The fluorescence of fluorescamine-amino acids. |
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Authors: | R F Chen P D Smith M Maly |
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Institution: | Laboratory of Technical Development, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20014 USA |
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Abstract: | Fluorescamine reacts with various amino acids to yield solutions exhibiting different amounts of fluorescence, and the fluorogenic reaction does not go to completion. To investigate these phenomena, we measured the lifetimes and quantum yields of the fluorescamine-amino acids and studied their rates of formation by stopped-flow fluorescence and transmission measurements. The quantum yields were similar (0.11 ± 0.03), as were the lifetimes (3.5 ± 0.5 ns). The only exceptions were the derivatives of tryptophan and cysteine, which were internally quenched. It was concluded that the chemical yields of the fluorescamine-amino acids varied greatly. Kinetic experiments showed wide variations in rates, with some amino acids requiring several seconds for reaction under some conditions. Proline, on the other hand, reacted rapidly, with a second order rate constant of 6.2 × 10?4 liter mol?1 s?1. Sequential additions of fluorescamine to amino acids were more efficient in producing the fluorescent product than the same amount of reagent added all at once; this suggested that fluorescamine was inactivated by a concentration-dependent process. A mechanism to explain the low chemical yields is proposed in which both base and amine catalyze the inactivation of fluorescamine. |
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Keywords: | To whom correspondence should be addressed |
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