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Frequency-Domain Lifetime
Fluorometry of Double-Labeled Creatine Kinase
M. GREGOR1, M. KUBALA2,3,
E. AMLER3, J. MEJSNAR1
1Department of Physiology and Developmental Biology,
Faculty of Science, 2Institute of Physics, Charles
University, 3Institute of Physiology, Czech Academy
of Sciences, Prague, Czech Republic
Received July 31, 2002
Accepted November 1, 2002
Summary
Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of
muscle energy metabolism, has been selected for studies of
conformational changes that underlie the cellular control of
enzyme activity. For fluorescence spectroscopy measurements, the
CK molecule was double-labeled with IAF
(5-iodoacetamidofluorescein) and ErITC (erythrosin
5'-isothiocyanate). Measurement of fluorescence resonance energy
transfer (FRET) from fluorescein to erythrosin was used to
obtain information about the donor-acceptor pair distance.
Frequency-domain lifetime measurements evaluate the
donor-acceptor distance in the native CK molecule as 7.8 nm. The
Förster radius equals 5.3 nm with the resolution range from 0.2
to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for
artificial conformational changes of the CK molecule with
high-salt concentration treatment. The transition distance,
defined by His-97 and Cys-283 and derived from a 3D model equals
0.766 nm for the open (inactive) form and 0.277 nm for the
closed (reactive) form of the CK molecule. In this way, the
resolution range of the used spectroscopy method is significant,
concerning the difference of 0.489 nm. Nevertheless, the CK
enzyme activity, assessed by the hexokinase-coupled assay, was
diminished down to 1 % of the activity of the native enzyme. EFL
is suitable for description of conformational behavior implied
from the regulation of creatine kinase. However, the observed
inhibition restricts EFL to studies of conformational changes
during natural catalytic activity.
Key words
Creatine kinase molecule • Conformational change • FRET •
Frequency-domain lifetime fluorometry
Reprint requests
Martin Gregor, Department of Physiology and Developmental
Biology, Faculty of Sciences, Charles University, Viničná 7,
CZ-128 44 Praha 2, Czech Republic, e-mail
gregor@natur.cuni.cz
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