Optimization of histamine radio enzyme assay with purified histamine-N-methyltransferase

The radio enzyme assay for histamine based on the transmethylation with purified histamine-N-methyltransferase and utilizing [3H-methyl]-S-adenosylmethionine as the methyl donor has been optimized to measure low histamine concentrations, for example in plasma. The pH-optimum for the assay is pH 8.3 in Tris-glycine buffer at 20 degrees C. An incubation time of 90 min is necessary using an enzyme concentration of 5.8 micrograms/ml. EDTA and dithiothreitol were included in the assay to keep the histamine-N-methyltransferase active as agents that oxidize -SH groups were found to be inhibitory to the reaction. The present assay is sensitive to about 0.5 nmol/l of histamine in a sample volume of 50 microliter (about 3 pg/sample).