| Paraoxonase(PON) is an important hydrolase. The activity of enzyme is rel ated to many human diseases, which is one of biomarkers, such as tumor, cardio vascular, liver etc.Therefore, the detection of arylesterase activity and study of P ON1 has a clinical significance for the early diagnosis. Acridinium estersare che miluminescent compounds, it can be used as a substrate of PON1 to measure th e activity of PON1. Therefore, four kinds of novel acridinium esters were synthe sized, and the luminescent properties as a PON1 substrate were investigated. Bas ed on one of the prepared acridinium ester, serum PON1 activators from traditio nal Chinese medicines were evaluated.The 9-(4-chlorophenoxycarbonyl)-10-methylacridinium triflate(Compound 1) was modified, and four acridinium esters were synthesized, which were 9-(4-tertbutylphenoxycarbonyl)-10-methylacridiniumtriflate(Compound 2), 9-(4-phenyloxyca rbonyl)-10-methylacridinium triflate(Compound3), 9-(4-methylphenoxycarbonyl)-10-methylacridiniumtriflate(Compound 4), and 9-(2,4-dimethylphenoxycarbonyl)-10-methylacridinium triflate(Compound 5) respectively. The chemiluminescence react ion of compound 5 was completed in 4 seconds, while the reactions for the othe r four compounds were completed in 0.8 seconds. The chemiluminescence efficie ncy of 5 acridinium is in the order as follows: 3>5>4>2>1. This result showed that the unsubstituted acridiniumester had the highest CL efficiency and a para substituent resulted in slight decrease in CL efficiency.Self-hydrolysis of five acridinium esters was evaluated. The results showed t hat the self hydrolysis of the five acridinium esters in Tris-HCl buffer(p H 7.5) at 25 °C was insignificant within 22 min. It means that all the five acridinium e sters in the buffer were stable enough for PON1 activity assay. The stock solutio ns of the acridinium esters in acetonitrile(1.25×10-5 mol/ L) were kept at a refr igerator(4 °C) for 12 weeks before dilution to ready-to-usesolutions(4.02×10-11 mol/ L). The stability of the acridinium estersranked in the following order: 5>2>4 >3>1.The rh PON1-catalyzed hydrolysis and serum-catalyzed hydrolysis of the acrid inium esters were determined. These kinetic constant values were in the same or der as that for the rh PON1-catalyzed hydrolysis: 1>3>4>2>5. As expected, all the five acridinium esters can be hydrolyzed by rh PON1(pure enzyme) and serum,respectively. Among them compound 5 showed the best stability. Compound 5 have two methyls in its phenol ring, and this increased the steric hindrance of c ompounds, thereby enhanced the stability of compound.Esterase D, lipase, and acetylcholinesterase are important serum carboxylester ases. So, their abilities for hydrolysis of the acridinium esters were investigated, respectively. The results showed that acetylcholinesterase did not hydrolyze the n ew acridinium esters. When the concentrations of esterase D and of the lipase w ere more than 300 times as of rh PON1, their catalytic abilities for hydrolyses of acridinium esters were in a similar level. It is wel acknowledged both Na+ and Ca2+ are activators of PON1. When adding the agonist of PON1, the specificity and sensitivity of substrate can be improved. As the inhibitors of PON1, EDTA can suppress the specific activity of PON1 in serum, among them the specificity of compound 5 was the best.The effects six traditional Chinese medicine on the activity of serum PON1 were measured. Results show that ginger, ginseng and Sanqi have activating effe cts on serum PON1. Due to the nature of luminescence, extracts of Ligusticum chuanxiong Hort has great interference on the measurement, and it in low conce ntrationhas no activating effect on serum PON1. However extracts of Ploydatin c ould not be screened by chemiluminescence method, because of it has componen ts which can hydrolyze acridinium ester, and Salvia miltiorrhiza almost has no ef fect on serum PON1. |
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