In sample pH measurement by ³¹P phosphate NMR: application to measure the intrinsic GTPase activity of Rab1a

A quick, noninvasive method based on ³¹P NMR spectroscopy to measure the pH of samples containing phosphate buffer is presented. By taking advantage of all four phosphate species populated at acidic, mildly acidic, mildly alkaline and alkaline pH, the method is applicable over a wide range of pH values, from 1 to 13. This tool is most precise at pH values near the pKa values, namely from 5.8 to 8.0 and 10.6 to 13.0. To facilitate its use, a web application is presented that calculates the pH value directly from phosphate ³¹P chemical shift (ppm) when sodium or potassium is the cation: ( https://rmni.iqf.csic.es/software/31phnmr/ ). In addition, the potential of this approach to monitor reactions which generate or consume H⁺ is illustrated by following the hydrolysis of GTP catalyzed by Ras-like protein in brain 1a (Rab1a), an essential protein linked to Parkinson's disease and tuberculosis. The intrinsic GTPase rate of Rab1a is found to be 3.3 ± 0.8·10^- 3 min at 37 °C, which places Rab1a amoung the Rabs with slow GTPase rates and the longer lived "active" GTP-bound states.