Redox Status of Bowman-Birk Inhibitor from Soybean Influence Its in vitro Antioxidant Activities

大豆Bowman-Birk蛋白酶抑制劑之氧化還原狀態影響其體外之抗氧化活性

黃冠中(Guan-Jhong Huang) ； 邱傳淞(Chuan-Sung Chiu) ； 吳介信(Chieh-Hsi Wu) ； 黃世勳(Shyh-Shyun Huang) ； 雨谷榮(Sakae Amegaya) ； 侯文琪(Wen-Chi Hou) ； 許明志(Ming-Jyh Sheu) ； 廖容君(Jung-Chun Liao) ； 陳怡娟(Yi-Chaun Chen) ； 林耀輝(Yaw-Huei Lin)

《Botanical Studies》 51卷4期 (2010/10) Pp. 431-437

摘要

大豆(Glycine max)是動物和人類營養之一個重要的蛋白質來源。Bowman-Birk蛋白酶抑制劑(BBI)為大豆種子中蛋白質含量排名第三之貯藏蛋白質，是一個重要的抗營養因子。將BBI在37℃下1 mM DTT溶液中培育2小時，然後直接加入Sephadex G-25凝膠管柱中純化。還原態之BBI經SDS-PAGE測定其分子量約0.8 kDa。本研究分析的項目有：總抗氧化能力、DPPH (1,1-dipheny-2-picrylhydrazyl)染色法、DPPH自由基清除活性、還原力、亞鐵離子螯合能力、抑制過氧化物形成能力和保護DNA免於氫氧自由基傷害。氧化態和還原態BBI在總抗氧化能力分析上在200 μg/mL時可達最高的抗氧化活性（以4.74±0.36和7.20±0.20 mM Trolox equivalent antioxidative value, TEAC, 分別表示）。在DPPH染色法中，12.5 μg/mL （實際使用量為0.6μg）開始具有抗氧化活性。像在總抗氧化能力、還原力、亞鐵離子螯合能力、抑制過氧化物形成能力和保護DNA免於氫氧自由基傷害分析還原態BBI比氧化態BBI具有較高的抗氧化能力。由實驗結果得知在一系列的體外分析試驗中還原態BBI比氧化態BBI具有較高的抗氧化能力。這些發現可提供BBI應用在治療其他各種疾病的一個分子基礎。

關鍵字

大豆 ； Bowman-Birk蛋白酶抑制劑 ； 抗氧化 ； 氧化還原狀態

並列摘要

Soybean (Glycine max) is a major protein source for animal and human nutrition. The Bowman-Birk protease inhibitor (BBI), ranking 3rd of protein contents among soybean seed storage proteins, is a major antinutritional factor. BBI was incubated with 1 mM DTT at 37℃ for 2 h and loaded directly onto a Sephadex G-25 gel column for purification. The molecular mass of the reduced form of BBI is ca. 8 kDa determined by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The methodology we used includes total antioxidant status, (1,1-diphenyl-2-picryl hydrazyl) DPPH staining, DPPH radical scavenging activity, reducing power method, Fe(superscript 2+)-chelating ability, FTC (ferric thiocyanate) method, and protection calf thymus DNA against hydroxyl radical-induced damage. The oxidized and reduced form of BBI with a concentration of 200 μg/mL exhibited the highest activity (expressed as 4.74±0.36 and 7.20±0.20 mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining the reduced form of BBI appeared as white spots when it was diluted to 12.5 μg/mL (a final amount of 0.6 μg). Like total antioxidant status, the reducing power, Fe(superscript 2+)-chelating ability, FTC activity and protection against hydroxyl radical-induced calf thymus DNA damage all showed that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI. The results suggested that the reduced BBI exhibited higher antioxidative activities than the oxidized BBI in a series of in vitro tests. These findings provide one of the molecular bases for BBI applications to treat various serious diseases.

並列關鍵字

Antioxidant ； Bowman-Birk inhibitor ； Redox status ； Soybean