Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
Anal Chim Acta. 2017 Jan 25;951:140-150. doi: 10.1016/j.aca.2016.11.036. Epub 2016 Nov 25.
The biology of hyaluronidase activity on age related turnover of the hyaluronic acid (HA) in skin dermis and epidermis has not been established. Elucidation of this phenomenon enables discovery of novel compounds for skin health. As a simple and green technique, capillary electrophoresis (CE) was used for the first time for the determination of the kinetic constants (K V and IC) of the enzymatic degradation of HA. Reaction products were identified using CE/high-resolution mass spectrometry (HRMS) after appropriate optimization. Best results in terms of signal sensitivity were obtained using 10 mM ammonium acetate (pH 9.0) BGE, a sheath liquid composed of methanol-water (80:20, v/v) with 0.02% (v/v) formic acid at 10 μL min and an ESI voltage at -4 kV. K and V were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL and 150.4 ± 0.1 nM s, respectively. They were also successfully obtained by CE/HRMS (n = 3) with K of 0.49 ± 0.02 mg mL and V of 155.7 ± 0.2 nM s. IC of a standard natural inhibitor, epigallocatechin gallate, was also determined by CE-UV/HRMS. Kinetic constant values obtained by CE compared well with literature which validated the developed CE-based assay. In addition, the activity of homemade tetrasaccharides of biotinylated chondroitin sulfate CS-A or CS-C (4- or 6- sulfated in a homogeneous or heterogeneous way) on the hydrolysis reaction of hyaluronidase was evaluated. Hyaluronidase was mostly dose-dependently inhibited by CS-A tetrasaccharides sulfated in a homogeneous way. Two trisaccharides from truncated linkage region of proteoglycans were also tested as inhibitors or activators. CE-based assay showed that even a small modification of one hydroxyl group changes the influence on hyaluronidase activity. CE-based assay can be used for the screening of natural and synthetic inhibitors of hyaluronidase activity for cosmetic and therapeutic applications.
透明质酸（HA）在皮肤真皮和表皮中的年龄相关周转率的透明质酸酶活性的生物学尚未确定。阐明这一现象可以发现用于皮肤健康的新型化合物。作为一种简单而绿色的技术，毛细管电泳（CE）首次用于确定 HA 酶促降解的动力学常数（K V 和 IC）。通过适当的优化，使用 CE/高分辨率质谱（HRMS）对反应产物进行鉴定。在信号灵敏度方面，使用 10 mM 乙酸铵（pH 9.0）BGE 获得最佳结果，鞘液由甲醇-水（80:20，v/v）组成，其中含有 0.02%（v/v）甲酸，流速为 10 μL/min，ESI 电压为-4 kV。使用 CE/UV 在 200 nm 处测定 K 和 V（n = 3），分别为 0.24±0.02 mg/mL 和 150.4±0.1 nM s。使用 CE/HRMS（n = 3）也成功获得了 K 和 V，分别为 0.49±0.02 mg/mL 和 155.7±0.2 nM s。还通过 CE-UV/HRMS 测定了标准天然抑制剂表没食子儿茶素没食子酸酯的 IC。CE 获得的动力学常数值与文献值吻合良好，验证了所开发的基于 CE 的测定法。此外，还评估了生物素化硫酸软骨素 CS-A 或 CS-C 的内消旋四糖（以均相或异相方式 4-或 6-硫酸化）对透明质酸酶水解反应的活性。透明质酸酶大部分被均相硫酸化的 CS-A 四糖剂量依赖性抑制。两种来自蛋白聚糖截断连接区的三糖也被用作抑制剂或激活剂进行测试。基于 CE 的测定法表明，即使一个羟基的微小修饰也会改变对透明质酸酶活性的影响。基于 CE 的测定法可用于筛选用于美容和治疗应用的透明质酸酶活性的天然和合成抑制剂。
