de Sousa Neto Diógenes, Salmon Carlos Ernesto Garrido, Alonso Antonio, Tabak Marcel
Instituto de Química de São Carlos, Universidade de São Paulo, Cx. Postal 780, CEP 13560-970, São Carlos, SP, Brazil.
Colloids Surf B Biointerfaces. 2009 Apr 1;70(1):147-56. doi: 10.1016/j.colsurfb.2008.12.026. Epub 2008 Dec 25.
EPR spectra of 5- and 16-doxyl stearic acid nitroxide probes (5-DSA and 16-DSA, respectively) bound to bovine serum albumin (BSA) revealed that in the presence of ionic surfactants, at least, two label populations coexist in equilibrium. The rotational correlation times (tau) indicated that component 1 displays a more restricted mobility state, associated to the spin labels bound to the protein; the less immobilized component 2 is due to label localization in the surfactant aggregates. For both probes, the increase of surfactant concentration leads to higher motional levels of component 1 followed by a simultaneous decrease of this fraction of nitroxides and its conversion into component 2. For 10mM cethyltrimethylammonium chloride (CTAC), the nitroxides are 100% bound to the protein, whereas at 10mM N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (HPS) and sodium dodecyl sulfate (SDS) the fractions of bound nitroxides are reduced to 18% and 86%, respectively. No significant polarity changes were observed in the whole surfactant concentration range for component 1. Moreover, at higher surfactant concentration, component 2 exhibited a similar polarity as in the pure surfactant micelles. For 16-DSA the surfactant effect is different: at 10mM of HPS and CTAC the fractions of bound nitroxides are 76% and 49%, respectively, while at 10mM SDS they are present exclusively in a micellar environment, consistent with 100% of component 2. Overall, both SDS and HPS are able to effectively displace the nitroxide probes from the protein binding sites, while CTAC seems to affect the nitroxide binding to a significantly smaller extent.
与牛血清白蛋白（BSA）结合的5-和16-二氧硬脂酸氮氧化物探针（分别为5-DSA和16-DSA）的电子顺磁共振光谱表明，至少在离子表面活性剂存在的情况下，两种标记物群体以平衡状态共存。旋转相关时间（tau）表明，组分1表现出更受限的迁移状态，这与结合到蛋白质上的自旋标记物有关；迁移性较小的组分2是由于标记物定位于表面活性剂聚集体中。对于两种探针，表面活性剂浓度的增加导致组分1的运动水平升高，随后该氮氧化物部分同时减少并转化为组分2。对于10mM十六烷基三甲基氯化铵（CTAC），氮氧化物100%与蛋白质结合，而在10mM N-十六烷基-N，N-二甲基-3-铵基-1-丙烷磺酸盐（HPS）和十二烷基硫酸钠（SDS）存在时，结合氮氧化物的比例分别降至18%和86%。在整个表面活性剂浓度范围内，未观察到组分1有明显的极性变化。此外，在较高的表面活性剂浓度下，组分2表现出与纯表面活性剂胶束相似的极性。对于16-DSA，表面活性剂的作用不同：在10mM的HPS和CTAC存在时，结合氮氧化物的比例分别为76%和49%，而在10mM SDS存在时，它们仅存在于胶束环境中，这与100%的组分2一致。总体而言，SDS和HPS都能够有效地将氮氧化物探针从蛋白质结合位点上置换下来，而CTAC似乎对氮氧化物结合的影响要小得多。
