Barros Ana E B, Carvalho Francisco A O, Alves Fernanda R, Carvalho José W P, Tabak Marcel
Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil.
Instituto de Química de São Carlos, Universidade de São Paulo, SP, Brazil; Universidade do Estado de Mato Grosso, MT, Brazil.
Int J Biol Macromol. 2015 Mar;74:327-36. doi: 10.1016/j.ijbiomac.2014.12.035. Epub 2014 Dec 26.
Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.
已在变性剂存在的情况下监测了圣保罗舌蛭细胞外血红蛋白（HbGp）的稳定性。使用了8-苯胺基-1-萘磺酸（ANS），并开展了光谱学和流体动力学研究。十二烷基三甲基溴化铵（DTAB）使ANS荧光发射强度增加，最大发射波长从517 nm蓝移至493 nm。观察到两个转变，分别在2.50和9.50 mmol/L的DTAB时出现，分别归因于ANS与前胶束聚集体和胶束的相互作用。与DTAB胶束相比，在氧合HbGp中，ANS与较少暴露于溶剂的蛋白质位点结合。在DTAB-HbGp-ANS三元体系中，在pH 7.0时，观察到蛋白质聚集、寡聚体解离和去折叠，而在pH 5.0时，则不存在聚集现象。DTAB诱导的去折叠过程显示出两个转变，一个是由于寡聚体解离，另一个可能是由于解离亚基的变性。此外，盐酸胍和尿素浓度分别高于1.5和4.0 mol/L时，会诱导HbGp完全变性，随着变性剂浓度达到1.0和5.0 mol/L时荧光猝灭，与ANS结合的氧合HbGp疏水斑块减少。我们的结果清楚地表明了在有和没有蛋白质存在的情况下，探针对表面活性剂的敏感性差异，以及对变性剂对HbGp去折叠影响的新见解。
