Gouveia Scott M, Tiffany John M
Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford, OX2 6AW, UK.
Biochim Biophys Acta. 2005 Dec 1;1753(2):155-63. doi: 10.1016/j.bbapap.2005.08.023. Epub 2005 Sep 21.
Human tear viscosity is poorly understood. Tears need to remain on the ocular surface for lubrication without causing damage to the surface epithelia due to drag when blinking. Whole tears are shear-thinning (non-Newtonian), which cannot be explained by the amount of mucin present, nor by individual proteins. Whole tears minus lipids become Newtonian. Though no free lipids had previously been found in collected tears, tear lipocalin (TL), a major tear protein, is known to bind lipids. In this study, we aimed to confirm whether there are any free lipids in collected tears, and to clarify the combined contribution of tear proteins to viscosity, including experiments on recombinant TL, both without (apo-TL) and with (holo-TL) bound lipid. We also investigated possible oligomer formation by holo- and apo-TL as a mechanism for viscosity using SDS-PAGE and analytical ultracentrifugation (AU). For comparison, we have included results for beta-lactoglobulin, a well-characterised lipocalin protein. No free lipids were detected in whole tears. Rheology showed that any protein combination that included lysozyme or lactoferrin was shear-thinning, as was apo-TL, though holo-TL was Newtonian (linear). Results from SDS-PAGE and AU showed apo-TL to be entirely monomeric, but holo-TL showed some dimerization. Both apo- and holo-beta-lactoglobulin exhibited a monomer-dimer equilibrium. We conclude that hetero-protein interactions, possibly electrostatic, involving lipid-binding-induced structural changes to TL, significantly contribute to the viscosity of human tears.
人们对人类泪液的黏性了解甚少。泪液需要留在眼表以起到润滑作用，同时在眨眼时不会因拖拽而对表面上皮造成损伤。全泪液呈剪切变稀特性（非牛顿流体），这无法用黏蛋白的含量或单个蛋白质来解释。去除脂质后的全泪液变为牛顿流体。尽管此前在收集的泪液中未发现游离脂质，但已知泪液视黄醇结合蛋白（TL），一种主要的泪液蛋白质，能够结合脂质。在本研究中，我们旨在确认收集的泪液中是否存在游离脂质，并阐明泪液蛋白质对黏性的综合贡献，包括对重组TL进行实验，分别研究未结合脂质的（脱辅基TL）和结合脂质的（全蛋白TL）情况。我们还使用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和分析超速离心（AU）研究了全蛋白TL和脱辅基TL可能形成的寡聚体作为黏性机制。为作比较，我们纳入了β - 乳球蛋白（一种特性明确的视黄醇结合蛋白）的结果。在全泪液中未检测到游离脂质。流变学表明，任何包含溶菌酶或乳铁蛋白的蛋白质组合以及脱辅基TL都呈剪切变稀特性，而全蛋白TL呈牛顿流体特性（线性）。SDS - PAGE和AU的结果显示脱辅基TL完全为单体形式，但全蛋白TL显示出一些二聚化现象。脱辅基和全蛋白β - 乳球蛋白均呈现单体 - 二聚体平衡。我们得出结论，涉及脂质结合诱导的TL结构变化的异源蛋白质相互作用（可能是静电作用）对人类泪液的黏性有显著贡献。
