摘要
αA水晶體蛋白源自於小熱休克蛋白，也被證明具有伴護活性，其在體內的主要功用之一是維持水晶體的透明度。白內障的產生與水晶體蛋白的活性有很大的關聯，而有許多研究指出在白內障患者的水晶體檢測出有較高濃度的金屬離子累積，特別是銅離子和鋅離子對於體內酵素功能有相當重要的影響，因此本篇實驗目的是比較αA水晶體蛋白和突變株D69R在銅、鋅離子存在下，其結構與伴護活性的差異。
首先CD光譜顯示銅離子均會造成二級或三級結構極大的改變，鋅離子則無。ANS和Tryptophan螢光光譜於銅離子存在時有淬熄現象，隨著銅離子濃度增加而強度減弱，鋅離子會使疏水性表面增加，銅鋅離子同時存在則近似銅離子單獨存在的情況。雖然D69R對銅離子有較敏感的選擇性，更容易被誘導沉澱，但伴護活性並未受影響，當銅鋅離子同時存在時，伴護活性卻有增加，而鋅離子明顯對αA水晶體蛋白和D69R有增加伴護活性的效果。尿素誘導變性實驗上也顯示鋅離子可以增加水晶體蛋白的穩定性。另外，分別將此兩種水晶體蛋白與銅離子混合後，發現高分子量的聚集體會隨時間增加而增加，但並不會影響其伴護活性。
這兩種金屬離子（銅、鋅）與αA水晶體蛋白的作用展現了完全不同的結果，即鋅離子不會造成αA水晶體蛋白結構改變，卻有助益伴護活性；銅離子存在下卻會改變蛋白的結構，但伴護活性不受影響。而伴護活性實驗中的金屬濃度，銅離子屬於過量、鋅離子約在正常範圍，比較其結果，可知鋅離子的存在確實會增加伴護活性，使用過量的銅離子則並不影響；當Asp69突變為Arg後，雖然對結構和伴護活性沒有太大影響，但在與金屬離子作用上仍顯現了差異，比起αA水晶體蛋白更容易被銅離子誘導產生聚集。

Abstract
The αA-crystallin is one of the small heat shock proteins, and was proved to have chaperone activity. It was found that many metal ions accumulated in cataract lens, so it is important to investigate the metal ions effect on crystallins, especially the copper and zinc ions which have critical effects on enzymes and proteins. In this study, we compare recombinant αA- and mutant D69R crystallins’ structure and chaperone activity under the presence of copper and zinc ions.
The CD spectra shows that copper ion could make great different on secondary and tertiary structures of both crystallins, while the zinc ion didn’t. And copper ion could quench the Tryptophan and ANS fluorescence, showing that the intensity decrease with the increase of copper concentration up to 50M; the zinc ion had no difference on Tryptophan but showed higher intensity of ANS fluorescence. When both ions coexisted, the ANS and Tryptophan fluorescence spectra were same with that of copper ion alone, indicating copper ion showed higher affinity to A-crystallin. Mutant D69R also have higher intensity on fluorescence than αA-crystallin, indicating that it has more hydrophobic sites and exposed Tryptophan site chain. Incubate copper ion with crystallin to monitor molecular weight distribution using size exclusive gel filtration, it was found that copper would induce high molecular weight aggregation. Chaperone activity results showed that for zinc the chaperone activity increased with the increase of its concentration, but not for copper.
In summary, zinc and copper ions have totally different behavior on αA- and D69R crystalline. Zinc ion did not cause significant change in its structure; however it enhanced its chaperone activity and stability. Copper ion caused significant change in its structure while no influence on its activity. The mutant D69R crystallin didn’t show obvious change in structure and chaperone activity but more tends to aggregate in the presence of copper ion than αA-crystallin.

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