Wooten J B, Cohen J S
Biochemistry. 1979 Sep 18;18(19):4188-91. doi: 10.1021/bi00586a023.
Hen egg white lysozyme has been prepared in which the C epsilon position of the single histidine residue is substituted by a deuterium atom as a nondisturbing stable isotope probe. The deuterium nuclear magnetic resonance (2H NMR) spectrum in H2O shows a broad resonance (500--1000 Hz) due to the histidine deuteron and a sharp signal from residual HOD. The line width of the deuterium signal increases with pH, reflecting the self-association of lysozyme which is known to involve this histidine [shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]. Correlation times calculated from spin-spin relaxation times (T2) derived from the 2H widths indicate that His-15 is restricted in motion and that lysozyme is predominantly dimerized at pH 7.5. Controls carried out with [epsilon-2H]imidazole showed a small pH dependence of the spin-lattice relaxation time (T1), which parallels the 2H chemical shift change upon ionization of the imidazole. Similar results cannot generally be observed by proton nuclear magnetic resonance (1H NMR) because of paramagnetic relaxation due to trace metal ion impurities. The pH dependence of the 2H T1 values indicates a change in the 2H quadrupole coupling constant upon protonation of the imidazole ring.
已制备出一种鸡蛋清溶菌酶，其中单个组氨酸残基的ε位被氘原子取代，作为一种无干扰的稳定同位素探针。在H₂O中的氘核磁共振（²H NMR）谱显示，由于组氨酸氘核产生宽共振（500 - 1000 Hz），以及来自残余HOD的尖锐信号。氘信号的线宽随pH值增加，这反映了溶菌酶的自缔合，已知这种自缔合涉及该组氨酸[Shindo, H., Cohen, J.S., & Rupley, J. A. (1977) Biochemistry 16, 3879]。根据由²H宽度得出的自旋 - 自旋弛豫时间（T₂）计算出的相关时间表明，His - 15的运动受限，并且溶菌酶在pH 7.5时主要形成二聚体。用[ε - ²H]咪唑进行的对照实验表明，自旋 - 晶格弛豫时间（T₁）对pH有较小的依赖性，这与咪唑电离时的²H化学位移变化相似。由于痕量金属离子杂质引起的顺磁弛豫，一般通过质子核磁共振（¹H NMR）无法观察到类似结果。²H T₁值对pH的依赖性表明，咪唑环质子化时²H四极耦合常数发生变化。
