UBC Theses and Dissertations
An improved assay procedure for dopamine-B-hydroxylase Holubitsky, Don J.
Two of the existing assay procedures for dopamine B-hydroxylase were studied with respect to their suitability in the measurement of low levels of activity. Specifically, the spectrophotometric method and the coupled radioenzymatic methods were assessed relative to their ability to detect DBH activity in the perfusate of isolated rat tail arteries incubated under conditions known to inhibit the sodium pump. The spectrophotometric method , was the initial choice because of its low cost, greater ease of performance, and the fact that DBH is assayed under saturating conditions. Although it was found that this method suffered from a lack of sensitivity, attempts were made to improve this. The blank value was reduced and stabilized by the introduction of a mixture of 5 X 10⁻⁴ M fusaric acid and 5 X 10⁻⁴ M EDTA, which produced routine blank levels of ΔA= 0.005, equal to the lowest reported literature values. Also, the effect of ADP activation was investigated for its suitability in the procedure. Despite a three-fold increase in measured DBH activity, however, this method was still not sensitive enough to detect the enzyme activity in tissue incubates, although these improvements made the method more suitable for the assessment of levels of DBH activity in the serum of laboratory animals. The second choice was the coupled radioenzymatic method of Molinoff, because of its inherently greater sensitivity. While initial experiments proved this procedure effective in the detection of the release of DBH from rat vas deferens under conditions known to stimulate maximum exocytosis it was felt that problems could be encountered in the measurement of DBH activities obtained under less than optimal conditiona. Therfore, the procedure was investigated with respect to maximizing the absolute sensitivity. Preliminary studies on the feasibility of extended incubation in the first step, led to a rationale in which the two enzymic reactions were isolated and studied separately. In the second step, it was found that tyramine as expected, inhibited the PNMT reaction, although with sigmoidal kinetics. Paradoxically, non-dialyzable and heat stable impurities that inhibited PNMT were found in commercial preparations of catalase, although the addition of ascorbate eliminated this effect. Bovine serum albumin was found to selectively activate PNMT in a highly concentration dependent manner, with a five-fold maximum activation resultant from the inclusion of 0.14% BSA in the sample aliquot. Blank mixture and reaction time were investigated, and a doubling of sensitivity was found to result from limiting reaction time for the second step to 25 minutes. The PNMT was also shown to be unsaturated with respect to SAM, and an increase in the total concentration of SAM by the addition of labelled and unlabelled SAM to a concentration 40 times normal, was found to make the second reaction linear with respect to octopamine concentration. Fin ally, the [PNMT] could be increased up to 20 times without affecting linearity, and this produced an increase in sensitivity in direct proportion to the increase in enzyme concentration. These modifications were sufficiently effective so as to allow an increase in the concentration of tyramine in the first reaction mixture without too much of a loss of activity. This made the DBH reaction linear with respect to both time and enzyme concentration, which greatly improves accuracy and correlatability of results. Trial experiments proved that the combination of all these modifications into an improved procedure resulted in an assay with an improvement in sensitivity of at least two orders of magnitude over the standard method, with vastly improved characteristics of time and concentration linearity. The suitability of this method for our planned tissue release studies was also confirmed. It is hoped that the improvement in sensitivity and linearity of this modified procedure will allow its use in new experimental situations.
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