A Chilling Dilemma

Back when I was at Chapel Hill, a fellow student asked my help in figuring out a strange phenomenon he observed with one of his proteins. It seemed that the protein was perfectly soluble at room temperature, but precipitated when it was cooled. Because proteins typically denature at higher, rather than lower, temperatures, he was mystified. Mostly out of luck, the first question I asked him proved to be the right one: "What buffer are you using?" As it happened, he was using Tris buffer, a very common buffer in the biosciences, but also one that changes pH significantly upon cooling. It was not temperature that was denaturing his protein, but rather a change in the alkalinity of the solution it was dissolved in. The change of buffer pH with temperature is one of the most subtle and least-discussed issues in biochemistry, but as in my friend's case, can often produce confusing effects that significantly complicate research. An article by scientists from the University of Illinois online now at Chemical Communications (click link for open access), however, aims to resolve this problem by producing a temperature-independent pH (TIP) buffer for biological applications.

The approach they employed was actually very simple: rather than search the ends of the earth for a buffering agent that would maintain its pH across a huge temperature spread, they just found two that had opposite reactions to chilling and mixed them together. The buffer system they ended up with contained 60% HEPES and 40% potassium phosphate and experienced a pH change of less than 0.07 ± 0.1 over a temperature range from 25 °C to -180 °C. Readers who have encountered this subject before will recognize that HEPES and K₂HPO₄ are already fairly pH-stable over most of the laboratory temperature range, but as the paper's first figure shows, the TIP maintains these benefits down to very low temperature. A colorimetric assay was used to track the pH to very low temperatures.

So, problem solved, right? Well, not exactly. This finding is very nice as far as it goes, and has some promise for labs that do a lot of work that involves freezing proteins—structural biology labs come to mind. But while it would be nice to be able to select buffers on the basis of pH characteristics alone, that's rarely feasible. Not all buffers work with all proteins. In some cases, this can be rationalized: proteins that bind ATP or nucleic acids often bind or interact with phosphate buffers, resulting in poor solubility or aberrant structural dynamics. In other cases the observations are harder to understand—it seems that some proteins just "don't like" some buffers. So long as you're dealing with very dilute proteins, this is not usually an issue, though the biochemistry is sometimes deranged as a result. However, when high protein concentrations are employed—again, this is characteristic of structural biology—incompatible buffer-protein combinations tend to result in crashed protein.

The good news is that, with the exception of proteins that normally interact with phosphate moieties, both of these buffers are fairly well tolerated by a wide spectrum of systems. So long as a nearly-neutral pH is desired, this buffer combination should be useful. Researchers wishing to safely put pH-sensitive small molecules into long-term cold storage are also likely to find this buffer a boon. Moreover, because the buffer components are common and inexpensive almost any lab will be able to use this approach.

Sieracki, N. A., Hwang, H-J., Lee, M.K., Garner, D.K. and Lu, Y. "A temperature independent pH (TIP) buffer for biomedical biophysical applications at low temperatures." Chem. Commun. 2008 DOI 10.1039/b714446f