A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation
A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation
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DOI：
10.1016/j.ab.2010.04.014
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发表时间：
2010-09-01
影响因子：
2.9
通讯作者：
Schiraldi, Chiara
中科院分区：
文献类型：
--
作者：
La Gatta, Annalisa;De Rosa, Mario;Schiraldi, Chiara
采用体积排阻色谱结合三重检测（在线激光散射、折光和粘度）（SEC-TDA）对牛睾丸透明质酸酶（BTH）催化水解过程中产生的透明质酸（HA）片段进行了研究。这种方法提供的主要优点是完整的流体动力学特性，而不需要进一步的实验。使用若干BTH量和增加的孵育时间来水解HA。根据重均和数均分子量（分别为M（w）和M（n））、多分散指数（M（w）/M（n））、流体动力学半径（R（h））和特性粘度（[η]）表征片段。然后直接导出Mark-Houwink-Sakurada（MHS）曲线（log [eta]与log M（w））。产生的片段覆盖整个范围的M（w）（10-900 kDa）和大小（R（h）= 4-81 nm），并呈现相当窄的摩尔质量分布（M（w）/M（n）= 1.6-1.7）。从MHS曲线，HA构象导致从无规卷曲向刚性棒结构的变化，同时降低M（w）。还监测了在BTH抑制剂存在下的HA酶促水解，揭示了抑制曲线受离子强度的影响。最后，来自SEC-TDA的动力学数据与来自流变学测量的数据的比较表明，在取决于水解条件的解聚速率的测定中，这两种方法具有不同的强度。(C)2010年爱思唯尔公司All rights reserved.
Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M(w) and M(n), respectively), polydispersity index (M(w)/M(n)), hydrodynamic radius (R(h)), and intrinsic viscosity ([eta]). The Mark-Houwink-Sakurada (MHS) curves (log [eta] versus log M(w)) were then derived directly. Fragments covering a whole range of M(w) (10-900 kDa) and size (R(h) = 4-81 nm) and presenting a rather narrow distribution of molar masses (M(w)/M(n) = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M(w). HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions. (C) 2010 Elsevier Inc. All rights reserved.