Biphasically Modulating The Activity Of Carboxypeptidase G2 With Ultrasound

BackgroundThe"CPG2-prodrug"therapy is limited by toxicity:the leak of active drugs into circulation leads to severe adverse events.Therefore,the activity of CPG2 should be under control to modulate the release of active drugs.However,no such a strategy is available.ObjectiveTo determine whether ultrasound can be used to modulate the activity of CPG2.MethodsCPG2 was subjected to insonation.The activity was determined,and then K_m,V_maxax and reversibility were explored.Monomer was analyzed with SDS-PAGE,PR-HPLC and MS,and dimer was assayed with SEC-HPLC.The secondary structure was analyzed with CD,and the conformational transformationwasdeterminedwithsynchronousfluorescence spectroscopy.The ultrasonic mechanisms were explored from the perspective of heat and cavitation.Results1.Under 10 W/cm²,the activity was improved when the insonation time was?400 s,with a peak value at L1?200 s?;insonation decreased the activity when the insonation time was?700 s.The activity was decreased under 20 W/cm² exposure;the decrease followed zero-order kinetics when the insonation time was?1000s,and displayed first-order kinetics when the insonation time was?1200 s.2.An increase or a slight decrease of activity attributable to 10 W/cm²was reversible,but the activity decrease due to 20 W/cm² was irreversible.3.L1 improved the CPG2 activity via increasing the specific activity.L2 or L3?20 W/cm² for 1200 or 3000 s?decreased the CPG2 activity via disassembling the dimer,degrading the monomer,inducing glycosylation,transforming conformation and decreasing the specific activity.4.Insonation induced a higher yield of free radicals in sonicated solution,but without temperature rise.Conclusion:UltrasoundcanbiphasicallymodulatetheCPG2activity.Ultrasonically enzymatic modulation was mediated by cavitation.