Abdul Latif Abdul Raziq, Kono Ryohei, Tachibana Hideki, Akasaka Kazuyuki
Department of Biotechnological Science, School of Biology-Oriented Science and Technology, Kinki University, Wakayama, Japan.
Biophys J. 2007 Jan 1;92(1):323-9. doi: 10.1529/biophysj.106.088120. Epub 2006 Sep 22.
We present here the first detailed kinetic analysis of the dissociation reaction of amyloid protofibrils by utilizing pressure as an accelerator of the reaction. The experiment is carried out on an excessively diluted typical protofibril solution formed from an intrinsically denatured disulfide-deficient variant of hen lysozyme with Trp fluorescence as the reporter in the pressure range 3-400 MPa. From the analysis of the time-dependent fluorescence decay and the length distribution of the protofibrils measured on atomic force microscopy, we conclude that the protofibril grows or decays by attachment or detachment of a monomer at one end of the protofibril with a monomer dissociation rate independent of the length of the fibril. Furthermore, we find that the dissociation reaction is strongly dependent on pressure, characterized with a negative activation volume DeltaV(odouble dagger) = -50.5 +/- 1.60 ml mol(-1) at 0.1 MPa and with a negative activation compressibility Deltakappa(double dagger) = -0.013 +/- 0.001 ml mol(-1) bar(-1) or -0.9 x 10(-6) ml g(-1) bar(-1). These results indicate that the protofibril is a highly compressible high-volume state, but that it becomes less compressible and less voluminous in the transition state, most probably due to partial hydration of the existing voids. The system eventually reaches the lowest-volume state with full hydration of the monomer in the dissociated state.
我们在此展示了对淀粉样原纤维解离反应的首次详细动力学分析，该分析利用压力作为反应的促进剂。实验是在由鸡溶菌酶的一种内在变性的、缺乏二硫键的变体形成的过度稀释的典型原纤维溶液上进行的，在3 - 400 MPa的压力范围内，以色氨酸荧光作为报告分子。通过对随时间变化的荧光衰减以及在原子力显微镜下测量的原纤维长度分布进行分析，我们得出结论：原纤维通过在其一端附着或脱离单体而生长或衰变，单体解离速率与纤维长度无关。此外，我们发现解离反应强烈依赖于压力，在0.1 MPa下其特征为负的活化体积ΔV(‡)= -50.5±1.60 ml mol⁻¹，以及负的活化压缩率Δκ(‡)= -0.013±0.001 ml mol⁻¹ bar⁻¹或 -0.9×10⁻⁶ ml g⁻¹ bar⁻¹。这些结果表明原纤维处于一种高度可压缩的高体积状态，但在过渡态时其可压缩性和体积变小，很可能是由于现有空隙的部分水合作用。该系统最终达到最低体积状态，此时解离态的单体完全水合。
