Measurement of the Binding of Adenosylcobalamin to Glutamate Mutase by Fluorescence Spectroscopy

Fluorescence spectroscopy is a fast, highly sensitive technique for investigating protein‐ligand interactions. Intrinsic protein fluorescence is usually occurred by exciting the proteins with 280‐295 nm ultraviolet light, and the light emission is observed approximately between 330‐350 nm. No emissi...

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Bibliographic Details
Published in:Journal of the Chinese Chemical Society (Taipei) 2012-11, Vol.59 (11), p.1478-1481
Main Authors: Lin, Heng-Ju, Hsu, Hui-Ju, Duann, Yeh-Fang, Chen, Hao-Ping
Format: Article
Language:English
Subjects:
Adenosylcobalamin
Fluorescence spectroscopy
Glutamate mutase
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Summary:Fluorescence spectroscopy is a fast, highly sensitive technique for investigating protein‐ligand interactions. Intrinsic protein fluorescence is usually occurred by exciting the proteins with 280‐295 nm ultraviolet light, and the light emission is observed approximately between 330‐350 nm. No emission light between 330‐350 nm can be observed when adenosylcobalamin (AdoCbl) is excited at 282 nm. The binding of AdoCbl to glutamate mutase was therefore investigated by fluorescence spectroscopy in this study. Our results show that direct measurement for determining the Kd of AdoCbl by fluorescence spectroscopy leads to significant errors. Here we report the source of error and a corrected method for measuring the binding of coenzyme B12 to glutamate mutase using fluorescence spectroscopy. No emission light between 330‐350 nm can be observed when adenosylcobalamin (AdoCbl) is excited at 282 nm. Our results show that direct measurement for determining the protein Kd of AdoCbl by fluorescence spectroscopy leads to significant errors. A corrected method for measuring the binding of coenzyme B12 to glutamate mutase using fluorescence spectroscopy was reported.
ISSN:0009-4536
2192-6549
DOI:10.1002/jccs.201100586