血卟啉衍生物光敏引起NAD(P)H氧化作用研究

血卟啉衍生物(HPD)光敏引起NAD(P)H氧化为中介的luminol化学发光(CL)与多种因素有关,如pH、激光功率密度、照光时间以及luminol、HPD以及底物NAD(P)H浓度等的改变都可引起CL的变化.当NAD(P)H浓度远远大于HPD浓度时,CL强度与HPD浓度成正比关系,这表明化学发光测定可以作为检测HPD光敏反应的指标,而且HPD敏化的NAD(P)H光氧化过程中化学发光的产生是与活性氧物质(ROS)有关的.选择性应用O_2、H_2O_2、OH、~1O_2的专一性清除剂研究ROS及~1O_2在HPD敏化的NAD(P)H氧化过程中的作用,其主要结果如下:(1)NAD(P)H光氧化为中介的lumlnol化学发光在水溶液中受到铜锌超氧化物岐化酶(CuZn-SOD)、过氧化氢酶(CAT)和甲酸钠(F)的抑制,在D_2O中受到His和Met的抑制.1μg/ml的CuZn-SOD抑制可达84%,但再增加酶量,抑制程度不再增加;CAT的抑制作用也出现类似情况;F的抑制作用较弱,在14—42mM浓度范围内,抑制程度不超过60%.(2)CuZn-SOD对化学发光的抑制作用随照光时间延长而持续下降,而CAT和F的抑制作用却随照光时间延长而有上升的趋势.His和Met在D_2O中的作用比较复杂.(3)观察伴随NAD(P)H光氧化失活时OD_(340)值的变化,发现ROS清除剂在H_2O中对失活有保护作用,而~1O_2猝灭剂His和Met在D_2O中对失活有保护作用.上述清除剂和猝灭剂

STUDIES ON THE NAD(P)H PHOTOOXIDATION SENSITIZED BY HEMATOPORPHYRIN DERIVATIVE

The luminol chemiluminesence in the photosensitized oxidation of NAD(P)H was found to be dependent upon many factors such as pH,laser density, irradiation time,concentrations of luminol, HPD and NAD(P)H.When the concentration of NAD(P)H was far more excessive as compared with that of hematoporphyrin derivative (HPD) , the intensity of CL was positively proportional to the concentration of HPD, which implied that the CL measurement can be used as an indicator for the detection of photosensitized oxidation and that the CL may be generated by the photooxidation of NAD(P)H in the presence of reactive oxygen species(ROS). By selectively using scavengers of O2,H2O2, OH and quenchors of 1O2 the actions of ROS and 1O2 involved in the photoensitized oxidation of NAD(P)H by HPD were obviously identified. The experimental results were briefly summerized as follows:(1) The CL emitted from the photooxidation of NAD(P)H was depressed by copper-zinc superoxide dismutase(CuZn-SOD) ,catalase(CAT)and sodium formate (F) in aqueous solution aa well as by histionine (Met) and methiorin(Met) in deuterium oxide. The inhibition percentage of CL by the addition of 1?g/ml Cu Zn-SOD was 84%,but that by adding the enzyme in excess did not increase any more.The inhibition style by CAT was the same as CuZn-SOD,and the inhibition by F was relatively weak,in the range of 14-42 mM F, the inhibition did not exceed 60%.(2) The inhibition of CL by CuZn-SOD was decreased along with the duration time of irradiation of laser,wherean that by CAT or F was increased in the same condition and the effects of His and Met were more complicated.(3) The inactictivation of NAD(P)H,indicated by the changes of OD340,was prevented by ROS scavengers in aqueous as well as by His and Met in deuterium oxide soultion.It has been demonstrated that a parallel ralationship between the inhibition of CL and the prevention of inactivation of NAD(P) H by ROS scavengers or 1O2 quenchors existed.