紫甘薯花色素与胰蛋白酶相互作用特性

Interaction between Purple Sweet Potato Anthocyanins and Trypsin

测定紫甘薯花色素与胰蛋白酶反应前后酶的催化活性、催化反应动力学并采用紫外光谱法、荧光光谱法和红外光谱法研究紫甘薯花色素与胰蛋白酶相互作用特性。结果表明:紫甘薯花色素对胰蛋白酶催化活性有明显的抑制作用,抑制类型为可逆的竞争性抑制,抑制常数Ki=6.16×10-4 mmol/L,当紫甘薯花色素与胰蛋白酶的物质的量比为140∶1,在37℃反应15 min,抑制率达到38.61%,而反应时间对催化活性的影响不明显;紫甘薯花色素可使胰蛋白酶的内源荧光猝灭,猝灭类型为静态猝灭,室温下猝灭常数Kq为1.73×1012 L/(mol·s),结合常数KA为3.88×104 L/mol,结合位点数n为0.86;热力学参数确定两者之间的作用力主要为氢键和范德华力;根据F?rster能量转移理论得出它们的结合距离为3.56 nm;红外光谱经过去卷积、二阶导数处理得知与紫甘薯花色素作用后胰蛋白酶的α-螺旋含量降低,β-折叠含量升高。

The interaction between purple sweet potato anthocyanins and trypsin was studied by measuring the catalytic activity and reaction kinetics through ultraviolet(UV) absorption, fluorescence and infrared(IR) spectroscopy. The results showed that purple sweet potato anthocyanins had an obvious inhibitory effect on trypsin catalytic activity, and the inhibition was reversible competitive inhibition with an inhibitory constant(Ki) of 6.16 × 10-4 mmol/L. When the reaction between purple sweet potato anthocyanins and trypsin with a mole ratio of 140:1 was carried out at 37 ℃ for 15 min, the inhibitory rate was 38.61%; however, the catalytic activity was not obviously affected by reaction time. Purple sweet potato anthocyanins treatment led to the quenching of intrinsic fluorescence of trypsin. The quenching of trypsin by purple sweet potato anthocyanins was probably a static quenching process with a quenching constant(Kq) of 1.73×1012 L/(mol·s), and a binding constant(KA) of 3.88×104 L/mol. The number of binding sites was 0.86. According to the thermodynamic parameters, hydrogen bonding and van der Waals force played a dominant role in the interaction between the anthocyanins and trypsin. The distance between donor and acceptor in anthocyanin-trypsin was calculated as 3.56 nm, based on the equations from Frster non-radiation energy transfer theory. The IR spectra revealed the conformational change of trypsin caused by binding, thus leading to a decrease of α-helix and an increase of β-fold.