Linked Life Data

11603835

Source:http://linkedlifedata.com/resource/pubmed/id/11603835

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
11
pubmed:dateCreated
2001-10-17
pubmed:abstractText
Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
1386-1425
pubmed:author
pubmed:issnType
Print
pubmed:day
14
pubmed:volume
57
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2135-44
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects.
pubmed:affiliation
MicroSpectroscopy Centre, Department of Biomolecular Sciences, Wageningen University, The Netherlands.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't