원보 : 글리코사미노글칸 분해 효소와 핵자기공명을 이용한 오염된 헤파린 시료의 분석

We examined the purity of six heparin samples by using heparinase, chondroitinase, 1H-NMR, and polyacrylamide gel electrophoresis. To obtain high molecular weight contaminants from heparin samples, heparinase I-digested samples were subjected to the exhaustive microcon filtration. The filtration process removed heparin-derived di- and oligosaccharides effectively. By combining chondroitinase ABC treatment and strong anion exchange-high performance liquid chromatography, the result showed all six samples contained chondroitin sulfate as a contaminant ranging from 1.3 to 14.9%. Among them, sample S3 showed the highest content of 14.9%, which was further analyzed by chondroitinase AC treatment to confirm chondroitin sulfate B (dermatan sulfate). 1H-NMR chemical shifts of N-acetyl groups clearly suggested the existence of chondroitin sulfate B (sample S3) and oversulfated chondroitin sulfate (samples S2 and S4) as contaminants. In addition, polyacrylamide gel electrophoresis was useful for qualitative detection on the sample`s purity. These results suggest that the tools of heparinase I and chondroitinase ABC in combination with NMR spectroscopy would give very useful information for investigation of heparin contaminants such as oversulfated chondroitin sulfate and dermatan sulfate in heparin samples.