Almutairi Fahad M, Cifre Jose-Gines Hernandez, Adams Gary G, Kök M Samil, Mackie Alan R, de la Torre Jose Garcia, Harding Stephen E
National Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, LE12 5RD, UK.
Department of Physical Chemistry, University of Murcia, Regional Campus of Excellence Mare Nostrum, 30071, Murcia, Spain.
Eur Biophys J. 2016 Jan;45(1):45-54. doi: 10.1007/s00249-015-1075-0. Epub 2015 Nov 23.
Mucins are the primary macromolecular component of mucus--nature's natural lubricant--although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 10(6) g mol(-1), in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 10(6) g mol(-1). Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length L p ~10 nm and mass per unit length, M L ~2380 g mol(-1) nm(-1), consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient.
粘蛋白是黏液（自然界的天然润滑剂）的主要大分子成分，尽管它们是特征不明的异质物质。流体动力学方法的最新进展为更好地了解其溶液性质提供了机会。在本研究中，结合使用了这些方法，以便从异质性和构象灵活性定量两方面，加深对猪肠粘蛋白（PIM）即MUC2粘蛋白制剂的理解。应用新的沉降平衡算法SEDFIT-MSTAR得出重均（基于整个分布）摩尔质量为7.1×10⁶ g·mol⁻¹，这与尺寸排阻色谱结合多角度光散射（SEC-MALS）的结果完全一致，后者得出的值为7.2×10⁶ g·mol⁻¹。沉降速度分布图显示粘蛋白具有很高的多分散性，使用扩展藤田算法获得了较宽的摩尔质量分布，这与SEC-MALS的洗脱曲线一致。将在线差示压力粘度测定法与SEC-MALS联用，以获得特性粘度[η]作为摩尔质量的函数。这些数据与沉降系数数据一起纳入全局构象算法HYDFIT，结果表明PIM具有柔性线性结构，其持久长度Lp约为10 nm，单位长度质量ML约为2380 g·mol⁻¹·nm⁻¹，这与从沉降系数的浓度依赖性得出的约1.2的威尔士-范霍尔德比率一致。
