کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
5131356 1490881 2017 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Hyaluronidase reaction kinetics evaluated by capillary electrophoresis with UV and high-resolution mass spectrometry (HRMS) detection
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Hyaluronidase reaction kinetics evaluated by capillary electrophoresis with UV and high-resolution mass spectrometry (HRMS) detection
چکیده انگلیسی


- Linearity and stability study of bovine testicular hyaluronidase activity by CE.
- Km and Vmax for hyaluronic acid hydrolysis by hyaluronidase obtained by CE-UV/HRMS.
- IC50 of epigallocatechin gallate toward hyaluronidase obtained by CE-UV/HRMS.
- Effect of chondroitin sulfate (CS) oligosaccharides on hyaluronidase evaluated by CE.
- Sulfate position and number in CS contribute to anti-hyaluronidase activity.

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 (Km, Vmax and IC50) 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−1 and an ESI voltage at −4 kV. Km and Vmax were determined (n = 3) using CE/UV at 200 nm as 0.24 ± 0.02 mg mL−1 and 150.4 ± 0.1 nM s−1, respectively. They were also successfully obtained by CE/HRMS (n = 3) with Km of 0.49 ± 0.02 mg mL−1 and Vmax of 155.7 ± 0.2 nM s−1. IC50 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.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytica Chimica Acta - Volume 951, 25 January 2017, Pages 140-150
نویسندگان
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