Mitrakos P, Macdonald P M
Department of Chemistry and Erindale College, University of Toronto, Mississauga, Ont., Canada.
Biochim Biophys Acta. 1998 Sep 23;1374(1-2):21-33. doi: 10.1016/s0005-2736(98)00128-x.
2H-NMR spectroscopy was used to investigate the effects of polyadenylic acid (PolyA) on three aminomethyl-deuterated cationic amphiphiles: specifically, N-[1-(2,3-dioleoyloxy)propyl]-N',N',N'-trimethylammonium chloride (DOTAP-gamma-d3), 3beta-[N-(N',N',N'-trimethylaminoethane)carbamoyl] cholesterol (TC-CHOL-gamma-d3), and cetyltrimethylammonium bromide (CTAB-gamma-d9). When mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and incorporated into lipid bilayer membranes, each of the cationic amphiphiles yielded 2H-NMR spectra consisting of a motionally averaged Pake powder pattern. The 2H-NMR quadrupolar splitting generally increased with increasing mole fraction of cationic amphiphile in the lipid bilayer with the exception of CTAB-gamma-d9. Adding PolyA caused the quadrupolar splitting to increase progressively in every case, until a 1:1 cation:anion charge ratio was achieved, after which the quadrupolar splitting changed no further. Deuterium NMR relaxation time measurements showed a parallel increase in T(qe)2 with increasing PolyA. The size of these changes produced by PolyA increased in the order: TC-CHOL < DOTAP < CTAB. NaCl addition reversed much, but not all, of the PolyA-related changes in 2H-NMR quadrupolar splittings and T(qe)2 relaxation times. A UV-based PolyA-membrane binding assay showed that salt addition caused PolyA desorption, and that the salt concentration required to do so increased in the order: TC-CHOL < DOTAP < CTAB. The results are consistent with an electrostatic binding of PolyA to the cationic lipid bilayer surface, accompanied by formation of a stoichiometric charge complex between PolyA and the cationic amphiphile, in which the cationic amphiphile retains considerable motional freedom. The strength of the complex increases in the order: TC-CHOL < DOTAP < CTAB.
采用2H-核磁共振光谱法研究了聚腺苷酸（PolyA）对三种氨基甲基氘代阳离子两亲物的影响：具体为N-[1-(2,3-二油酰氧基)丙基]-N',N',N'-三甲基氯化铵（DOTAP-γ-d3）、3β-[N-(N',N',N'-三甲基氨基乙烷)氨基甲酰基]胆固醇（TC-CHOL-γ-d3）和十六烷基三甲基溴化铵（CTAB-γ-d9）。当与1-棕榈酰-2-油酰基-sn-甘油-3-磷酸胆碱（POPC）混合并掺入脂质双层膜中时，每种阳离子两亲物产生的2H-核磁共振光谱均由动态平均的帕克粉末图样组成。除CTAB-γ-d9外，脂质双层中阳离子两亲物摩尔分数增加时，2H-核磁共振四极分裂通常也会增加。添加PolyA导致每种情况下四极分裂逐渐增加，直至达到1:1的阳离子:阴离子电荷比，此后四极分裂不再变化。氘核磁共振弛豫时间测量表明，随着PolyA增加，T(qe)2也呈平行增加。PolyA产生的这些变化大小顺序为：TC-CHOL < DOTAP < CTAB。添加NaCl可逆转大部分但并非全部与PolyA相关的2H-核磁共振四极分裂和T(qe)2弛豫时间变化。基于紫外的PolyA-膜结合试验表明，添加盐会导致PolyA解吸，所需盐浓度顺序为：TC-CHOL < DOTAP < CTAB。结果表明，PolyA与阳离子脂质双层表面发生静电结合，同时PolyA与阳离子两亲物形成化学计量电荷复合物，其中阳离子两亲物保留了相当大的运动自由度。复合物强度顺序为：TC-CHOL < DOTAP < CTAB。
