LUMINESCENCE QUENCHING BY NITROXIDE SPIN LABELS IN AQUEOUS-SOLUTION - STUDIES ON THE MECHANISM OF QUENCHING

被引:67
作者
MATKO, J [1 ]
OHKI, K [1 ]
EDIDIN, M [1 ]
机构
[1] JOHNS HOPKINS UNIV,DEPT BIOL,BALTIMORE,MD 21218
关键词
D O I
10.1021/bi00118a010
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42-degrees-C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants. The extremely high sensitivity of the quenching rate to the separation distance in the range of 0.5-2 nm should make nitroxide-conjugated antibodies paired with fluorophore-conjugated antibodies a useful fluorophore-quencher system for studying lateral organization and association of membrane proteins on cell surfaces.
引用
收藏
页码:703 / 711
页数:9
相关论文
共 56 条
[11]   9-FLUORENYLMETHOXYCARBONYL AMINO-PROTECTING GROUP [J].
CARPINO, LA ;
HAN, GY .
JOURNAL OF ORGANIC CHEMISTRY, 1972, 37 (22) :3404-&
[12]   PARALLAX METHOD FOR DIRECT MEASUREMENT OF MEMBRANE PENETRATION DEPTH UTILIZING FLUORESCENCE QUENCHING BY SPIN-LABELED PHOSPHOLIPIDS [J].
CHATTOPADHYAY, A ;
LONDON, E .
BIOCHEMISTRY, 1987, 26 (01) :39-45
[13]   PHOTOPROCESSES IN DIPHENYLPOLYENES .2. EXCITED-STATE INTERACTIONS WITH STABLE FREE-RADICALS [J].
CHATTOPADHYAY, SK ;
DAS, PK ;
HUG, GL .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1983, 105 (20) :6205-6210
[14]   DISTANCE, STEREOELECTRONIC EFFECTS, AND THE MARCUS INVERTED REGION IN INTRAMOLECULAR ELECTRON-TRANSFER IN ORGANIC RADICAL-ANIONS [J].
CLOSS, GL ;
CALCATERRA, LT ;
GREEN, NJ ;
PENFIELD, KW ;
MILLER, JR .
JOURNAL OF PHYSICAL CHEMISTRY, 1986, 90 (16) :3673-3683
[15]  
DEPETRIS S, 1978, METHODS MEMBRANE BIO, V9, P1
[16]   A THEORY OF SENSITIZED LUMINESCENCE IN SOLIDS [J].
DEXTER, DL .
JOURNAL OF CHEMICAL PHYSICS, 1953, 21 (05) :836-850
[17]  
EDIDIN M, 1982, J CELL BIOL, V95, P458, DOI 10.1083/jcb.95.2.458
[18]  
FORSTER T, 1949, Z NATURFORSCH A, V4, P321
[19]   QUENCHING OF AROMATIC TRIPLET-STATES IN SOLUTION BY NITRIC-OXIDE AND OTHER FREE-RADICALS [J].
GIJZEMAN, OL ;
KAUFMAN, F ;
PORTER, G .
JOURNAL OF THE CHEMICAL SOCIETY-FARADAY TRANSACTIONS II, 1973, 69 (05) :727-737
[20]   OXYGEN QUENCHING OF AROMATIC TRIPLET-STATES IN SOLUTION .2. [J].
GIJZEMAN, OL ;
KAUFMAN, F .
JOURNAL OF THE CHEMICAL SOCIETY-FARADAY TRANSACTIONS II, 1973, 69 (05) :721-726