Dimicoli J L, Nakache M, Lhoste J M
Biochim Biophys Acta. 1979 Dec 7;571(2):294-304. doi: 10.1016/0005-2744(79)90099-8.
Porcine pancreatic elastase (EC 3.4.21.11) has been immobilized on polyacrylamide beads using glutaraldehyde ad bridging reagent without important loss of catalytic activity. A nitroxide spin label, 1-oxyl-2,2,5,5-tetramethyl-4-piperidinyl-ethylphosphonofluoridate, reacting covalently with the serine-195 residue of the active centre of free elastase was used as a conformational and dynamical electron spin resonance probe. This signal is quenched by (Cu2+) which bind specifically at the active site at a distance of 7 A from the nitroxide group. This distance is not significantly affected by the fixation on the solid support. The electron spin resonance lineshape analysis indicates some mobility of the spin label with respect to the native protein. This restricted motion, which is pH dependent, is not noticeably modified by the immobilization of the enzyme. This immobilization has therefore induced no large conformational change of the protein in the vicinity of the active centre. Thermal denaturation of elastase in homogeneous solution is irreversible. Immobilization on the polyacrylamide beads results in 70% reversibility, but the temperature of denaturation is not modified.
猪胰弹性蛋白酶（EC 3.4.21.11）已使用戊二醛作为桥联试剂固定在聚丙烯酰胺珠上，催化活性没有显著损失。一种氮氧化物自旋标记物，1-氧基-2,2,5,5-四甲基-4-哌啶基-乙基膦酰氟化物，与游离弹性蛋白酶活性中心的丝氨酸-195残基共价反应，用作构象和动态电子自旋共振探针。该信号被（Cu2+）淬灭，（Cu2+）特异性结合在距氮氧化物基团7埃的活性位点。该距离不受固定在固体支持物上的显著影响。电子自旋共振线形分析表明自旋标记物相对于天然蛋白质有一定的流动性。这种受限制的运动依赖于pH值，酶的固定化对其没有明显改变。因此，这种固定化在活性中心附近没有引起蛋白质的大的构象变化。弹性蛋白酶在均相溶液中的热变性是不可逆的。固定在聚丙烯酰胺珠上导致70%的可逆性，但变性温度没有改变。
