Correction of right-angle fluorescence measurements for the absorption of excitation radiation

Holland, John F.; Teets, Richard E.; Kelly, P.M.; Timnick, Andrew.

doi:10.1021/ac50014a011

Cited by 140 publications

(73 citation statements)

References 18 publications

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“…[14-17, 23 -281 and references therein) which outline the problem of inner-filter effects and discuss how they may be reduced. It is possible to reduce the inner-filter effects at source [25,26], but at least in the case of physiological samples where the signal is already relatively low, it is more practicable to apply a correction factor [14,15,23,241, of which that of Parker [14] given in Eqn (1) seems the most apt. and after (U----0) applying the correction procedure [14].…”

Section: Consideration Of the Inneryilter Effectsmentioning

confidence: 99%

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol—cytochrome‐c reductase by the binding of ubiquinone

Samworth

Esposti

Lenaz

1988

European Journal of Biochemistry

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1. The quenching by ubiquinone (Q) of the intrinsic fluorescence of tryptophan residues within ubiquinolcytochrome-c reductase (complex 111) has been exploited to provide direct information on the interaction between these two components of the mitochondrial respiratory chain.2. The fluorescence quenching data have been corrected for inner filter effects and interpreted using the classical Stern-Volmer and modified Stern-Volmer plots. The latter of these plots allows computation of both the dissociation constant (Kd) of complex formation between ubiquinone and complex 111, and the percentage of fluorophores accessible to quenching.3. It is found that different Q homologues bind to complex 111 with different affinities depending upon the length of the isoprenoid chain: 2,3-dimethoxy-5-methyl-6-decyl-l ,Cbenzoquinone, an analogue of Q2, exhibits the same Kd as Q2. Furthermore, the accessibility of fluorophores to quenching was lower for Q1 than for the other quinones tested.4. The binding affinity of Qz to complex I11 depends upon the redox state of the enzyme. 5. Addition of the complex I11 inhibitor, antimycin, has very little effect on the binding affinity or on the 6. Addition of the inhibitor myxothiazol has a similar effect to reducing complex I11 with ascorbate. 7. Reconstitution of complex I11 into asolectin lipid vesicles gives similar qualitative results to the enzyme in accessibility of fluorophores to the quencher.solution regarding both the redox state and the addition of inhibitors.It is well established that ubiquinones are involved in the electron transport between flavoprotein dehydrogenases (e.g. succinate dehydrogenase) and the ubiquinol -cytochrome-c oxidoreductase (also known as complex 111) in the mitochondrial respiratory chain [l -41. However, there is some uncertainty over how ubiquinone (Q) functions; for example, does it function as a homogeneous mobile pool [4, 51 or as a protein-bound prosthetic group [6,7]? It is possible that this point, and others, can be clarified by a direct investigation of the binding/dissociation of ubiquinone with its redox partners in the mitochondrial respiratory chain. To date, the main body of work concerning ubiquinone has concentrated on a dynamic o r kinetic approach [2-5, 8, 91. To our knowledge the binding properties of ubiquinone have not been addressed directly, despite the fact that Q-binding proteins have been isolated from different respiratory chain complexes of beef heart mitochondria [6, 71.Here we present a novel approach to the process of Qbinding to the respiratory chain complex, with reference in particular to the binding of ubiquinone to complex 111. It should be noted that the substrate of complex 111 is actually

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Section: Consideration Of the Inneryilter Effectsmentioning

confidence: 99%

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol—cytochrome‐c reductase by the binding of ubiquinone

Samworth

Esposti

Lenaz

1988

European Journal of Biochemistry

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“…Sample dilution (Guilbault, 1990;Lakowicz, 1983) is the most popular approach, but it may cause changes: in conformation, bonding, solvation, and the degree of association. Also other chemical events may alter absorption-fluorescence processes and, there by, introduce large unknown errors (Holland, 1977). IFEs can distort fluorescence emission spectral profiles.…”

Section: Resultsmentioning

confidence: 99%

Inner Filter Effect in the Fluorescence Emission Spectra of Room Temperature Ionic Liquids with-β-Carotene

Pawlak¹,

Skrzypczak²,

Bialek-Bylka³

2011

Applications of Ionic Liquids in Science and Technology

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“…The 1,6-diphenyl-1,3,5-hexatriene (0.5/~g/ml) was incorporated into the VLDL (50/.tg lipid/m1) resuspended in phosphate-buffered saline, (pH 7.4) as in [ 11 ]. The computer-centered spectrofluorimeter in [ 12,13] was usedto measure absorbance, absorbance corrected fluorescence (corrected for the inner filter effect), relative fluorescence efficiency, corrected fluorescence emission, and light scattering corrections as in [14,15]. Excitation for ~-parinaric acid and 1,6-diphenylhexatriene was measured at 313 nm and 362 nm, respectively, while emission was measured at 420 nm and 424 nm, respectively.…”

Section: Methodsmentioning

confidence: 99%

Investigation of the surface structure of the very low density lipoprotein using fluorescence probes

1979

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Correction of right-angle fluorescence measurements for the absorption of excitation radiation

Cited by 140 publications

References 18 publications

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol—cytochrome‐c reductase by the binding of ubiquinone

Quenching of the intrinsic tryptophan fluorescence of mitochondrial ubiquinol—cytochrome‐c reductase by the binding of ubiquinone

Inner Filter Effect in the Fluorescence Emission Spectra of Room Temperature Ionic Liquids with-β-Carotene

Investigation of the surface structure of the very low density lipoprotein using fluorescence probes

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