TY - JOUR
T1 - Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. A potent and selective fluorescent inhibitor of butyrylcholinesterase
AU - Brimijoin, Stephen
AU - Mintz, Keith P.
AU - Prendergast, Franklyn G.
N1 - Funding Information:
Acknowledgements--This work was supported in part by Grant NS 11855 from the National Institutes of Health and by Grant PCM 79-11492 from the National Science Foundation. The encouragement and material aid from Drs. K. G. Mann and M. E. Nesheim are gratefully acknowledged. The synthesis of N-methyl acridinium was carried out in the Laboratory of Organic Chemistry in the Mayo Clinic Division of Gastroenterology under the supervision of Dr. Gerald Carlson, whose direction is sincerely appreciated. We thank Luanne Wussow for preparing the figures and for expert editorial assistance.
PY - 1983/2/15
Y1 - 1983/2/15
N2 - Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 × 10-7 M) but not of acetylcholinesterase (IC50 = 4 × 10-4 M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a Kd of 4.5 × 10-7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.
AB - Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase (IC50 = 2 × 10-7 M) but not of acetylcholinesterase (IC50 = 4 × 10-4 M). For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate Ki unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a Kd of 4.5 × 10-7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.
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U2 - 10.1016/0006-2952(83)90495-1
DO - 10.1016/0006-2952(83)90495-1
M3 - Article
C2 - 6830632
AN - SCOPUS:0020671708
SN - 0006-2952
VL - 32
SP - 699
EP - 706
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 4
ER -