人類血液Haptoglobin之抗氧化角色

Abstract

人類血液中之Haptoglobin (Hp)，與血型分類相似，可以分為三種表現型：1-1、2-1、與2-2。然而在這些Hp表現型之結構與功能的關係，由於其複雜的結構與困難繁複的純化步驟，目前的瞭解仍十分有限。在此我們發展一個可以純化每一種Hp表現型的簡便方法。首先將血漿通過已結合可專一辨識Hp之單株抗體，將所得到之Hp再通過gel filtration管柱，藉由SDS-PAGE分析，所得到之Hp純度可大於95%，並可保有其本身所具有之醣基成分與血紅素結合能力。經由Circular dichroism分析，Hp 1-1 (29%) 之α-helix組成比例高於2-1 (22%) 與2-2 (21%)。這個方法較現有Hp的純化方法有了明顯的改善與進步。為了進一步瞭解Hp在in vitro之抗氧化活性，thiobarbituric acid-reactive substances (TBARS) assay用來估計在脂質過氧化反應中Hp的抗氧化活性。Hp在銅離子所誘發之脂質過氧化反應中表現了極強之抗氧化能力。此外，在另一種親水性自由基產生者2,2’-azobis(2-amidinopropane)-dihydrochloride (AAPH) 所誘發的脂質過氧化反應中， Hp亦具有相似之抗氧化特性，因此推測Hp可能也扮演著自由基清除者的角色。為了更進一步研究結構對於其抗氧化特性的影響，carboxymethylation被用來阻絕在Hp中cysteine間雙硫鍵的形成，有趣地，經過修飾後的Hp反而較native Hp表現出更強之抗氧化能力，因此推論在native的構形中，抗氧化domain可能並未完全暴露在外。為了更深入研究Hp在細胞內的抗氧化角色，我們將Hp的cDNA放入含有CMV啟動子之pcDNA 3.0載體中，並轉殖至本身不會表現Hp之Chinese Hamster Ovary (CHO) 細胞中，發現確實可以增加該細胞對於氧化壓力的耐受度，在添加雙氧水的培養條件下24小時，其耐受度較未轉殖之細胞高出1倍。因此Hp在in vitro與ex vivo的研究中皆表現出極佳之抗氧化能力。最後我們分析了目前普遍使用之抗氧化活性檢測方法，並且闡釋如何研發與設計防止動脈硬化之強效抗氧化藥物。首先抗氧化藥物必須可以專一地LDL結合。第二，必須具有高度之bioavailability。文中並針對這些抗氧化作用機制與分析程序之原理與策略進行討論。

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the Hp phenotypes has not been studied in detail due to their complicated structures and difficult isolation procedures. In the present study, we developed a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound Hp was eluted and further chromatographied on a HPLC. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS polyacrylamide gel electrophoresis. It retained the carbohydrate moiety and hemoglobin-binding ability. Circular dichroic spectra showed that the α-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%) and 2-2 (21%). The procedures described here represent a significant improvement in current purification methods for each Hp phenotypes. To investigate in vitro antioxidant role of Hp, thiobarbituric acid-reactive substances (TBARS) was used to estimate antioxidant activity of Hp in low-density lipoprotein (LDL) lipid peroxidation. We demonstrated that Hp molecule was an extremely potent antioxidant activity in Cu2+-induced LDL peroxidation. Using 2,2’-azobis(2-amidinopropane)-dihydrochloride (AAPH), a hydrophilic decomposed radical initiator, it produced a similar antioxidant effect of Hp against LDL oxidation suggesting a free radical-scavenging role of Hp. To study the structural effect in its antioxidant activity, carboxymethylation that alters the overall structure of Hp by blocking the formation of disulfide linkages between cysteine residues was used for the evaluation. Interestingly, carboxymethylated Hp exerting higher antioxidant potency than that of native Hp indicated that the antioxidant domain of Hp might not be fully exposed. To investigate antioxidant role of Hp on the cellular level, the cDNA of Hp 1-1 was cloned, constructed (containing the pcDNA3.0 vector with CMV promoter) and transfected to Chinese Hamster Ovary (CHO) cells expressing no Hp. These transfected CHO cells were able to express Hp 1-1 and significantly (P<0.001) elevated the tolerance against the oxidative stress. The elevation was about twice-higher than that normal CHO cells when challenged with hydrogen peroxide for 24 h. Thus, Hp plays a provocative antioxidant role as demonstrated in our in vitro and ex vivo studies. Finally, we analyzed commonly used analytical methods for measuring the antioxidant potency and outlined the critical steps as how to evaluate and design a potent antioxidant agent that can be used for the intervention of atherosclerosis. We conclude that an antioxidant should be first targeted and incorporated into human LDL. Second, the candidate compound should possess high bioavailability. The rationale and strategy for the analytical procedures are discussed.