Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A
Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A
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DOI：
10.1179/096805102125000263
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发表时间：
2002-01-01
期刊：
影响因子：
--
通讯作者：
Brandenburg, K
中科院分区：
文献类型：
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作者：
Jürgens, G;Müller, M;Brandenburg, K
采用多种物理技术和生物学试验研究了细菌内毒素、深粗糙突变型脂多糖LPS Re和“内毒素原则”脂质A与重组人血清白蛋白（rHSA）的相互作用。采用傅里叶变换红外光谱法和差示扫描量热法研究了白蛋白对内毒素酰基链熔融行为的影响。此外，还研究了对内毒素官能团的影响，特别是其方向，包括与多粘菌素B的竞争实验。此外，研究了内毒素与rHSA结合对蛋白质二级结构的影响。结果表明，非静电结合，LPS的主链方向没有变化，而rHSA的二级结构只有轻微的变化。相应地，通过电泳迁移率测量的rHSA引起的内毒素的电荷中和量仅表现出表面电位的轻微降低。根据这些测量和等温滴定量热法，脂质：蛋白质结合化学计量估计为[LPS]：[rHSA]，10：1摩尔。通过X-射线小角散射测定内毒素的聚集体结构，发现立方结构变为非层状结构的复杂变化。荧光共振能量转移法未检测到rHSA对脂多糖结合蛋白诱导的内毒素嵌入磷脂脂质体的影响。最后，LPS诱导的人单核细胞的细胞因子的产生仅在高摩尔rHSA过量时略有增加，而鲎试验中变形细胞裂解物的凝固由于rHSA与LPS的结合而产生了复杂的变化。
The interaction of bacterial endotoxins, deep rough mutant lipopolysaccharide LPS Re and the 'endotoxic principle' lipid A, with recombinant human serum albumin (rHSA) was investigated with a variety of physical techniques and biological assays. With Fourier-transform infrared spectroscopy and differential scanning calorimetry, the influence of albumin on the acyl chain melting behavior of the endotoxins was measured. Also, the effect on the functional groups of the endotoxins, in particular with respect to their orientation, was studied, including competition experiments with polymyxin B. Furthermore, the influence of endotoxin binding to rHSA on the protein's secondary structure was investigated. The results indicate a non-electrostatic binding with no change of the backbone orientation of LPS and only a slight change of the secondary structure of rHSA. Correspondingly, the amount of charge neutralization of the endotoxins due to rHSA measured by the electrophoretic mobility exhibited only a slight reduction of the surface potential. From these measurements and isothermal titration calorimetry, the lipid:protein binding stoichiometry was estimated to [LPS]:[rHSA], 10:1 molar. The determination of the aggregate structure of the endotoxins by X-ray small-angle scattering exhibited a complex change of a cubic into a non-lamellar structure. No influence of rHSA on endotoxin intercalation into phospholipid liposomes induced by lipopolysaccharide-binding protein could be detected by fluorescence resonance energy transfer. Finally, the LPS-induced cytokine production of human mononuclear cells was only slightly increased at high molar rHSA excess, while the coagulation of amebocyte lysate in the Limulus test yielded a complex change due to rHSA binding of LPS.