[Zurück]

M. Schleeger, C. Wagner, M. Vellekoop, B. Lendl, J. Heberle:
"Time-resolved flow-flash FT-IR difference spectroscopy: the kinetics of CO photodissociation from myoglobin revisited";
Analytical and Bioanalytical Chemistry, 394 (2009), 7; S. 1869 - 1877.

Fourier-transform infrared (FT-IR) difference
spectroscopy has been proven to be a significant tool in
biospectroscopy. In particular, the step-scan technique
monitors structural and electronic changes at time resolutions
down to a few nanoseconds retaining the multiplex
advantage of FT-IR. For the elucidation of the functional
mechanisms of proteins, this technique is currently limited
to repetitive systems undergoing a rapid photocycle. To
overcome this obstacle, we developed a flow-flash experiment
in a miniaturised flow channel which was integrated
into a step-scan FT-IR spectroscopic setup. As a proof of
principle, we studied the rebinding reaction of CO to
myoglobin after photodissociation. The use of microfluidics
reduced the sample consumption drastically such that a
typical step-scan experiment takes only a few 10 ml of a
millimolar sample solution, making this method particularly
interesting for the investigation of biological samples that
are only available in small quantities. Moreover, the flow
cell provides the unique opportunity to assess the reaction
mechanism of proteins that cycle slowly or react irreversibly.
We infer that this novel approach will help in the elucidation
of molecular reactions as complex as those of vectorial ion
transfer in membrane proteins. The potential application to
the oxygen splitting reaction of cytochrome c oxidase is
discussed.

http://dx.doi.org/10.1007/s00216-009-2871-0