Jullian C, Brunet J E, Thomas V, Jameson D M
Instituto de Quimica, Universidad Catolica de Valparaiso, Chile.
Biochim Biophys Acta. 1989 Aug 31;997(3):206-10. doi: 10.1016/0167-4838(89)90188-x.
The hemin moiety of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was removed and the apoprotein reconstituted with the fluorescent protoporphyrin IX. Steady-state and time-resolved fluorescence properties of the HRP(desFe) adduct were examined; the multifrequency phase and modulation method was utilized for lifetime and dynamic polarization studies. The emission spectrum of HRP(desFe) had maxima at 633 and 696 nm. The lifetime of this emission was characterized by a single exponential decay of 16.87 ns at 22 degrees C. Debye rotational relaxation times for HRP(desFe) were determined using both static (Perrin plot) and dynamic (differential phase and modulation fluorometry) methods; these two approaches gave values of 96 and 86 ns, respectively. A spherical protein of HRP's molecular weight and partial specific volume would be expected to have a Debye rotational relaxation time, at 22 degrees C, in the range of 50 to 60 ns, depending upon the extent of hydration. Hence our results indicate that HRP(desFe) is asymmetric; the global rotational relaxation times observed are consistent with those of a prolate ellipsoid with an axial ratio of 3:1.
去除了辣根过氧化物酶（供体：过氧化氢氧化还原酶，EC 1.11.1.7）的血红素部分，并用荧光原卟啉IX重构了脱辅基蛋白。研究了HRP(desFe)加合物的稳态和时间分辨荧光特性；采用多频相位和调制方法进行寿命和动态极化研究。HRP(desFe)的发射光谱在633和696 nm处有最大值。在22℃下，该发射的寿命以16.87 ns的单指数衰减为特征。使用静态（佩林图）和动态（差相和调制荧光法）两种方法测定了HRP(desFe)的德拜旋转弛豫时间；这两种方法分别给出了96和86 ns的值。对于HRP分子量和偏比容的球形蛋白质，预计在22℃下德拜旋转弛豫时间在50至60 ns范围内，这取决于水合程度。因此，我们的结果表明HRP(desFe)是不对称的；观察到的整体旋转弛豫时间与轴比为3:1的长椭球体一致。
