Volpi N
Department of Biologia Animale, University of Modena, Italy.
Biochim Biophys Acta. 1997 Oct 20;1336(3):455-64. doi: 10.1016/s0304-4165(97)00058-5.
Heparins with different structures, charge density and molecular mass were evaluated for their capacity to induce structural and functional alterations of bovine trypsin in a low ionic strength buffer (20 mM Tris-HCl pH 7.4). Unfractionated heparin, and slow and fast moving heparin species increased the fluorescence peak emission of trypsin to the same extent (about +40.0%), whilst partially desulfated and re-N-acetylated heparin with a charge density of 1.47 modified the fluorescence at 330 nm by about +27% and natural heparan sulfate with a sulfate-to-carboxyl ratio < 1 by about +13%. Heparin fractions with narrow polydispersity and the same charge density (produced by chemical depolymerization in the presence of free radicals and further gel-permeation chromatography) having molecular mass lower than about 6000 interact with trypsin to a less extent, even though fractions with molecular mass of about 4500 and 3600 partially retain this property. No modification of fluorescence peak emission of trypsin with heparin was appreciable when the ionic strength of the buffer was increased to 0.3 mM NaCl. An altered ability to reduce cytochrome c was observed for heparins of different charge density; fragments with molecular mass lower than approximately 4000 were also unable to produce superoxide. Trypsin was degraded into fragments by heparin and derivatives after 3 h incubation at 37 degrees C. After electrophoresis in polyacrylamide-gels the trypsin bands disappeared and fragments with lower molecular mass were more evident. This effect depended on the molecular mass of heparin, and was more evident for unfractionated heparin and for a heparin fraction with a molecular mass of 7820. The esterolytic activity of trypsin was inhibited to the same extent by heparin derivatives of various structure and charge density while activity undermet minor changes in the presence of heparin fractions of Mr lower than 4000.
在低离子强度缓冲液（20 mM Tris-HCl，pH 7.4）中，评估了具有不同结构、电荷密度和分子量的肝素诱导牛胰蛋白酶结构和功能改变的能力。未分级肝素以及慢速和快速移动的肝素种类使胰蛋白酶的荧光峰值发射增加到相同程度（约 +40.0%），而电荷密度为1.47的部分脱硫和再N-乙酰化肝素使330 nm处的荧光改变约 +27%，硫酸乙酰肝素与羧基之比 < 1的天然硫酸乙酰肝素使荧光改变约 +13%。具有窄多分散性和相同电荷密度（通过在自由基存在下化学解聚并进一步进行凝胶渗透色谱法产生）且分子量低于约6000的肝素级分与胰蛋白酶的相互作用程度较小，尽管分子量约为4500和3600的级分部分保留了该特性。当缓冲液的离子强度增加到0.3 mM NaCl时，肝素对胰蛋白酶荧光峰值发射的修饰不明显。观察到不同电荷密度的肝素还原细胞色素c的能力有所改变；分子量低于约4000的片段也无法产生超氧化物。在37℃孵育3小时后，胰蛋白酶被肝素及其衍生物降解为片段。在聚丙烯酰胺凝胶中电泳后，胰蛋白酶条带消失，分子量较低的片段更明显。这种效应取决于肝素的分子量，对于未分级肝素和分子量为7820的肝素级分更为明显。各种结构和电荷密度的肝素衍生物对胰蛋白酶的酯解活性抑制程度相同，而在分子量低于4000的肝素级分存在下，活性发生轻微变化。
