.
52.BA, C. and P. JM., Structural and functional changes in the alpha A-crystallin R116C mutant in hereditary cataracts. Biochemistry, 2000. 39: p. 15791–15798.
53.NP, S., C.-S. M, and B. S, Abraham EC Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function. Biochemistry, 2000. 39: p. 1420 –1426.
54.CD, H., K. S, and P. JM, A transgenic mouse model for human autosomal dominant cataract. Invest Ophthalmol Vis Sci., 2006. 47: p. 2036 –2044.
55.Y, L., Z. X, and L. L, A novel alpha B-crystallin mutation associated with autosomal dominant congenital lamellar cataract. Invest Ophthalmol Vis Sci., 2006. 47: p. 1069 –1075.
56.Smulders, R.H., et al., The mutation Asp69→Ser affects the chaperone-like activity of αA-crystallin. Eur.J.Biochem, 1995. 232: p. 834-838.
57.Derham, B.K., P.J. Muchowski, and J.I. Clark, Chaperone function of mutant versions of αA- and αB-crystallin prepared to pinpoint chaperone binding sites. Eur.J.Biochem., 2001. 268: p. 713-721.
58.A, K. and A. EC, Interaction of C-terminal truncated human alphaA-crystallins with target proteins. PLoS One., 2008. 3(9): p. 3175.
59.Andley, U.P., et al., Cloning ,expression, and chaperone-like activity of human αA-crystallin. J.Biol. chem., 1996. 271: p. 31973-31980.
60.Sharma, K.K., et al., Identification of 1,1 '-bi(4-anilino)naphthalene-5,5 '-disulfonic acid binding sequences in alpha-crystallin. J. Biol. Chem., 1998. 273(25): p. 15474-15478.
61.Sharma, K.K., et al., Synthesis and characterization of a peptide identified as a functional element in alpha A-crystallin. J. Biol. Chem., 2000. 275(6): p. 3767-3771.
62.Sreelakshmi, Y. and K.K. Sharma, Interaction of alpha-lactalbumin with mini-alpha A-crystallin. J. Protein Chem. , 2001. 20(2): p. 123-130.
63.Santhoshkumar, P. and K.K. Sharma, Phe71 Is Essential for Chaperone-like Function in αA-crystallin. J. Biol. Chem., 2001. 276(50): p. 47094 –47099.
64.黃秀慧, 大白鼠αA水晶體蛋白突變株F71W與F71R之功能及結構之研究. 2006, 國立成功大學化學所: 台南.
65.T.Murata and Y. Taura, study of trace metallic elements in the lens. Ophthal.Res, 1975. 7: p. 8-14.
66.Stanojevic, A., V. Hristil, and M. Cuperlovic, Macro and microelements in the cataractous eye lens. Ophthalmic Res., 1987. 19: p. 230–234.
67.V. Rasi , S., Constantini, and A. Moramarco, Inorganic element concentrations in cataractous human lenses. Ann. Ophthalmol, 1992. 24: p. 459–464.
68.Balaji, M., K. Sasikala, and T. Ravindran, Copper levels in human mixed, nuclear brunescance, and posterior subcapsular cataract. Br. J. Ophthalmol., 1992. 76: p. 668–669
69.Marini, I., et al., α-crystallin-link molecular chaperone against the thermal denaturation of lens aldose reductase：the effect of divalent metal ions. Biochem. Biophys. Res. Commun, 1995. 212: p. 413-420.
70.Racz, P. and A. Erdöhelyi, Cadmium, lead and copper concentrations in normal and senile cataractous human lenses. Ophthalmic Res., 1988. 20: p. 10-13.
71.Cekic, O., Copper, lead, cadmium and calcium in cataractous lenses. Ophthalmic Res., 1998. 30: p. 49-53.
72.Valle, L.J.d., et al., Calcium-induced decrease of the thermal stability and chaperone activity of α-crystallin. Biochimica et Biophysica Acta 2002. 1601: p. 100– 109.
73.Liao, J.-H., et al., Ditopic Complexation of Selenite Anions or Calcium Cations by Pirenoxine:An Implication for Anti-Cataractogenesis. Inorg. Chem., 2011. 50: p. 365–377.
74.G, D. and J. TJ., Calcium and the physiology of cataract .Ciba Found Symp., 1987. 106: p. 132-52.
75.David, L.L. and T.R. Shearer, Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest. Ophthalmol. Vis. Sci., 1984. 25: p. 1275–1283.
76.Sanderson, J., J.M. Marcantonio, and G. Duncan, A human lens model of cortical cataract: Ca2+-induced protein loss, vimentin cleavage and opacification. Invest. Ophthalmol. Vis. Sci., 2000. 41: p. 2255–2261.
77.Marcantonio, J.M., G. Duncan, and H. Rink, Calcium-induced opacification and loss of protein in the organ-cultured bovine lens. Exp. Eye Res., 1986. 42: p. 617–630.
