Hutcheon G A, Parker M C, Moore B D
Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, G1 1XL, UK.
Biotechnol Bioeng. 2000 Nov 5;70(3):262-9.
A novel deuterium ((2)H) NMR technique as developed for measuring the total number of deuterons exchanged by lyophilised protein samples following hydrogen-deuterium (H-D) exchange. Using this methodology differences in the H-D exchange behaviour of the proteolytic enzyme subtilisin Carlsberg hydrated either in air or an organic solvent were probed as a function of hydration. At low thermodynamic water activity (a(w)), the degree of H-D exchange increased rapidly with hydration (from anhydrous to a(w) 0.22). At a(w) 0.22, subtilisin powders hydrated in air were found to have reached an H-D exchange level comparable to that found upon aqueous dissolution and in agreement with previous studies using lysozyme. Lyophilised subtilisin hydrated in either dichloromethane (DCM) or diisopropyl ether (DIPE) showed a pattern of exchange (vs. a(w)) comparable to that found for powders hydrated in air. However, subtilisin hydrated in n-hexane showed a significant reduction in H-D exchange at all a(w) studied. Control experiments demonstrated that the reduction in H-D exchange observed for subtilisin in n-hexane was not a kinetic effect. This lower level of exchange in n-hexane implies that hydrated subtilisin Carlsberg has a lower conformational motility and more rigid protein matrix.
一种新的氘（(2)H）核磁共振技术，用于测量冻干蛋白质样品在氢-氘（H-D）交换后交换的氘核总数。使用这种方法，研究了在空气或有机溶剂中水合的蛋白水解酶枯草杆菌蛋白酶卡尔伯格变体的H-D交换行为随水合作用的差异。在低热力学水活度（a(w)）下，H-D交换程度随水合作用迅速增加（从无水状态到a(w) 0.22）。在a(w) 0.22时，发现空气中水合的枯草杆菌蛋白酶粉末达到了与水溶液溶解时相当的H-D交换水平，这与之前使用溶菌酶的研究结果一致。在二氯甲烷（DCM）或二异丙醚（DIPE）中水合的冻干枯草杆菌蛋白酶显示出与空气中水合粉末相当的交换模式（相对于a(w)）。然而，在正己烷中水合的枯草杆菌蛋白酶在所有研究的a(w)下H-D交换都显著减少。对照实验表明，在正己烷中观察到的枯草杆菌蛋白酶H-D交换减少不是动力学效应。正己烷中这种较低的交换水平意味着水合的枯草杆菌蛋白酶卡尔伯格变体具有较低的构象运动性和更刚性的蛋白质基质。
