Real time analysis of phosphate transitions from adenosine triphosphate

Abigail H. Casey; Gregory E. Triplett

doi:10.1117/12.2575994

5 March 2021 Real time analysis of phosphate transitions from adenosine triphosphate

Abigail H. Casey, Gregory E. Triplett

Proceedings Volume 11663, Integrated Sensors for Biological and Neural Sensing; 116630Y (2021) https://doi.org/10.1117/12.2575994
Event: SPIE BiOS, 2021, Online Only

Conference Poster

Abstract

Protein phosphorylation is responsible for a broad range of signal transduction roles such as muscular movement and growth, metastatic behavior, mental cognition and metabolic rate. This process follows an enzymatic reaction mechanism, where the enzyme or kinase facilitates the transfer of the phosphate group from adenosine triphosphate (ATP) to the substrate protein. To better understand signal transduction, we designed and fabricated microfluidic devices, integrated them with our confocal Raman spectrometer, and monitored real time signal changes from ATP. In this study, we loaded the microfluidic device with Casein in MgCl2 solution, and fed into the chamber a solution of PKA/cAMP/ATP in MgCl2 using Tygon tubing attached to 1 ml syringes with 30g needles attached. At time, t=0, the reactor is preloaded with the Casein MgCl2 solution, and ATP is absent from the chamber. The PKA/cAMP/ATP in MgCl2 solution was allowed to flow at a rate of 0.005 mg/min until the POx peaks between 1000-1600 /cm appeared in the Raman spectra. At this point, the fluid flow was stopped and the reaction progressed while Raman spectra was collected over the wavenumber range 1000 – 1600 /cm to monitor phosphate bonds. Results align with calculations from mass transfer analysis. The isoelectric point of phosphorylated Casein, dephosphorylated Casein, and PKA as reported from literature [1] were observed from the reaction performed in the microfluidic reactor. This confirmed real time observation of phosphorylation of Casein through catalysis of PKA and phosphate donation from ATP.

Conference Presentation

Citation Download Citation

Abigail H. Casey and Gregory E. Triplett "Real time analysis of phosphate transitions from adenosine triphosphate", Proc. SPIE 11663, Integrated Sensors for Biological and Neural Sensing, 116630Y (5 March 2021); https://doi.org/10.1117/12.2575994

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