| 摘要: | 在大腸桿菌(M15)中大量表現重組蛋白質sporaminB，然後利用鎳離子螯合之親和性管柱純化。Sporamin B經SDS-PAGE分析其分子量約為26kDa。本研究分析的項目有：總抗氧化能力、DPPH(1, 1-diphony-2-picrythydrazyl)染色法、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。Sporamin B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4.21±0.0078mM Troloxe equivalent antioxidative value，TEAC，表示）。在DPPH染色法中，25mg sporamin B/mL（實際使用量為75µg）開始具有抗氧化活性。在所有分析項目中，重組之sporamin B蛋白質都具有顯著的的抗氧化活性。利用胃蛋白酶水解sporamin B時，小分子的胜肽會隨著水解時問增加。24小時後抗氧化活性（對DPPH之清除能力）可以從19%(0h)增加到29%(24h)。利用電腦模擬胃蛋白酶水解sporamin B蛋白質的結果，八種人工合成具有抗氧化活性胜肽：SNIP，VRL，SYCQ，GTEKC，RF，VKAGE，AH，KIEL，利用DPPH自由基清除率測定其ICS。為8.36，4.23，0.206，0.0884，9.72，14.9，13.8和24.9mM。結果發現胜肽上具有半胱胺酸基者具有很好的抗自由基活性。
Recombinant sporamin B overproduced in E. coli (M15) was purified by Ni(superscript 2+)-chelated affinity chromatography. The molecular mass of sporamin B is ca. 26kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Total antioxidant status, 1, 1-dipheny-2-picrylhydrazyl (DPPH) staining, reducing power method, Fe2 -chelating ability, ferric thiocyanate (FTC) method, and protecting calf thymus DNA against hydroxyl radical-induced damage were studied. The sporamin B protein with a concentration of 100µg/mL exhibited highest activity (expressed as 4.21±0.0078mM Trolox equivalent antioxidative value, TEAC) in total antioxidant status test. In the DPPH staining sporamin B appeared as a white spot when the concentration was diluted to 25 mg sporamin B/mL (with an absolute amount of 75µg). Like total antioxidant status, the reducing power, Fe(superscript 2+) -chelating ability, FTC activity and protection calf thymus DNA against hydroxyl radical-induced damage all showed that sporamin B polypeptides have significant antioxidant activities. It was found that antioxidant activities of sporamin B increased from 19% (0 h) to about 29% (24 h) after 24 h hydrolysis by pepsin. Smaller peptides increased with hydrolytic times. Eight peptides for testing antioxidative activity were synthesized according to peptic hydrolysis simulation. The obtained SNIP, VRL, SYCQ, GTEKC, RF, VKAGE, AU, KIEL showed IC50 values of 8.36, 4.23, 0.206, 0.0884, 9.72, 14.9, 13.8 and 24.9mM, respectively, when scavenging activity of DPPH radicals (%) was measured. These findings mean that cysteine residue is most important in antiradical activities. It was suggested that sporamin B might contribute to its antioxidant activities against hydroxyl and peroxyl radicals. |
