Intermolecular Association Provides Specific Optical and NMR Signatures for Serotonin at Intravesicular Concentrations
Suman Nag 1, Jayaprakash Balaji 1, Perunthiruthy K. Madhu 1 and Sudipta Maiti 1*
1 Tata Institute of Fundamental Research
* To whom correspondence should be addressed. E-mail: maiti{at}tifr.res.in.
Submitted on September 6, 2007
Revised on October 30, 2007
Accepted on 20 December 2007
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Abstract |
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Neurotransmitter vesicles contain biomolecules at extraordinarily high concentrations (hundreds of mM). Such concentrations can drive intermolecular associations, which may affect vesicular osmolarity and neuronal signaling. Here we investigate whether aqueous serotonin (a monoamine neurotransmitter) forms oligomers at intravesicular concentrations, and whether these oligomers have specific spectroscopic signatures which can be potentially used for monitoring neuronal storage and release. We report that as serotonin concentration is increased from 60 µM to 600 mM, the normalized fluorescence spectrum of serotonin displays a growing long-wavelength tail, with an iso-emissive point at 376 nm. The fluorescence decay is mono-exponential with a lifetime of 4 ns at low concentrations, but is multi-exponential with an average lifetime of 0.41 ns at 600 mM. A 600 mM serotonin solution has 30% less osmolarity than expected for monomeric serotonin, indicating oligomer formation. The proton NMR chemical shifts move upfield by as much as 0.3 ppm at 600 mM compared 10 mM, indicating a stacking of the serotonin indole moieties. However, no intermolecular cross peak is evident in the two dimensional NMR Rotating Frame Overhauser Effect SpectroscopY (ROESY) spectrum even at 600 mM, suggesting that oligomeric structures are possibly weakly coupled. The appearance of a single peak for each proton suggests that the rate of interconversion between the monomeric and the oligomeric structures is faster than 240 Hz. A stop-flow kinetic experiment also confirms that the rate of dissociation is faster than 100 ms. We conclude that serotonin forms oligomers at intravesicular concentrations but becomes monomeric quickly upon dilution. NMR signatures of the oligomers provide potential contrast agents for monitoring the activity of serotonergic neurons in vivo.
Key Words:
5-hydroxy Tryptamine, serotonin, serotonin NMR, serotonin lifetime, serotonin oligomer, vesicular serotonin
