A Novel Trypsin Inhibitor from Sweet Potato (Ipomoea Batatas Lam.) Leaves and its Synthesized Peptides with Antioxidant Activities in Vitro

甘薯葉中新型胰蛋白酶抑制因子和其合成之胜肽含有抗氧化的活性

黃冠中(Guan-Jhong Huang) ； 林穎志(Ying-Chih Lin) ； 鄧正賢(Jeng-Shyan Deng) ； 陳顯榮(Hsien-Jung Chen) ； 廖容君(Jung-Chun Liao) ； 黃世勳(Shyh-Shyun Huang) ； 張淑貞(Shu-Jen Chang) ； 林耀輝(Yaw-Huei Lin)

《Botanical Studies》 53卷2期 (2012/04) Pp. 215-222

摘要

在大腸桿菌（M15）中大量表現重組蛋白質SPLTI-a，然後利用鎳離子螯合之親和性管柱純化。SPLTI-a經SDS-PAGE分析其分子量約為8 kDa。本研究利用不同的抗氧化方法評估（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）。SPLTI-a在總抗氧化能力分析上在100μg/mL時可達最高的抗氧化活性（以2.12±0.02 mM Trolox equivalent antioxidative value，TEAC，表示）。在所有分析項目中，重組之SPLTI-a蛋白質都具有顯著的的抗氧化活性。利用胰蛋白酶水解SPLTI-a時，小分子的胜肽會隨著水解時間增加。24小時後抗氧化活性（對DPPH之清除能力）可以從18%（0h）增加到35%（24h）。利用電腦模擬胰蛋白酶水解SPLTI-a蛋白質的結果，四種人工合成具有抗氧化活性胜肽：VR，STIEK，ITDGK和EYIFDR，利用DPPH自由基清除率測定其IC50為5.83，3.75，2.65，and 0.73mM。結果發現胜肽上具有酪胺酸基者具有很好的抗自由基活性。本篇文章建議，SPLTI-a可能有助於抗氧化活性。

關鍵字

抗氧化活性 ； 葉子之胰蛋白酶抑制因子 ； 重組蛋白質 ； 甘藷

並列摘要

Recombinant SPLTI-a [sweet potato leaf trypsin inhibitor-a] overproduced in E. coli (M15) was purified by Ni(superscript 2+)-chelated affinity chromatography. The molecular mass of SPLTI-a is ca. 8000 Da as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SPLTI-a was examined using different antioxidative models (Total antioxidant status, reducing power method, Fe(superscript 2+)-chelating ability, ferric thiocyanate (FTC) method, and protecting calf thymus DNA against hydroxyl radical-induced damage). The SPLTI-a protein with a concentration of 100 μg/mL exhibited highest activity (expressed as 2.12 ± 0.02 mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. Like total antioxidant status, the reducing power, Fe(superscript 2+)-chelating ability, FTC activity and protecting calf thymus DNA against hydroxyl radical- induced damage all showed that SPLTI-a polypeptide has significant antioxidant activities. It was found that the antioxidant activity increased after 24 h hydrolysis of SPLTI-a by trypsin from 18% (0 h) to about 35% (24 h). Accumulation of shorter peptides increased along the longer trypsin incubation. The obtained VR, STIEK, ITDGK, and EYIFDR showed IC50 (concentration for 50% inhibition) values of 5.83, 3.75, 2.65, and 0.73 mM, respectively, when scavenging activity of DPPH radicals (%) was measured. These findings mean that tyrosine residue is most important in antiradical activities. It was suggested that SPLTI-a possess antioxidant activities.

並列關鍵字

Antioxidant activity ； Leaf trypsin inhibitor ； Recombinant protein ； Sweet potato