Enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate and guanosine 3',5' cyclic monophosphate in biological fluids.

Simple, rapid and sensitive competitive enzyme immunoassays for the estimation of adenosine 3',5' cyclic monophosphate (cAMP) and guanosine 3',5' cyclic monophosphate (cGMP) in human plasma and urine are described. Specific antisera to each nucleotide were raised in rabbits by immunization with succinyl cyclic nucleotide--human serum albumin conjugates. For the assay, specific antibodies were incubated with a mixture of succinyl cyclic nucleotide labelled with horseradish peroxidase together with unlabelled standard or sample. The antibody-bound enzyme conjugate was separated from free hapten by anti-rabbit (IgG) sera immobilized to a microtitre plate. Activity of the bound enzyme conjugate was determined with tetramethylbenzidine. The assays were capable of detecting levels as low as 2 fmol of cAMP and cGMP. Good correlations were obtained between values generated by enzyme immunoassay and radioimmunoassay.

Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2.

1. 2-Furoyl-CoA hydroxylase of Pseudomonas putida F2 has been purified 60-fold by a combination of (NH(4))(2)SO(4) fractionation, DEAE-cellulose chromatography and agarose chromatography. 2. The purified enzyme catalyses the formation of 5-hydroxy-2-furoyl-CoA, which tautomerizes to form 5-oxo-Delta(2)-dihydro-2-furoyl-CoA. 3. The enzyme has a requirement for an electron acceptor that can be satisfied by a membrane preparation from 2-furoate-grown Ps. putida F2 or by artificial electron acceptors, and so presumably the incorporated oxygen atom is derived from water rather than molecular oxygen. 4. The enzyme is a large protein with a molecular weight of 3.27x10(6) and is disrupted to form inactive subunits in the presence of 0.2% (w/v) sodium dodecyl sulphate. It has a pH optimum of 8.5-9.5, a K(m) for 2-furoyl-CoA of 20.2mum and an absorption spectrum with a trough at 265nm and a single peak at 273nm. No absorption peaks are detectable in the visible region of the spectrum. 5. The enzyme is resistant to the effects of a wide range of potential inhibitors, but is inhibited by the copper-chelating agents bathocuproin and cuprizone, though not by sodium diethyldithiocarbamate. 6. Flavins are absent and the iron content does not show a sustained increase during purification. The copper content of the protein increases in close correlation with the increase in specific activity during purification. 7. A catalytic sequence for the hydroxylation of 2-furoyl-CoA by a copper protein is proposed.