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Scientists have known since the 1930s that the pH of chemical buffers
that are used to maintain the pH of lab samples can change as those
samples are cooled, with some buffers raising and others lowering pH
in the cooling process.
Freezing is a standard method for extending the shelf life of
biological specimens and pharmaceuticals, and biological samples are
routinely cooled to slow chemical reactions in some experiments. Even
tiny changes in the acidity or alkalinity of a sample can influence
its properties, Lu said.
�We like to freeze proteins, nucleic acids, pharmaceutical drugs and
other biomolecules to keep them a long time and to study them more
readily under very low temperatures using different spectroscopic
techniques and X-ray crystallography,� Lu said. �But when the pH
changes at low temperature, the sample integrity can change.�
Graduate student Nathan Sieracki demonstrated this by repeatedly
freezing and thawing oxacillin, a penicillin analog used to treat
infections.
�After one freeze-thaw 50 percent of the drug was dead in several of
the buffers investigated,� Sieracki said.
Sieracki was able to demonstrate that the loss of activity was due to
changes in pH and not a result of the temperature changes.
To find a buffer that would maintain a stable pH at varying
temperatures, Sieracki first evaluated the behavior of several
commonly used buffers over a range of temperatures. He saw that some
buffers became more alkaline at lower temperatures while others grew
more acidic.
These observations led to an obvious methodology: �Why don�t we just
mix them together?� Sieracki said.
Little by little, he varied the proportions of the combined buffers
until he found a formula that exhibited minimal pH changes at a
variety of temperatures. Instead of registering changes of 2 or more
pH units while cooling, which was typical of some standard buffers,
the new formula changed less than 0.2 pH units during cooling, he said.
�We�re canceling out 100-fold changes in proton concentration and
bringing them down within an order of magnitude,� Sieracki said.
The creation of a temperature-independent pH (TIP) buffer could have
broad implications for new � and previously published � research, Lu
said.
�We�re not in the business of looking at the literature and correcting
other mistakes,� he said. �But some of the conclusions from previous
studies could be on shaky ground if a buffer was used that changed pH
dramatically at low temperatures.�
The new buffer is immediately useful for biological research, and
Sieracki said he is confident that a similar buffer could be made for
use in many fields, such as biochemistry, biophysics, chemical biology
and biomedical research.
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