以DCAP-P为底物的酸性磷酸酶连续监测法的建立

A continuous-monitoring procedure of acid phosphatase activity with 2,6-dichloro-4-acetylphenylphosohpric acid as substrate

目的 :建立以2,6 -二氯 -4 -乙酰基苯基磷酸盐(DCAP -P)为底物的一种新的酸性磷酸酶(ACP)的测定方法。方法 :在BTS -310半自动生化分析仪上研究连续监测法 ,建立实验参数。结果 :本法测定DCAP吸收峰为326nm ;ACP的Km=0.139mmol/L ;340nm时DCAP的摩尔消光系数为14440L/(mol×cm) ;缓冲液浓度0.1mol/L ,最适pH5.4 ,底物浓度2.0mmol/L ,延滞期60s,线性反应期15min ;线性范围为0～1188u/L ;血红蛋白(Hb)<115.62mg/L对测定结果无影响 ;黄疸对测定结果无影响 ;本法总ACP与PAP(前列腺酸磷酶 )的参考值范围分别为12.58～19.42u/L、2.52～9.04u/L。结论 :此方法重复性好 ,线性反应期长 ,线性范围广 ,干扰因素少 ,适用于自动生化分析。

Objective: To establish a new determination method for the measuring of acid phosphatase activity (ACP) with 2,6-dichloro-4 acetylphenylphosphoric acid as substrate. Methods: With the help of a semiautomatic analyzer, researched a continuous-monitoring procedure and set up experimental parameters. Results: When useing this assay, the wave-length of DCAP's absorption was 326nm, and the Km of ACP was 0.139mmol/L. The molecular extinction coefficient of DCAP at 340nm was 14440L/(mol×cm) and the concentration of citrate buffer was 0.1 mol/L. During the analytic process, we found that the optimum pH of enzyme was 5.4 and linear range was 0～1188 u/L. The time of linear reaction was 900 seconds. Serum total ACP and PAP (Prostatic acid phosphatase )were 12.58～19.42u/L and 2.52～9.04 u/L respectively,based on results from 30 health adults. Conclusion: The method was practical in its repetition and convenience , saved time and wasn't liable to be affected by bilirubin in serum.It is especially suited to the use of automatic analyzers.