Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor.
Solvent exchange of buried water and hydrogen exchange of peptide NH groups hydrogen bonded to buried waters in bovine pancreatic trypsin inhibitor.
复制标题
牛胰蛋白酶抑制剂中埋藏水的溶剂交换和与埋藏水氢键合的肽 NH 基团的氢交换。
DOI：
10.1021/bi00390a040
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Woodward,C
中科院分区：
文献类型：
--
作者：
Tüchsen,E;Hayes,JM;Ramaprasad,S;Copie,V;Woodward,C
明尼苏达大学生物化学系，圣保罗，明尼苏达州55108摘要：用高精度同位素比质谱法测定了牛胰胰蛋白酶抑制剂（BPTI）、胰蛋白酶和胰蛋白酶-BPTI复合物中标记埋水的溶剂交换。埋水通过蛋白质在180富集水中的平衡来标记。然后，蛋白质样品通过凝胶过滤与正常同位素组成的水进行快速透析并储存。交换180标记洗脱与蛋白质在10-300秒是由h2o - co2平衡技术确定的。BPTI、胰蛋白酶和BPTI-胰蛋白酶以及溶菌酶和羧肽酶在10-15秒前完成与溶剂水的交换。当交换透析和储存在pH值> 2.5下进行时，胰蛋白酶-BPTI和胰蛋白酶，而不是游离BPTI，具有相当于一个180原子的缓慢交换（300秒后和几天前）。这个氧可能与胰蛋白酶的特定位点共价结合。当交换透析和储存在pH 1.1下进行时，胰蛋白酶- bpti复合物与每分子相当于3到7个180个原子相关联，这显然是由于低pH下羧基的非特异性共价lsO标记。除了埋藏水的180交换外，埋藏NH基团H键与埋藏水的氢同位素交换也被测量。它们的碱催化交换速率常数的顺序为：晶体中暴露于溶剂的NH基团（静态可及性> 0）和氢键主链O，它们的^，与模型化合物相似。它们的交换速率常数对pH值的依赖性表明，与水的直接交换可能显著地促进了它们所观察到的交换速率。
Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108 Received October 7, 1986; Revised Manuscript Received March 19, 1987 abstract: Solvent exchange of, 80-labeled buried water in bovine pancreatic trypsin inhibitor (BPTI), trypsin, and trypsin-BPTI complex is measured by high-precision isotope ratio mass spectrometry. Buried water is labeled by equilibration of the protein in 180-enriched water. Protein samples are then rapidly dialyzed againstwater of normal isotope composition by gel filtration and stored. The exchangeable 180 label elutingwith the protein in 10-300 s is determined by an H20-C02 equilibration technique. Exchange of buried waters with solvent water is complete before 10—15 s in BPTI, trypsin, and BPTI-trypsin, as well as in lysozyme and carboxypeptidase measured as controls. When in-exchange dialysis and storage are carried out at pH> 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 180 atom that exchanges slowly (after 300 s and before several days). This oxygen is probably covalently bound to a specific site in trypsin. When in-exchange dialysis and storage are carried out at pH 1.1, the equivalent of three to seven 180 atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent lsO labeling of carboxyl groups at low pH. In addition to 180 exchange of buried waters, the hydrogen isotope exchange of buried NH groups H bonded to buried waters was also measured. Their base-Catalyzed exchange rate constants are on the order of NH groups that in the crystal are exposed to solvent (static accessibility> 0) and hydrogen-bonded main chain O, and their,^,, is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may sig-nificantly contribute to their observed exchange rate.