探討血漿中Haptoglobin: 生理功能及純化方法

Abstract

人類haptoglobin分成三種表現型:1-1，2-1和2-2。 先前，我們利用血紅素亦或是單株抗體(8B1-3A)所製作之親合性管柱純化之Hp仍有大量apoA-Ι所污染。針對不純的問題，我們發展出一套利用親合性管柱之新式純化流程。首先，我們製作及純化單株抗體抗α次單元(3H8)。血漿通過親合性層析管柱與抗體結合後，利用含0.12 M NaCl之phosphate緩衝液(pH 7.4)沖洗後，在使用含0.04%十二烷基磺酸鈉緩衝液(pH 7.4)再次沖洗結合於管柱上的最主要的雜蛋白質apoA-Ι。最後，以含0.1%十二烷基磺酸鈉緩衝液(pH 11)將Hp沖提下來，並以1M Tris-HCl 緩衝液(pH 6.8)中和。純化之Hp具有高純度去除apoA-Ι之污染，並且仍具有與血紅色結合之生化功能。純化之Hp三種表現型的純度皆可到達95%以上。此新式純化方式具有高效率及高純度。其設計原理及最佳化過程皆詳述於內文中。 豬Hp是一個急性蛋白質，它於血漿中的濃度在發炎或感染時會急速升高。Hp之主要功能為負責與血漿中游離血紅素結合以抑制其氧化壓力。我們研究台灣蘭嶼迷你豬(n=43)的Hp表現型，發現它們皆為同型二聚體的結構(β-α-α-β)相近於人類Hp1-1表現型。利用西方墨點法及凝膠過濾管柱分析，我們發現有25%的蘭嶼迷你豬血漿中具有極低或是沒有Hp表現(<0.05 mg/ml)。其cDNA轉譯成胺基酸之序列與普通家豬(NP_999165)比較分析後發現identity高達99.7%。蘭嶼迷你豬(n=43)於血漿中之平均濃度為0.21 ± 0.25 mg/ml，明顯低於普通家豬0.78 ± 0.45 mg/ml。另外，25%的蘭嶼迷你豬其血漿Hp濃度低於0.05 mg/ml。再者，具低濃度與具高濃度之迷你豬其血紅素新陳代謝速率並無顯著差異。迷你豬其心血管系統近似於人類，因此被視為重要臨床前實驗動物，可用於動脈硬化、心導管及心臟移植方面的試驗。然而，迷你豬是否可作為發炎相關疾病之實驗動物仍未知。因此，此研究對於蘭嶼迷你豬作為發炎相關疾病之實驗動物提供了完整的參考性價值。

Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we have isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp β-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we developed a novel affinity column procedure using a mAb prepared against α-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl, 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then pre-washed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HCl, pH 6.8. As a result, the isolated Hp was devoid of apoA-I and able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1 or 2-2 was greater than 95% with a yield greater than 50%. The procedure described here is significantly improved in time consuming, recovery and purity. The rationale, design, and optimization for each step are described in detail.