邻苯二甲酸酐化学修饰对辣根过氧化物酶催化活性的影响

通过采用邻苯二甲酸酐(PA)对辣根过氧化物酶(HRP)的蛋白链进行修饰, 研究了PA化学修饰对HRP的稳定性、催化活性、活性中心结构、酶对底物的亲合性和专一性等催化性质的影响. 结果显示: 酸性条件下(pH=3), 4小时后PA-HRP的催化活性比天然HRP提高了7.5%;碱性条件下(pH=10), 4小时后PA-HRP的催化活性比天然HRP提高了27%. PA-HRP的K_m值为8.16 (mmol/L), 小于天然HRP的K_m值12.99 (mmol/L), 而PA-HRP的k_(cat)/K_m值为7.86(10~4(L/ mmol· min)大于天然HRP的k_(cat)/K_m的6.70(10~4(L/ mmol· min). 这些催化活性和动力学数据表明了PA-HRP与天然HRP相比其稳定性、酶对底物的亲和性和专一性得到了提高. 紫外-可见光谱、拉曼光谱数据显示: 修饰剂PA 改变了天然HRP血红素周围的微环境, 对酶蛋白分子的活性中心结构并没有影响. 差示光谱显示PA修饰剂可以提高酶对底物的亲和力.

Effects of Phthalic Anhydride Modification on Horseradish Peroxidase Properties

horseradish peroxidase (HRP) was modified by phthalic anhydride( PA). The stability and activity of native and modified HRP in water was investigated. The results showed that the activities of PA-HRP have been in-creased by 7.5% and 27% more than native HRP at pH =3 and 10, respectively, after 4 hours. In order to inves-tigate the molecular mechanism of the stability, native and PA-HRP were studied using kinetics methodology. The K_m of PA-HRP (K_m =8.16 nunol/L) was lower than native HRP (K_m = 12.99 mmol/L) and that K_(cat)/K_m of PA-HRP( K_(cat)/K_m=7.86×10~4L/ mmol min ) was greater than native HRP ( K_(cat)/K_m = 6.70×10~4 L/ mmol min ). These results indicate that the chemical modification of HRP increased the affinity and specificity. Absorbance spectra and Resonance Raman spectra indicated that the modification changed the environment of heme. The different spectra of native or PA-modified HRP with phenol in phosphate buffer, PA-HRP showed a stronger affinity than native HRP.