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Arch Biochem Biophys. 2008 Jul 1;475(1):14-7. doi: 10.1016/ Epub 2008 Apr 7.

p-dimethylaminocinnamaldehyde derivatization for colorimetric detection and HPLC-UV/vis-MS/MS identification of indoles.

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Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Lawrence, KS 66045, USA.


Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was identified as unsubstituted indole, which facilitated refinement of two alternate conformations in the CYP2A13 crystal structure active site.

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