Tüchsen E, Hayes J M, Ramaprasad S, Copie V, Woodward C
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1987 Aug 11;26(16):5163-72. doi: 10.1021/bi00390a040.
Solvent exchange of 18O-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 18O-enriched water. Protein samples are then rapidly dialyzed against water of normal isotope composition by gel filtration and stored. The exchangeable 18O label eluting with the protein in 10-300 s is determined by an H2O-CO2 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 greater than or equal to 2.5, trypsin-BPTI and trypsin, but not free BPTI, have the equivalent of one 18O 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 18O atoms per molecule is associated with the trypsin-BPTI complex, apparently due to nonspecific covalent 18O labeling of carboxyl groups at low pH. In addition to 18O 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 greater than 0) and hydrogen-bonded main chain O, and their pH min is similar to that for model compounds. The pH dependence of their exchange rate constants suggests that direct exchange with water may significantly contribute to their observed exchange rate.
通过高精度同位素比质谱法测量牛胰蛋白酶抑制剂（BPTI）、胰蛋白酶和胰蛋白酶 - BPTI复合物中18O标记的埋藏水的溶剂交换。通过在富含18O的水中使蛋白质平衡来标记埋藏水。然后通过凝胶过滤将蛋白质样品快速透析到具有正常同位素组成的水中并储存。通过H2O - CO2平衡技术测定在10 - 300秒内与蛋白质一起洗脱的可交换18O标记。在BPTI、胰蛋白酶和BPTI - 胰蛋白酶中，以及在作为对照测量的溶菌酶和羧肽酶中，埋藏水与溶剂水的交换在10 - 15秒之前完成。当在pH大于或等于2.5的条件下进行交换后透析和储存时，胰蛋白酶 - BPTI和胰蛋白酶，但游离的BPTI没有，有一个18O原子等效物缓慢交换（在300秒后且在数天之前）。这个氧可能共价结合到胰蛋白酶中的一个特定位点。当在pH 1.1的条件下进行交换后透析和储存时，每个分子有三到七个18O原子等效物与胰蛋白酶 - BPTI复合物相关联，显然是由于在低pH下羧基的非特异性共价18O标记。除了埋藏水的18O交换外，还测量了与埋藏水形成氢键的埋藏NH基团的氢同位素交换。它们的碱催化交换速率常数与晶体中暴露于溶剂（静态可及性大于0）并与主链O形成氢键的NH基团的交换速率常数相当，并且它们的pH最小值与模型化合物的相似。它们的交换速率常数对pH的依赖性表明与水的直接交换可能对观察到的交换速率有显著贡献。
