Does low fluorophore pKa = low pH sensitivity?

Probes & Prep
1

Hi,

Simple question : does low fluorophore pKa = low pH sensitivity ?
Does for example a pKa of 4 mean that the fluorphore will be stable above pH 4 ?

@talley

Thanks a lot

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pKa is the pH at which fluorescence intensity drops to 50% of it’s maximal value… so yeah, a low pKa suggests the fluorophore is more acid-tolerant.

Note though, that’s a bit of an oversimplification: it’s not a binary “on/off” above and below the pKa value. It’s a sigmoidal change, and the slope of that fluorescence-vs-pH curve (the Hill coefficient) also determines pH sensitivity. @joachimgoedhart may have more to say on the topic… he was on a recent paper that discussed this nicely… here’s an excerpt from that paper:

… pH quenching curves can be described by a Hill fit that gives a pKa value and a Hill coefficient. The pH robustness can easily be misinterpreted by only looking at pKa values. However, FPs with a low pKa and a low Hill coefficient do not show a pH range in which the fluorescence remains constant and are still pH-sensitive. On the other hand, FPs with a low pKa and a high Hill coefficient are pH-insensitive as these FPs have a plateau at pH values above the pKa. Thus, pH-insensitive FPs are better identified based on these two parameters combined. In addition, pH sensitivity has always been described in vitro , neglecting the effect of cytolosic components on the pH quenching. How representative in vitro pH sensitivity is for the in vivo performance is unknown.
Botman et al (2019). Scientific Reports

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For fluorescent proteins it is usually true that a low pKa means high tolerance to acid.
Note that FPs can have multiple pKa’s and that (as mentioned by Talley) and that the Hill coefficient may be relevant for the response to pH. Therefore, it’s best to check the fluorescence in response to a range of pH values (these data are often unavailable).

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