超声波对大豆胰蛋白酶抑制剂活性及二级结构的影响

Different Effects of Ultrasound on Two Types of Soybean Trypsin Inhibitors in Activity and Structures

研究了Kunitz型大豆胰蛋白酶抑制剂(KSTI)和Bowman-Birk型大豆胰蛋白酶抑制剂(BBTI)经超声波处理后的活性、巯基含量、远紫外圆二色谱的变化。发现KSTI和BBTI的单一吸收负峰在200nm波长处,KSTI的[θ]200nm=-2545deg·cm2·d/mol,, 其二级结构由22.5%β折叠,16.25%β转角和61.4%无规卷曲组成;BBTI的[θ]200nm= -797deg·cm2·d/mol,由52.6%β折叠和47.4%无规卷曲组成。用65%振幅的超声波场处理11 min 后,KSTI 的[θ]200nm值变为-1827 deg·cm2·d/mol,处理20min后,其β转角和无规卷曲的含量分别下降至10.8%和54%,而β折叠的含量则增加至35.2%,同时有约71.5%的二硫化键转变成巯基,对胰蛋白酶的抑制活性则被钝化55%;而BBTI的二级结构则表现稳定,65%振幅的超声波场处理20min后,[θ]200nm值只改变为-700 deg·cm2·d/mol,其β折叠、无规卷曲结构以及对胰蛋白酶的抑制活性基本上不受影响,仅有5.29%的二硫键转化成巯基。超声波可能是通过影响KSTI的二硫键,使其二级结构发生变化,从而影响其活性。

The effects of ultrasonic fields on the activities and secondary structures of Kunitz trypsin inhibitor (KSTI) andBowman-Birk trypsin inhibitor (BBTI) from soybean were studied by measuring the changes of inhibitory activities, sulfhydrylbonds and far-UV circular dichroism (CD) spectra. It was found that the far-UV CD spectra of both SKTI and BBTI show asingle negative peak at around 200nm. The negative minimum [θ]200nm of SKTI was measured as -2545 deg·cm2·d/mol andthe secondary structure was composed of β-sheet (22.5%), β-turn (16.2%) and random (61.4%). The negative minimum [θ]200nm of BBTI was measured as -797 deg·cm2·d/mol and the secondary structure was composed only of β-sheet (52.6%)and random (47.4%). By the treatment at 65% amplitude of ultrasonic field and 11min period, [θ]200nm of KSTI was changedas-1827 deg·cm2·d/mol. When the treatment period was extended to 20 min, The β- turn and random coil content of KSTIwere decreased to 10.8% and 54% respectively and β-sheet was increased to 35.2%. About 71.5% of disulfide bonds werefound transformed to sulfhydryl bonds and 55% of inhibitory activity inactivated. Whereas BBTI show more stable than KSTIunder ultrasonic fields in its inhibitory activity and secondary structure. With 20min period treatment at 65% amplitude ofultrasonic field.The [θ]200nm of BBTI was only changed as -700 deg·cm2·d/mol. The β-sheet, random structures and theinhibitory activity of BBTI were kept unchanged basically and only about 5.29% of disulfide bonds were transformed. Hence itis suggested that ultrasonic fields lead the reduction and breakdown of disulfide bonds of SKTI, and then lead the alteration of thesecondary structures of KSTI, and finally, inactivate the inhibitory activity of KSTI.