No abstract

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.

A series of 50 neurochemicals, important for the investigation of neurotransmission processes, has been labelled with tritium, using in all 8 methods. Tritium nuclear magnetic resonance spectroscopy has been employed to determine the positions and extent of labelling in these neurochemicals which can be important for their use in tracer studies. The specificity of the labelling processes is discussed.

N-Succinimidyl propionate [ NSP] was synthesised and characterised as a pale y e l l o x waxy s o l i d . Rates of hydrolysis i n b u f f e r s range pH 5-9 were determined. Optimum conditions f o r r e a c t i o n with l y s i n e were within t h e range pH 7 t o 8. A p r o t e i n , wheat germ a g g l u t i n i n was l a b e l l e d a t pH 7.5 a t room temperature f o r two hours. Rates of r e a c t i o n of o t h e r s u b s t r a t e s under these conditions were i n v e s t i g a t e d . Key Words: P r o t e i n l a b e l l i n g , t r i t i u m , k i n e t i c s .

The complete analysis of the tritium distribution in tritiated methyl groups in labelled compounds is achieved by a direct method for the first time, through 'H NMR spectroscopy aided by resolution enhancement.