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Illustration of the cleavage of
proteins near a titanium dioxide surface: when illuminated with
ultraviolet light, hydroxyl radicals are formed in water near the
semiconductor's surface and cut proteins at the location of the
amino acid proline.
Image by NIST |
Because most proteins are very large, complex
molecules made up of hundreds or thousands of amino acids, they
usually must be cut up into more manageable pieces for analysis. Today,
this most commonly is done by using special enzymes called “proteases”
that sever the chains at well-known locations. The protease trypsin,
for example, cuts proteins at the locations of the amino acids lysine
and arginine. Analyzing the residual fragments can identify the
original protein. But enzymes are notoriously fussy, demanding fairly
tight control of temperature and acidity, and the enzymatic cutting
process can be time-consuming, from a matter of hours to days.
For a “radically” different approach, the NIST
group turned to a semiconductor material, titanium dioxide. Titanium
dioxide is a photocatalyst - when exposed to ultraviolet light its
surface becomes highly oxidizing, converting nearby water molecules
into hydroxyl radicals, a short-lived, highly reactive chemical
species.** In the NIST experiments, titanium dioxide coatings were
applied to a variety of typical microanalysis devices, including
microfluidic channels and silica beads in a microflow reactor. Shining
a strong UV light on the area, in the presence of a protein solution,
creates a small “cleavage zone” of hydroxyl radicals that rapidly cut
nearby proteins at the locations of the amino acid proline.
Although development work remains to be done,
according to the researchers, the NIST photocatalysis technique offers
several advantages over conventional enzyme cleavage of proteins. It’s
not particularly sensitive to temperature or acidity, and needs no
additional reagents other than dissolved oxygen in the solution. It’s
a simple arrangement, easy to incorporate into a wide range of
instruments and devices, and titanium dioxide, an inorganic material,
will last virtually forever in a broad range of conditions - enzymes
have to be treated carefully and stored in temperature-controlled
environments. The target amino acid, proline, is relatively sparse in
most proteins, but it’s found at key locations, such as sharp turns in
the molecule, that aid analysis. And it’s fast - in trials with the
protein angiotensin I, the team obtained detectable cleavage patterns
in as little as 10 seconds. |
