title: p-dimethylaminocinnamaldehyde derivatization for colorimetric detection and HPLC-UV/vis-MS/MS identification of indoles.
abstract: 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.
authors.affiliation: Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Lawrence, KS 66045, USA.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Porubsky
authors.forename: Emily E
authors.initials: EE
authors.lastname: Scott
authors.forename: Todd D
authors.initials: TD
authors.lastname: Williams
mesh_terms: D000171:Acrolein / Q000031:analogs & derivatives* / Q000737:chemistry; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002934:Cinnamates; D003124:Colorimetry; D006860:Hydrogen Bonding; D007211:Indoles / Q000737:chemistry*; D008024:Ligands; D013058:Mass Spectrometry; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D012015:Reference Standards; D013056:Spectrophotometry, Ultraviolet; D014867:Water / Q000737:chemistry
chemical_list: D002934:Cinnamates; D007211:Indoles; D008024:Ligands; D014867:Water; D000171:Acrolein; C018523:4-dimethylaminocinnamaldehyde; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; C012843:cinnamaldehyde