78.Shukla, N., J.K. Moitra, and R.C. Trivedi, Determination of lead, zinc, potassium, calcium, copper and sodium in human cataract lenses. Sci. Total Environ., 1996. 181: p. 161-165.
79.Cook, C.S. and a.P.J. Bentley, Copper exchanges and toxicity in the rabbit lens in vitro. Exp. Eye Res., 1986. 42(2): p. 107-116.
80.Swanson, A. and A.W. Trusdale, Elemental analysis in normal and cataractous human lens tissue. Biochem. Biophys. Res. Commun, 1971. 45: p. 1488–1496.
81.Mocchegianai, E., M. Boemi, and P. Fumelli, Zinc-dependent low thymic hormone level in type I diabetes. Diabetes 1989. 38: p. 932–937.
82.Fabe, J.S., B.H. Grahn, and P.G. Paterson, Zinc concentration of selected ocular tissues in zinc-deficient rats. BTER, 2000. 75(1-3): p. 43-52.
83.Nourmohammadi, I., M. Modarress, and F. Pakdel, Assessment of Aqueous Humor Zinc Status in Human Age-Related Cataract. Ann Nutr Metab, 2006. 50: p. 51–53.
84.Grahn, B.H., et al., Zinc and the Eye. J AM COLL NUTR, 2001. 20(2): p. 106–118.
85.Karmakar, S. and K.P. Das, Stabilization of oligomeric strcture of α-crystallin by Zn2+ through intersubunit bridging. Biopolymers, 2010. 95(2): p. 105-116.
86.Coi, A., et al., A modeling study of αB-crystallin in complex with zinc for seeking of correlations between chaperone-like activity and exposure of hydrophobic surfaces. Int J Biol Macromol., 2005. 36: p. 208–214.
87.Biswas, A. and K.P. Das, Zn2+ Enhances the Molecular Chaperone Function and Stability of R-Crystallin. Biochemistry, 2008. 47: p. 804-816.
88.GÜNDÜZ, G., et al., Levels of Zinc and Magnesium in Senile and Diabetic Senile Cataractous Lenses. Biol. Trace Elem. Res. , 2003. 95: p. 107-112.
89.Ortwerth, B.J. and H.L. James, Lens Proteins Block the Copper-Mediated Formation of Reactive Oxygen Species during Glycation Reactions in Vitro. Biochem Biophys Res Commun, 1999. 259: p. 706–710.
90.JR, H., et al., Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses. Invest. Ophthalmol. Vis. sci., 2003. 44(2): p. 672-679.
91.Garner, B., et al., Redox availability of lens iron and copper: implications for HO•generation in cataract. Redox Rep, 1999. 4(6): p. 313-315.
92.Ganadu, M.L., et al., Effects of divalent metal ions on the αB-crystallin chaperone-like activity: spectroscopic evidence for a complex between copper(II) and protein. J. Inorg. Biochem., 2004. 98: p. 1103-1109.
93.Moschini, R., et al., Chaperone-like activity of α-crystallin toward aldose reductase oxidatively stressed by copper ion. Arch Biochem Biophys, 2006. 453: p. 13–17.
94.Ahmad, M.F., et al., Selective Cu2+ Binding, Redox Silencing, and Cytoprotective Effects of the Small Heat Shock Proteins αA- and αB-Crystallin. J. Mol. Biol., 2008. 382: p. 812–824.
95.Raju, M., et al., Identification and characterization of a copper-binding site in αA-crystallin. Free Radical Biol. Med, 2011. 50: p. 1429–1436.
96.Singh, D., et al., Synergistic effects of metal ion and the pre-senile cataract-causing G98R αA-crystallin: self-aggregation propensities and chaperone activity. Mol Vis, 2009. 15: p. 2050-2060.
97.Maiti, M., M. Kono, and B. Chakrabarti, Heat-induced changes in the conformation of α- and /β-crystallins:unique thermal stability of α-crystallin. FEBS Lett., 1988. 236(1): p. 109-114.
98.WK, S. and O. PR, On the thermal stability of alpha-crystallin: a new insight from infrared spectroscopy. Biochemistry, 1995. 34(30): p. 9655-9660.
99.Das, K. and W. K.Surewicz, Temperature-induced exposure of hydrophobic surfaces and its effect on the chaperone activity of α-crystallin. FEBS Lett., 1995. 369: p. 321-325.
100.Brahms, S. and J. Brahms, Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J. Mol . BioI. , 1980. 138: p. 149-178.
101.Wetlaufer, D.B., Ultraviolet spectra of proteins and amino acids. Adv. Protein Chem. , 1962. 17: p. 303-390.
102.Matulis, D., et al., 1-Anilino-8-Naphthalene Sulfonate as a Protein Conformational Tightening Agent. Biopolymers, 1999. 49: p. 451–458.
103.http://en.wikipedia.org/wiki/Dithiothreitol.