Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

FTIR Spectroscopy of Calcium-Binding Proteins 59
ture is the amide I backbone mode, which occurs in the region 1610–1700 cm –1 .
Studies of proteins in the amide I region are complicated by the fact that the
bending vibration of water absorbs very strongly near 1640 cm –1 . As a consequence,
for transmission measurements in the amide I region very short path
length cells of 3–8 µm are needed. Experimentally, it is simpler to obtain protein
spectra in deuterium oxide (D 2O) solution than in H 2O solution. The infrared
bands of D 2O compared to those of H 2O occur at lower wavenumbers
because of the downshifted vibrations of the heavier deuterium atoms. This
isotopic effect creates a region of relatively low absorbance between 1400 and
1800 cm –1 , a window for observing the weak infrared bands of the dissolved
protein. Much longer path lengths of 40–80 µm may then be used (see Note 3).
2.2.3. Design of the IR Cells
Flowthrough demountable cells with luer-lock fittings and spacers covering
path lengths of 6 to 200 µm are often used for protein FTIR measurements.
These cells are available from virtually any infrared accessories supplier, but
they have some disadvantages in practice such as being difficult to clean and
giving rise to accidental injection of gas bubbles. To circumvent these problems,
we use custom-made IR cells of different design that consist of a flat cover disk
(typically made of CaF 2) and a second disk of the same material (sample disk),
with the center deepened to form a recessed parallel surface surrounded by a
trough (see Fig. 1). The trough prevents direct contact of the sample with the
outer part of the disk. Pressing the cover disk onto the sample disk seals the cell,
and this is sufficient to prevent the evaporation of water for many hours at room
temperature. These windows are fitted into a metal jacket through which heating
or cooling liquid from an external bath can circulate. For measurements at
high temperatures and/or long-time experiments, the sealing surface of the disks
is lubricated with mineral oil prior to filling and assembling of the cell. Depending
upon the diameter and the depth of the recessed surface of the window (i.e.,
the path length of the cell), only a few microliters are required to fill the cell.
Moreover, this type of cell is easy to fill with a solution, and can be assembled
and disassembled (most of the solution can be recovered), and cleaned between
measurements. It provides a constant path length, which is very difficult to achieve
with conventional tin or teflon spacers.
2.2.4. Attenuated Total Reflection (ATR) Sampling Technique
For ATR measurements, the sample is prepared on the surface of an infrared
transparent crystal. The IR beam is guided through the crystal in such a way
that some total reflections take place at the surface. Because the IR beam penetrates
slightly into the surrounding medium, the deposition of an infrared
absorber on the crystal surface causes the infrared light to be partially absorbed.