甲殼類生物的呼吸蛋白為血藍蛋白(hemocyanin)，是一種含銅蛋白，為甲殼生物血淋巴中含量最豐富的蛋白質。本研究分析紅腳蟳(Scylla olivacea)成蟳血淋巴中四個血藍蛋白分子的生理生化特性，此四個血藍蛋白分別為十二隅體血藍蛋白(24S hemocyanin)、六隅體血藍蛋白(16S hemocyanin)、雌性特有血藍蛋白(Female-specific hemocyanin: FSH)與非呼吸蛋白(Non-respiratory protein: NRP)。24S 與16S 血藍蛋白為成體的通用呼吸蛋白，兩者之間可互相轉換，24S 血藍蛋白在pH 8.3、且具充足Ca2+的溶液中維持十二隅體的型態；在pH 值8.9，或Ca2+不充足的溶液中，24S 血藍蛋白解離成16S 血藍蛋白或單元體。將溶液酸鹼值調降到8.3，且添加充足的鈣離子，僅有少量的16S 血藍蛋白再結合成24S 血藍蛋白，顯示由六隅體結合成十二隅體需要酸鹼值與鈣離子以外的條件。純化自血淋巴液的16S 血藍蛋白次單元體之間有雙硫鍵的鍵，而24S 血藍蛋白的次單元體之間並共價結構，因此可確認血淋巴液中的16S 血藍蛋白並非全由24S 血藍蛋白解離而來，且在自然狀態下，紅腳蟳血淋巴液內應同時存在著24S 與16S 血藍蛋白。雌性特有血藍蛋白之次單元體分子量約為79 kDa，經生化分析確認其含Cu、且具有與氧氣結合能力。由氧分壓曲線結果得知：雌性特有血藍蛋白的氧氣結合能力稍弱於16S 血藍蛋白。免疫分析結果發現Anti-FSH 血清可辨識16S 血藍蛋白，這說明雌性特有血藍蛋白與16S 血藍蛋白的結構有相似之處。由於雌性特有血藍蛋白出現於卵成熟母蟳、卵及初生幼體之中，因而推測雌性特有血藍蛋白的功能在於輔助這些個體由外界取得雌性特有血藍蛋白充足的氧氣。非呼吸蛋白為不帶銅、且不具與氧氣接合能力的蛋白質，普遍存在於各性別的成蟳之血淋巴液中，含量頗高但個體間差異很大。非呼吸蛋白之次單元體分子量約75 kDa，雷同於紅腳蟳血藍蛋白次單元體分子量，且其次單元體中的一個可被Anti-FSH 血清辨識，表示此次單元體結構與血藍蛋白相似，因而推斷非呼吸蛋白應屬於血藍蛋白類似分子。醣蛋白染色分析顯示非呼吸蛋白的碳水化合物含量高於雌性特有血藍蛋白，更遠高於16S 與24S 血藍蛋白。紅腳蟳的非呼吸蛋白之功能仍未知，有待進一步確認。

Hemocyanin, a copper containing protein, is the respiratory protein of Crustacea. Here, 4 major hemocyanin molecules in the hemolymph of Scylla olivacea were purified and studied. Both two-hexamer (24S) and hexamer (16S) form of hemocyanin are present in all adult’s hemolymph. In vitro, 24S hemocyanin dissociates into two 16S hemocyanin in the absence of calcium under high pH value (> 8.9). Little of 16S hemocyanin assembles spontaneously into 24S hemocyanin after calcium is added. Both 16S and 24S hemocyanins were purified from hemolymph, the 16S hemocyanin differs from the 24S hemocyanin in the presence of intersubunit disulfide bonds, it is inferred that not all native 16S hemocyanin dissociates from 24S hemocyanin. Besides the 24S and 16S hemocyanin, there are two massive proteins in the hemolymph of ovary-maturing female S. olivacea. One is female-specific hemocyanin (FSH), which occurs in the hemolymph of ovary-maturing females, but not in the hemolymph of juveniles of either sex or in adult males. FSH is confirmed as a hemocyanin due to its copper content and oxygen binding ability. FSH is also found in ovary, embryo and early-stage zoea, and is proposed to be an important hemocyanin that supplies enough oxygen for ovary-maturing female crab, egg, embryo and early-stage zoea. Another massive hemolymph protein is a non-respiratory protein (NRP) that is present in hemolymph of adults of both sex, the molecular mass of its subunits is similar to hemocyanin and FSH, and one of subunits can be slightly recognized by anti-FSH antibody. NRP is not a respiratory protein, there is neither copper containing nor absorbance of 340nm. FSH and NRP, especially NRP, contain more carbohydrates than the 16S hemocyanin.

謝辭…………………………………………………………………… 一
中文摘要……………………………………………………………… 二
英文摘要……………………………………………………………… 三
目錄…………………………………………………………………… 四
表目次………………………………………………………………… 五
圖目次………………………………………………………………… 六
略稱對照……………………………………………………………… 八
前言…………………………………………………………………… 1
材料與方法…………………………………………………………… 9
結果…………………………………………………………………… 17
討論…………………………………………………………………… 23
參考文獻……………………………………………………………… 35
表……………………………………………………………………… 44
圖……………………………………………………………………… 45
附錄…………………………………………………………………… 59

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