al-Hakim A, Linhardt R J
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
Appl Theor Electrophor. 1991;1(6):305-12.
Glycosaminoglycans (GAGs) including chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate types I (corneal) and II (cartilage) added to buffer, plasma and urine were enzymatically depolymerized. Enzymes, including chondroitin ABC lyase (chondroitinase ABC), heparin lyase (heparinase), heparan sulfate lyase (heparitinase), endo-beta-galactosidase and keratanase were used to depolymerize each GAG. Depolymerized GAGs and GAG mixtures were fractionated using gradient polyacrylamide gel electrophoresis. Staining with alcian blue dye resulted in a distinctive and well resolved banding pattern for each GAG. When these same gels were silver stained, an increase in detection sensitivity of 1000-fold was obtained. Picogram quantities of an oligosaccharide standard in buffer could be detected with silver staining while nanogram quantities could be detected in urine or plasma. The banding pattern observed for each depolymerized GAG was well resolved from contaminants found in these biological fluids and from intact GAGs. Endogenous GAGs present in samples of human urine and plasma were first concentrated and then enzymatically depolymerized. Chondroitin or dermatan sulfates, heparan sulfate and keratan sulfate were each detected in both concentrated plasma and urine samples.
将包括硫酸软骨素、硫酸皮肤素、硫酸乙酰肝素、肝素以及I型（角膜）和II型（软骨）硫酸角质素在内的糖胺聚糖（GAGs）添加到缓冲液、血浆和尿液中，然后进行酶促解聚。使用包括软骨素ABC裂解酶（软骨素酶ABC）、肝素裂解酶（肝素酶）、硫酸乙酰肝素裂解酶（乙酰肝素酶）、β-内半乳糖苷酶和角质素酶在内的酶对每种GAG进行解聚。使用梯度聚丙烯酰胺凝胶电泳对解聚后的GAGs和GAG混合物进行分级分离。用阿尔辛蓝染料染色后，每种GAG都呈现出独特且清晰分辨的条带模式。当对这些相同的凝胶进行银染时，检测灵敏度提高了1000倍。缓冲液中皮克级的寡糖标准品可用银染检测到，而尿液或血浆中的纳克级寡糖标准品也能被检测到。每种解聚后的GAG所观察到的条带模式与这些生物流体中发现的污染物以及完整的GAGs都能很好地分辨开来。首先对人尿液和血浆样本中存在的内源性GAGs进行浓缩，然后进行酶促解聚。在浓缩的血浆和尿液样本中均检测到了硫酸软骨素或硫酸皮肤素、硫酸乙酰肝素和硫酸角质素。
