Linhardt R J, Turnbull J E, Wang H M, Loganathan D, Gallagher J T
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
Biochemistry. 1990 Mar 13;29(10):2611-7. doi: 10.1021/bi00462a026.
We have examined the activities of different preparations of heparin and heparan sulfate lyases from Flavobacterium heparinum. The enzymes were incubated with oligosaccharides of known size and sequence and with complex polysaccharide substrates, and the resulting degradation products were analyzed by strong-anion-exchange high-performance liquid chromatography and by oligosaccharide mapping using gradient polyacrylamide gel electrophoresis. Heparinase (EC 4.2.2.7) purified in our laboratory and a so-called Heparinase I (Hep I) from a commercial source yielded similar oligosaccharide maps with heparin substrates and displayed specificity for di- or trisulfated disaccharides of the structure----4)-alpha-D-GlcNp2S(6R)(1----4)-alpha-L-IdoAp2S( 1----(where R = O-sulfo or OH). Oligosaccharide mapping with two different commercial preparations of heparan sulfate lyase [heparitinase (EC 4.2.2.8)] indicated close similarities in their depolymerization of heparan sulfate. Furthermore, these enzymes only degraded defined oligosaccharides at hexosaminidic linkages with glucuronic acid:----4)-alpha-D-GlcNpR(1----4)-beta-D-GlcAp(1----(where R = N-acetamido or N-sulfo). The enzymes showed activity against solitary glucuronate-containing disaccharides in otherwise highly sulfated domains including the saccharide sequence that contains the antithrombin binding region in heparin. A different commercial enzyme, Heparinase II (Hep II), displayed a broad spectrum of activity against polysaccharide and oligosaccharide substrates, but mapping data indicated that it was a separate enzyme rather than a mixture of heparinase and heparitinase/Hep III. When used in conjunction with the described separation procedures, these enzymes are powerful reagents for the structural/sequence analysis of heparin and heparan sulfate.
我们检测了来自肝素黄杆菌的不同肝素和硫酸乙酰肝素裂解酶制剂的活性。将这些酶与已知大小和序列的寡糖以及复合多糖底物一起孵育，然后通过强阴离子交换高效液相色谱法以及使用梯度聚丙烯酰胺凝胶电泳的寡糖图谱分析所得的降解产物。在我们实验室中纯化的肝素酶（EC 4.2.2.7）和一种来自商业来源的所谓肝素酶I（Hep I），与肝素底物产生了相似的寡糖图谱，并且对结构为----4)-α-D-葡糖胺-2S(6R)(1----4)-α-L-艾杜糖醛酸-2S( 1----（其中R = O-磺基或OH）的二硫酸化或三硫酸化二糖具有特异性。用两种不同的商业硫酸乙酰肝素裂解酶制剂[类肝素酶（EC 4.2.2.8）]进行的寡糖图谱分析表明，它们在硫酸乙酰肝素解聚方面非常相似。此外，这些酶仅在与葡糖醛酸的己糖胺键处降解特定的寡糖：----4)-α-D-葡糖胺-R(1----4)-β-D-葡糖醛酸-(1----（其中R = N-乙酰氨基或N-磺基）。这些酶对其他高度硫酸化区域中含孤立葡糖醛酸的二糖具有活性，包括肝素中含抗凝血酶结合区域的糖序列。另一种商业酶肝素酶II（Hep II）对多糖和寡糖底物表现出广泛的活性，但图谱数据表明它是一种单独的酶，而不是肝素酶和类肝素酶/肝素酶III的混合物。当与所述分离程序结合使用时，这些酶是用于肝素和硫酸乙酰肝素结构/序列分析的强大试剂。
