Mock D M, Lankford G, Horowitz P
Department of Pediatrics, University of Iowa, Iowa City 52242.
Biochim Biophys Acta. 1988 Aug 31;956(1):23-9. doi: 10.1016/0167-4838(88)90293-2.
The environment of the biotin binding site on avidin was investigated by determining the fluorescence enhancement of a series of fluorescent probes that are anilinonaphthalene sulfonic acid derivatives. Of the compounds tested, 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS) exhibited the greatest enhancement under the conditions used (which would reflect both molar fluorescence enhancement and binding affinity) and exhibited more than 95% reversal upon addition of biotin. Thus, 2,6-ANS was chosen for more detailed characterization of the interaction with avidin. Only a single class of binding sites for 2,6-ANS was identified; the mean value for the Kd was 203 +/- 16 microM (X +/- 1 S.D.), and the molar ratio of 2,6-ANS binding sites to biotin binding sites was approx. 1. These results provide evidence that the biotin binding site and the 2,6-ANS binding site are at least partially overlapping, but the possibility that the probe binding site is altered by a conformational change induced by biotin binding cannot be excluded. At excitation = 328 nm and emission = 408 nm, the molar fluorescence of the bound probe was 6.8 +/- 1.0 microM-1 and that of the free probe was 0.061 +/- 0.008 microM-1 giving an enhancement ratio (molar fluorescence of bound probe/molar fluorescence of free probe) of 111 +/- 22. Upon binding, the wavelength of maximum fluorescence decreases. These findings also provide evidence that the fluorescence enhancement associated with the interaction of 2,6-ANS and avidin reflects the environment of the biotin binding site. The Kosower's Z factor, an empirical index of apolarity, was 82.1 for the 2,6-ANS binding site on avidin. This value reflects a degree of apolarity that is similar to apolar environments observed for substrate binding sites on several enzymes; although not the dominant factor, this environment may contribute to the strong binding of biotin.
通过测定一系列作为苯胺萘磺酸衍生物的荧光探针的荧光增强，研究了抗生物素蛋白上生物素结合位点的环境。在所测试的化合物中，2-苯胺萘-6-磺酸（2,6-ANS）在所用条件下表现出最大的增强（这将反映摩尔荧光增强和结合亲和力），并且在添加生物素后表现出超过95%的荧光逆转。因此，选择2,6-ANS对其与抗生物素蛋白的相互作用进行更详细的表征。仅鉴定出一类2,6-ANS的结合位点；Kd的平均值为203±16μM（X±1标准差），2,6-ANS结合位点与生物素结合位点的摩尔比约为1。这些结果提供了证据，表明生物素结合位点和2,6-ANS结合位点至少部分重叠，但不能排除生物素结合诱导的构象变化改变探针结合位点的可能性。在激发波长为328nm和发射波长为408nm时，结合探针的摩尔荧光为6.8±1.0μM-1，游离探针的摩尔荧光为0.061±0.008μM-1，增强比（结合探针的摩尔荧光/游离探针的摩尔荧光）为111±22。结合时，最大荧光波长减小。这些发现还提供了证据，表明与2,6-ANS和抗生物素蛋白相互作用相关的荧光增强反映了生物素结合位点的环境。抗生物素蛋白上2,6-ANS结合位点的Kosower's Z因子（一种非极性的经验指标）为82.1。该值反映的非极性程度与在几种酶的底物结合位点观察到的非极性环境相似；虽然不是主要因素，但这种环境可能有助于生物素的强结合。
