ABSTRACT
OBJECTIVE:To establish a simple and rapid ion-pair-RP-HPLC method for the determination of intracellular concentration of ara-CTP.METHODS:Mononuclearcells were separated from the whole blood,centrifugated and injected for determination on Symmetry C18 column with the mobile phase consisted of 0.1 mol?L-1 KDP buffer solution and 0.01 mol?L-1 tetrabutylammonium hydrogen sulfate(pH=2.7)at a flow rate of 1.0 mL?min-1 with the UV wavelength set at 278 nm.RESULTS:The linear range of ara-CTP was 0.35-9.12 ?g?mL-1(r=0.998 6)with its detection limit at 0.35 ?g?mL-1.The recoveries of ara-CTP at low,middle and high concentrations,and the intra-day and inter-day RSDs were all up to the standards.CONCLUSION:The established method is simple,rapid,stable,reproducible and applicable for the monitoring of intracellular concentration of ara-CTP.
ABSTRACT
The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.