花豆種子中純化出一種Bowman-Birk型蛋白酶抑制劑及生化特性分析

Purification and Biochemical Characterization of a Bowman- Birk-type Protease Inhibitor from Phaseolus coccineus Seeds

以花豆（Phaseolus coccineus）種子為材料，利用硫酸銨分割、DEAE-cellulose 52陰離子交換樹脂及trypsin-Sepharose 4B親和性管柱等方法，純化出一種花豆胰蛋白酶抑制劑（Phaseolus coccineus trypsin inhibitor，PCTI）。以15％ SDS-PAGE分析純度及利用MALDITOF質譜儀分析PCTI的分子量，得到其分子量為8.92 kDa，屬於Bowman-Birk型蛋白酶抑制劑。進一步研究PCTI的性質，PCTI在溫度到達80℃及100℃時，其抑制胰蛋白酶的殘留活性仍保持在80％以上，顯示其對溫度具有高度的耐受性；PCTI在不同pH的緩衝溶液處理下，其抑制胰蛋白酶的殘留活性仍保持在80％以上，可知其結構的穩定性不受pH值的改變而有所影響；在不同濃度的DTT（還原劑）處理下，抑制胰蛋白酶的殘留活性隨著DTT濃度升高而下降，顯示維持PCTI活化位（active site）的構形穩定與其分子內雙硫鍵的存在有顯著的相關性。PCTI抑制胰蛋白酶作用其莫耳數比為1：1。以酵素動力學Lineweaver-Burkdouble reciprocal plot及Dixon plots分析，顯示PCTI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）值為8.96 x 10-9 M。 This study used methods such as ammonium sulfate precipitation, DEAE-cellulose 52 anionexchange resin and trypsin-Sepharose 4B affinity columns to purify and extract trypsin inhibitor fromPhaseolus coccineus (PCTI). 15% SDS-PAGE and MALDI-TOF mass spectrometers were used to analyzethe molecular weight of PCTI, which was 8.92 kDa, belonging to Bowman-Birk-type proteinase inhibitor.Further investigation on the nature of PCTI found that its residue activity for inhibiting trypsin was stillmaintained above 80% when its temperatures reached 80℃ and 100℃, showing its high tolerance totemperature. PCTI’s residue activity for inhibiting trypsin was also maintained above 80% when it wastreated by buffer solutions with different pH values, suggesting that its structural stability would notchange in regard to pH values. When treated with different concentrations of DTT (a reducing agent),PCTI’s residue activity for inhibiting trypsin decreased with increasing DTT concentration, showingthat the maintenance of the conformational stability of PCTI active site was significantly correlated todisulfide bonds inside the molecule. The mole ratio of PCTI to trypsin is one to one. The enzyme kineticsLineweaver-burk double reciprocal plot and Dixon plots analysis show that the inhibition effect of PCTIon trypsin is of competitive inhibition, with the inhibition constant (ki) being 8.96 x 10-9 M.