Schinkel J E, Downer N W, Rupley J A
Biochemistry. 1985 Jan 15;24(2):352-66. doi: 10.1021/bi00323a018.
The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison with the powder measurements are in good agreement with published data. Rank order was proven the same for all pHs by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pHs and was complex in that the rate increased, but not monotonically, with increased ionic strength.
通过在25℃下，从溶液中的蛋白质以及不同水合水平的粉末样品中进行氚的外交换，测定了溶菌酶不稳定氢的交换速率，pH值分别为2、3、5、7和10。对于所有pH值，粉末样品的交换对水合水平的依赖性是相同的。随着水合作用的增加，交换速率急剧增加，直到达到一个恒定的交换速率，该速率在水含量高于0.15 g H₂O/g蛋白质（120 mol H₂O/mol蛋白质）时保持不变。这种水合水平对应于蛋白质表面被单层水覆盖不到一半的情况。对于水含量更高的蛋白质粉末，未观察到额外的氢交换。结合其他溶菌酶水合数据[Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18 - 22]考虑，这一观察结果表明蛋白质内部动力学与表面性质没有强烈耦合。使用粉末样品可通过调节水蒸气压来控制水活性。交换速率对水活性的依赖性约为四级。该级数与pH无关，并且在剩余未交换氢的量为114至8 mol/溶菌酶mol时保持恒定。这些结果表明，所有主链酰胺的蛋白质氢交换速率决定步骤相似，并且涉及的水分子很少。粉末样品通过等压平衡进行水合，水合半衰期约为1小时，或者通过添加溶剂快速达到最终水合。通过等压平衡缓慢水合的样品比通过添加溶剂快速水合至相同水含量的样品表现出更多的交换。这种差异可以用盐和pH对几乎干燥的蛋白质的影响来解释。预计在等压平衡过程中，这些影响会更强烈。为与粉末测量进行比较而进行的溶液氢交换测量与已发表的数据吻合良好。通过溶液pH跃变实验证明，所有pH值下的排序相同。在pH 2和pH 5下，对添加盐浓度高达1.0 M的蛋白质溶液，研究了离子强度对氢交换的影响。两种pH下离子强度的影响相似，且很复杂，即随着离子强度的增加，速率增加，但不是单调增加。
