Lee C S, Ru M T, Haake M, Dordick J S, Reimer J A, Clark D S
Department of Chemical Engineering, University of California, Berkeley, California, 94720, USA.
Biotechnol Bioeng. 1998 Mar 20;57(6):686-93.
Multinuclear NMR spectroscopy has been used to study water bound to subtilisin Carlsberg suspended in tetrahydrofuran (THF), with the water itself employed as a probe of the hydration layer's physicochemical and dynamic characteristics. The presence of the enzyme did not affect the intensity, chemical shift or linewidth of water (up to 8% v/v) added to THF, as measured by 17O- and 2H-NMR. This finding suggests that hydration of subtilisin can be described by a three-state model that includes tightly bound, loosely bound, and free water. Solid-state 2H-NMR spectra of enzyme-bound D2O support the existence of a non-exchanging population of tightly bound water. An important implication is that the loosely-bound water is the same as free water from an NMR viewpoint. This loosely bound water must also be the water responsible for the large increase in catalytic activity observed in previous hydration studies.
多核核磁共振光谱已被用于研究悬浮在四氢呋喃（THF）中的枯草杆菌蛋白酶卡尔伯格变体所结合的水，水本身被用作水合层物理化学和动态特性的探针。通过17O和2H核磁共振测量发现，添加到THF中的水（体积分数高达8%）的强度、化学位移或线宽不受酶存在的影响。这一发现表明，枯草杆菌蛋白酶的水合作用可以用一个三态模型来描述，该模型包括紧密结合水、松散结合水和自由水。与酶结合的D2O的固态2H核磁共振光谱支持存在一个不发生交换的紧密结合水群体。一个重要的推论是，从核磁共振的角度来看，松散结合水与自由水是相同的。这种松散结合水也必定是先前水合研究中观察到的催化活性大幅增加所涉及的水。
