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Structural and functional modifications of bovine trypsin by heparins Influence of heparin molecular mass and structure

机译:肝素对牛胰蛋白酶的结构和功能修饰肝素分子量和结构的影响

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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°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.
机译:评价了具有不同结构,电荷密度和分子量的肝素在低离子强度缓冲液(20 mM Tris-HCl pH 7.4)中诱导牛胰蛋白酶结构和功能改变的能力。未分级的肝素和慢速移动的肝素种类在相同程度上(约+ 40.0%)增加了胰蛋白酶的荧光峰值发射,而部分脱硫和再N-乙酰化的肝素的电荷密度为1.47,则在330 nm处改变了荧光约+ 27%的天然硫酸乙酰肝素和硫酸盐与羧基之比<1的天然硫酸乙酰肝素约+ 13%。分子量小于约6000的具有窄多分散度和相同电荷密度(在自由基存在下通过化学解聚反应和进一步的凝胶渗透色谱法生产)的肝素级分与胰蛋白酶的相互作用程度较小,即使分子量为大约4500和3600部分保留了此属性。当缓冲液的离子强度增加到0.3 mM NaCl时,用肝素对胰蛋白酶的荧光峰发射没有任何修饰。对于不同电荷密度的肝素,观察到还原细胞色素c的能力发生了改变。分子量低于约4000的片段也无法产生超氧化物。在37°C孵育3小时后,胰蛋白酶被肝素和衍生物降解为片段。在聚丙烯酰胺凝胶中电泳后,胰蛋白酶带消失,分子量较低的片段更明显。该作用取决于肝素的分子量,对于未分级的肝素和分子量为7820的肝素级分更明显。不同结构和电荷密度的肝素衍生物在相同程度上抑制胰蛋白酶的酯水解活性。肝素分数低于4000时,肝素酶活性未见轻微变化。

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