Oliveira Marilene Silva, Moreira Leonardo Marmo, Tabak Marcel
Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, USP, C.P.780, 13560-970 São Carlos, SP, Brazil.
Int J Biol Macromol. 2008 Mar 1;42(2):111-9. doi: 10.1016/j.ijbiomac.2007.10.002. Epub 2007 Oct 7.
The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16-19kDa forming a monomer (d) and a trimer (abc), and around 36 non-heme structures, named linkers (L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms (d(1) and d(2)) of identical proportions with masses of 16,355+/-25 and 16,428+/-24Da, respectively, and two minor isoforms (d(3) and d(4)) with masses around 16.6kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d(1) and d(2), both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS-HbGp interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta 1770 (2007) 506-517.].
圣保罗巨蚓（Glossoscolex paulistus）的巨型细胞外血红蛋白（HbGp）由大约144个亚基组成，这些亚基含有分子量在16 - 19kDa范围内的血红素基团，形成一个单体（d）和一个三聚体（abc），以及大约36个非血红素结构，称为连接体（L）。最近进行了基质辅助激光解吸/电离飞行时间（MALDI - TOF - MS）分析，以直接获取关于氧合形式的HbGp中不同亚基分子量的信息。该技术证明了HbGp与广泛研究的陆生蚯蚓血红蛋白（HbLt）之间的结构相似性。实际上，检测到HbGp单体d有两种主要的同型异构体（d(1)和d(2)），比例相同，分子量分别为16,355±25Da和16,428±24Da，以及两种次要的同型异构体（d(3)和d(4)），分子量约为16.6kDa。在本工作中，通过MALDI - TOF - MS研究了阴离子十二烷基硫酸钠（SDS）和阳离子十六烷基三甲基氯化铵（CTAC）对HbGp寡聚结构的影响，以评估离子表面活性剂与HbGp之间的相互作用。用该技术获得的数据表明，阳离子表面活性剂CTAC与单体d的两种同型异构体d(1)和d(2)在整个蛋白质以及纯分离单体中都有有效的相互作用。结果表明，每个单体同型异构体最多结合10个CTAC分子。不同的是，SDS - HbGp系统获得的质谱表明，添加阴离子表面活性剂SDS不会导致单体亚基的任何质量增加，这表明与CTAC - HbGp相互作用相比，SDS - HbGp相互作用可能明显不太有效。蛋白质约为5.5的酸性pI可能是造成这种行为的原因。从观察到的质量增加判断，这项工作的结果还表明两种表面活性剂与连接体链以及三聚体都有一些相互作用。我们的数据与最近的一项光谱研究一致，该研究表明在生理pH下CTAC与HbGp之间有很强的相互作用[P.S.圣地亚哥等人，《生物化学与生物物理学报》1770（2007）506 - 517]。
