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).
Harvima
R J
RJ
Department of Dermatology, University of Kuopio, Finland.
Harvima
I T
IT
Fräki
J E
JE
D000818:Animals; D006632:Histamine / Q000097:blood*; D006637:Histamine N-Methyltransferase; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology; D008780:Methyltransferases; D051381:Rats; D014316:Tritium
D014316:Tritium; D006632:Histamine; D008780:Methyltransferases; D006637:Histamine N-Methyltransferase