微量热法研究10-23脱氧核酶切割反应的热动力学

摘要

选用乙型肝炎病毒（HBV）前区C/C区2031位点的序列作为底物RNA片段,并合成相应的10-23脱氧核酶,然后用微量热法研究10-23脱氧核酶的热动力学。在近生理条件下,通过微量热法测定10-23脱氧核酶切割反应的热动力学参数。根据热流曲线得出底物RNA不同浓度的反应初速率v0,再作出v-[S]曲线,通过Lineweaver-Burk作图得出10-23脱氧核酶切割反应的最大速率vmax为2.5×10-9mol/（L.s）、米氏常数Km为0.045nmol/L、催化常数Kcat为0.107min-1。%The sequence of 2031 site in anterior HBV C/C area was selected as the substrate RNA fragment to synthesize the corresponding 10-23 DNAzyme.Under mimic physiological conditions,the thermodynamics of 10-23 DNAzyme was evaluated by microcalorimic method.Heat-flow curves were plotted to obtain the initial reaction rate（v0） at various concentrations of the substrate RNA.The maximum rate（vmax）,Michaelis-menten constant（Km） and catalytic constant（Kcat） of 10-23 DNAzyme were obtained from the established Lineweaver-Burk plot of 1/v0 against 1/[S] to be 2.5 × 10-9 mol/（L·s）,0.045 nmol/L and 0.107 min-1,respectively.