Development of a spectrophotometric assay for cyclase activity.
We describe the development of a rapid colorimetric assay for soluble guanylate cyclase (sGC) activity adapted for a 96-well microplate. The assay greatly decreases the analysis time and cost over traditional methodologies based on radio- and immunoassays and high-performance liquid chromatography (HPLC) separations. The method does not demonstrate any significant interference with chemicals commonly used for sGC purification and reaction kinetics. The assay converts the inorganic pyrophosphate produced in the cyclase reaction to inorganic phosphate, which is then measured using a modified Fiske-Subbarow assay. We used the assay to compare the reaction kinetics of preparations of sGC from a commercial source with those from our lab with Mg(2+)-guanosine 5'-triphosphate (GTP) or Mn(2+)-GTP as a substrate. The commercial preparation was found to have a specific activity of around 1.5 micromol/min/mg, which is significantly lower than expected, as was the fold-activation upon addition of nitric oxide (NO). Our laboratory preparation had a higher specific activity that was consistent with results from HPLC assays. We determined that the human isoform of sGC is more active in the basal and NO forms with Mn(2)-GTP as a substrate than Mg(2+)-GTP, a feature more similar to rat lung sGC than the more commonly studied bovine lung.
Sousa
Eduardo H S
EH
Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA 94132, USA.
Garay
Paula A
PA
Tinianow
Jeff N
JN
Gerber
Nancy Counts
NC
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D011756:Diphosphates / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006160:Guanosine Triphosphate / Q000737:chemistry; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry; D010710:Phosphates / Q000138:chemical synthesis; D012680:Sensitivity and Specificity; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D011756:Diphosphates; D010710:Phosphates; D009569:Nitric Oxide; D008345:Manganese; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D008274:Magnesium