A general fluorescence-based coupled assay for S-adenosylmethionine-dependent methyltransferases.
We have developed a simple and sensitive fluorescence-based two-step coupled enzyme assay to report the activity of S-adenosylmethionine-dependent methyltransferases. This assay relies on a fluorescein-cystamine-methyl red (FL-S-S-MR) reporter molecule that can be activated by thiols. In the absence of thiols, fluorescence from the reporter is quenched through fluorescence resonance energy transfer between the two chromophores. In this report, we use catechol-O-methyltransferase with the addition of S-adenosylhomocysteine hydrolase to produce the thiol homocysteine. The presence of homocysteine leads to disulfide bond cleavage in the cystamine tether and fluorescence dequenching as the uncoupled chromophores are diluted into the surrounding media. The sensitivity and specificity of FL-S-S-MR to thiols enabled detection of <or=1 microM concentrations of homocysteine, suggesting that this assay is sensitive enough to detect biologically relevant amounts of homocysteine. We believe that this fluorescence reporter approach may be generalizable to all enzymatic or chemical assays that produce thiols.
Wang
Caihua
C
Department of Chemistry, The Purdue Cancer Center, Purdue University, West Lafayette, IN 47907-2084, USA.
Leffler
Scott
S
Thompson
David H
DH
Hrycyna
Christine A
CA
D003538:Cystamine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*; D005452:Fluoresceins / Q000138:chemical synthesis / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006710:Homocysteine / Q000032:analysis; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013438:Sulfhydryl Compounds; C501533:fluorescein-cystamine-methyl red; D006710:Homocysteine; D011497:Protein O-Methyltransferase; D003538:Cystamine