Biochemistry I - Histidine Titration By NMR

Using NMR to Determine pKa

This page simulates the measurement of pKa values using NMR spectroscopy. The absorption frequencies, or chemical shifts, of resonances in the histidine sidechain are sensitive to the protonation state of the histidine, showing one shift for the deprotonated state (δH) and another for the protonated state (δHA). When there is a mixture of both states, the exchange between protonated and deprotonated is fast such that the observed shift is just the weighted average of the two:
δobs=fHAδHA+fAδA.
The fraction protonated can be obtained at any pH using the following equation:
fHA=(δobs-δA)/(δHA-δA).
To obtain the pKa you should:
  1. Enter a pH, from pH=8 to 2, in steps of 0.5, value and obtain the chemical shift (δ) at that pH.
  2. Plot the chemical shift versus pH, to obtain the chemical shifts for the deprotonated and protonated states.
  3. Convert your chemical shifts to fraction protonated, and plot fraction protonated versus pH to estimate the pKa.
1. Enter a value for pH in this box:
pH =
2. For the above value of pH, the observed shift is:
δobs =
    (Each measured frequency has a small "experimental error" added to it.)

10.09.17