鵲豆種子中Bowman-Birk型的胰蛋白酶抑制劑之純化及特性分析

Purification and Characterization of a Bowman-Birk-type Trypsin Inhibitor from the Seeds of Lablab purpureus

林旻慶(Min-Ching Lin) ； 王海龍(Hai-Lung Wang) ； 洪志宏(Chih-Hung Hung)

《健康管理學刊》 14卷2期 (2016/12) Pp. 69-82

摘要

本實驗以鵲豆（Lablab purpureus）種子為材料，利用硫酸銨分割、Sephadex G-50、DEAE-cellulose 52陰離子交換樹脂及trypsin-Sepharose 4B親和性管柱，可以從鵲豆種子中純化出一種胰蛋白酶抑制劑（Lablab purpureus trypsin inhibitor），簡稱LPTI。以MALDI-TOF質譜儀分析LPTI，得知其分子量為8.24 kDa，依其分子量大小是屬於Bowman-Birk type胰蛋白酶抑制劑。進一步對此蛋白的性質研究，發現LPTI在溫度到達90及100℃時，其抑制胰蛋白酶的殘留活性仍有90%左右，對溫度具有非常高的耐受性；LPTI在不同pH的緩衝溶液處理下，活性仍保有88%以上，可知其結構的穩定性不受pH值的影響而有所改變；在不同濃度的DTT處理下，抑制胰蛋白酶的活性隨著DTT濃度升高而下降，故維持LPTI活化位置(active site)的構形穩定與分子內雙硫鍵的存在有明顯的相關性。LPTI抑制胰蛋白酶作用其莫耳數比為1：1。利用Lineweaver-burk double reciprocal plot及Dixon plots分析顯示，LPTI對胰蛋白酶的抑制是屬於競爭性抑制作用，抑制常數（inhibition constant，Ki）為1.9×10^(-9) M。

關鍵字

鵲豆 ； 胰蛋白酶抑制劑 ； Bowman-Birk

並列摘要

With the Lablab purpureus seeds as the test materials, this experiment utilized ammonium sulfate precipitation, Sephadex G-50, DEAE-cellulose 52 ion exchange resin and affinity chromatography with a trypsin-Sepharose 4B column to purify a kind of Lablab purpureus trypsin inhibitor (LPTI). The MALDI-TOF mass spectrometer was used to analyze the LPTI. With a molecular weight of 8.24 kDa, it belongs to the Bowman-Birk type trypsin inhibitor. Further study on the nature of the protein found that the residue activity of the trypsin still remained about 90% when the LPTI was under temperature of 90℃ to 100℃, which showed very high temperature tolerance. When LPTI was treated with buffer solution of different pH values, the trypsin activity remained above 88%, which showed that its structural stability was unchanged under the effect of pH values. When treated by different concentrations of DTT, the activity of the inhibiting trypsin decreases with the increasing DTT concentration, so the conformational stability for maintaining the active site of the LPTI is obviously correlated to the existence of intramolecular disulfide bonds. LPTI inhibits bovine trypsin activity in 1:1 molar ratio. The Lineweaver-burk double reciprocal plot and Dixon plot analyses showed that the inhibition effect of LPTI on the trypsin belongs to competitive inhibition, with the inhibition constant ki being 1.9 × 10^(-9) M.

並列關鍵字

Lablab purpureus ； trypsin inhibitor ； Bowman-Birk