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J. Biol. Chem., Vol. 275, Issue 51, 39900-39906, December 22, 2000
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From the Department of Chemistry, National Changhua University of
Education, Changhua 50058, Taiwan, Republic of China
Neocarzinostatin is a potent enediyne antitumor
antibiotic complex in which a chromophore is noncovalently bound to a
carrier protein. The protein regulates availability of the drug by
proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of
the protein with special emphasis on their relation to the release of
the chromophore from holoneocarzinostatin. The effect of the
Release of the Neocarzinostatin Chromophore from the Holoprotein
Does Not Require Major Conformational Change of the Tertiary and
Secondary Structures Induced by Trifluoroethanol*
helix-inducing agent, TFE, on all the 
-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and 1H NMR studies. By using binding of
anilinonaphthalene sulfonic acid as a probe, we observed that the
protein exists in a stable, partially structured intermediate state
around 45-50% TFE, which is consistent with the results from
tryptophan fluorescence and circular dichroism studies. The native
state is stable until 20% TFE and is half-converted into the
intermediate state at 30% TFE, which starts to collapse beyond 50%.
High pressure liquid chromatographic analysis of the release of the
chromophore caused by TFE treatment at 0 °C suggests that the
release process, which occurs below 20% TFE, does not result from an
observable conformational change in the protein. Kinetic measurements
of the release of chromophore at 25 °C reveal that TFE does
stimulate the rate of release, which increases sharply at 15% and
reaches a maximum at 20% TFE, although no major secondary or tertiary
structural change of the carrier protein is observed under these same
conditions. Our data suggest that chromophore release results from a
fluctuation of the protein structure that is stimulated by TFE.
Complete release of the chromophore occurs at TFE concentrations where
no overall observable unfolding of the apoprotein is seen. Thus, the
results suggest that denaturation of the protein by TFE is not a
necessary step for release of the tightly bound chromophore.
*
This work was supported by Laboratory Grant DOH88-HR-807
from National Health Research Institutes and Individual Grant NSC 88-2113-M-018-001 from the National Science Council, The Executive Yuan, Republic of China.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Chemistry,
National Changhua University of Education, Changhua 50058, Taiwan,
Republic of China. Fax: 886-4-7211178 or 886-4-7211190; E-mail:
chdhchin@cc.ncue.edu.tw.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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