Measuring & setting the pH of the Reverse Micelle Water Core

See Marques et al. (2014) J. Phys. Chem. B here for a detailed explanation of this topic.

Important Fact: Reverse micelles are constantly colliding and exchanging their water cores. Estimates by Halle and others place this exchange on the microsecond time scale. Thus, though individual RM particles are "nanoscale" they are averaged.

Difficulty of setting & measuring pH in RMs

How To Overcome This

To measure the effective (average) pH of the reverse micelle water core we have adapted a method previously developed for aqueous solution. We use simple, unlabeled buffer molecules that show distinct proton chemical shift perturbations within a distinct pH range due to their varying pKa's. By using as few as two of these buffer molcules (namely acetate and imidazole) one can determine the ensemble pH of a reverse micelle mixture anywhere within the pH range we generally operate (4 to 10).

Choose a buffer molecule(s) that meet a few desired criteria:

  1. the buffer molecule being used doesn't significantly interact with your desired surfactant molecule (this can be easily determined by dissolving a minimal -well below the CMC- amount of surfactant in an aqueous solution of the buffer molecule to see if there are any significant proton chemical shift perturbations)
  2. the proton chemical shift of the buffer molecule doesn't overlap with the other significant components of the reverse micelle ensemble (water, protein, surfactant, or solvent)
  3. the buffer molcules has a pKa near your desired pH

We have examined four buffer molecules that span the entire pH range from 4-10 which don't interact with any of the current surfactant molecules we use and for the most part show no chemical shift overlap with anything in the RM mixture. This is described in the Marques et al (2014) JPCB paper provided above.

Buffer Molecule pKa (in Water!) pH Indicator Range Chemical Shift Range (ppm)
Imidazole (H2) 7.0 5.5 - 8.0 7.70 - 8.70
Imidazole (H4/5) 7.0 5.5 - 8.0 7.10 - 7.50
Tris + 8.0 6.5 - 10.0 3.50 - 3.75
Formate 3.8 4.0 - 5.5 8.38 - 8.45
Acetate 4.75 4.0 - 6.5 1.90 - 2.05

+ - the chemical shift of tris overlaps throughout the whole pH range with the LDAO/10MAG RM mixture

Of course, you are welcome to try any other buffer molecules you'd like.

So it seems easy, right? Just stick your favorite buffer molecule in your RM mixture at your favorite pH and make sure the chemical shift matches what it would be in aqueous solution, right?.... Wrong!! There are multiple considerations before you can get started...

Consideration # 1: Some Surfactants are Buffers themselves

There are those surfactant mixtures which do not require any consideration of surfactant pH at all. For example, the pH of the CTAB or DTAB in CTAB/hexanol or DTAB/hexanol mixtures does not need to be adjusted prior to making a reverse micelle sample. CTAB and DTAB are not "pH-active," at least in the pH ranges we're working at. However, a number of important surfactant molecules that we use in the lab are pH-sensitive, namely AOT and LDAO.

HSQC spectra of uniformily 15N-labeled ubiquitin in aqueous solution at different pH and in various reverse micelle mixtures are shown below. Aqueous ubiquitin at pH 5 (A) was encapsulated in 10MAG/LDAO reverse micelles without prior pH equilibration of the surfactants (B). Similarly, aqueous ubiquitin at pH 7 (C) was encapsulated in AOT without prior pH equilibration of the surfactant (D). This shows that putting aqueous ubiquitin at pH 5 looks like it's closer to pH 7 when encapsulated into an LDAO/10MAG mixture without prior pH adjustment and vice versa for aqueous ubiquitin at pH 7 into AOT.
pH effect

In fact, no matter what pH is of a protein sample injected into an unadjusted AOT- or LDAO/10MAG reverse micelle mixture, you will get a sample at pH 5 or 7, respectively. What do we do!?

In general, when dissolved in water (at very low concentrations to avoid micelle formation) AOT has a pH of ~5. In order to adjust the pH, dissolve a small amount (~1mg/mL) of AOT in water. Then adjust the surfactant solution to the desired pH, freeze the solution, and lyophilize. After the surfactant is completely dry, redissolve in water and repeat the process until the aqueous solution of AOT is approximately your desired pH. This process needs to repeated anywhere from three to five times depending how far away from pH 5 you want to get. Since the process can be relatively arduous, do this in volumes of 20+ mL to have a stock of pH adjusted AOT for future use. Please note: the appearance of dry AOT above pH 8 is more coarse-grained than its typical pasty appearance.

In general, when dissolved in water (again, at very low concentrations) LDAO has a pH between 7 and 8 depending on the manufacturer and batch. In the majority of the RM mixtures containing the zwitterionic LDAO, it is accompanied by a nonionic surfactant (10MAG) or sometimes a positively charged surfactant (DTAB). Since 10MAG is almost completely insoluble in water, we generally do pH adjustments in 12% ethanol. We usually prepare enough amount of surfactant for two or three RM samples. Dissolve all of the surfactants in some amount (e.g. 1ml/RM sample equivalent) of 12% ethanol. This solution may be a bit cloudy but that is fine. Adjust the sample to the desired pH, evenly alliquot the mixture into separate glass vials (one per RM mixture equivalent), freeze the vials with the black caps sealed shut, twist the cap open just a bit (enough to let air escape but not enough to fall off), and lyophilize. Please note: 12% ethanol has a lower freezing temperature than water so you should leave 1mL surfactant solutions in dry ice/ethanol for at least five minutes. Performing this process once is enough to have surfactants at your desired pH (i.e. - you don't have to repeat the process multiple times like with AOT).