p-dimethylaminocinnamaldehyde derivatization for colorimetric detection and HPLC-UV/vis-MS/MS identification of indoles.
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.
Porubsky
Patrick R
PR
Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Dr., Lawrence, KS 66045, USA.
Scott
Emily E
EE
Williams
Todd D
TD
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
D002934:Cinnamates; D007211:Indoles; D008024:Ligands; D014867:Water; D000171:Acrolein; C018523:4-dimethylaminocinnamaldehyde; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human; C012843:cinnamaldehyde