Aoshima H, Naito A, Hatano H
Int J Pept Protein Res. 1976;8(2):131-9. doi: 10.1111/j.1399-3011.1976.tb02489.x.
Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.
胃蛋白酶用N-(1-氧代-2,2,6,6-四甲基-4-哌啶基)溴乙酰胺进行自旋标记，标记位点可能在活性部位，即一个反应性天冬氨酸的β-羧基上。自旋标记胃蛋白酶的光谱表明自旋探针被强烈固定（相关时间大于或等于10^(-8)秒）。自旋标记胃蛋白酶在不同温度下进行热变性，并在不同时间采集电子顺磁共振（e.p.r.）光谱。根据不同温度下的e.p.r.光谱估算的变性速率表明，在pH 3.5时自旋标记胃蛋白酶的热变性焓和熵分别为48.0±4.9千卡/摩尔和214.7±14.5熵单位。加入浓氢氧化钠或pH 6.0的1 M醋酸盐缓冲液会使自旋标记胃蛋白酶的e.p.r.光谱变尖锐，这表明自旋探针通过碱性变性变得可移动。加入尿素会导致蛋白质展开，且随着尿素浓度的增加而增加，尽管在e.p.r.光谱中仅观察到构象变化的轻微转变。
