铽离子荧光探针对酶与金属离子间能量转移和结合位的研究

Terbium(Ⅲ) Luminescence Probes:Determination of Energy Transfer Distance Between Metal Ion and Tryptophan in Proteins and Postulation of Their Binding Sites

应用荧光光度法研究了Ｔｂ^（３＋）与牛胰盼氧核糖核酸酶（ＢＰＤ），枯草杆菌α－淀粉酶（ＢＳα－Ａ）的络合发光现象．实验表明，ＢＰＤ和ＢＳα－Ａ分别在ｐＨ＝７～８和５～６范围内与Ｔｂ^（３＋）络合，并发射Ｔｂ^（３＋）的特征荧光，Ｔｂ^（３＋）与ＢＰＤ和ＢＳα－Ａ的络合比分别为２：１和４：１．并应用Ｆｏｒｓｔｅｒ理论测定了Ｔｂ^（３＋）与ＢＰＤ和ＢＳα－Ａ之间能量传递的距离Ｒ分别为１．３９ｎｍ和１．４８ｎｍ，其临界距离Ｒ０分别为０．３３６ｎｍ和０．３９０ｎｍ．推测出Ｔｂ^（３＋）络合在ＢＰＤ的１７８位Ｔｒｐ和ＢＳα－Ａ上的１８８和３２１位Ｔｒｐ．

The luminescence properties of the complex of Tb(Ⅲ)with bovine pancreatic deoxyribonuclease(BPD),bacillus subtilis α-amylase(BSα-A) were studied by using the fluorescence method.It was found that BPD, BSα-A can coordinated with Tb(Ⅲ)at pH=7～8 and 5～6 respectively, and then emits a strong characteristic fluorescence of Tb(Ⅲ).The complex ratios for Tb (Ⅲ) and BPD, BSα-A were 2:1 and 4:1 respectively.In this paper,Forster theory was used to determine the energy transfer distance between BPD,BSα-A energy-donor and Tb (Ⅲ) energy-acceptor.The values of the critical distance for 50% energy transfer,R0,of 0.336 nm and 0.390 nm were obtained. The donor-acceptor energy transfer distances,R,estimated from the measured efficiency were 1.39 nm,1.48 nm respectively.It is inferred that the binding sites between Tb (Ⅲ) and BPD, BSα-A were Trp-178, Trp-188 and Trp-321,respectively.