Fluorometric Analytical Methods
Published on Aug 15, 2016
Fluorometry has been firmly established as method for the sensitive, specific and economic detection of trace quantities of substances. Generally, a fluorometric method has the potential of being 10—100 fold more sensitive than a colorimetric procedure, since dyes have a molar absorptivity of i05.' By coupling a fluorometric read-out with a catalytic or a non-stoichiometric chemical process even greater enhanced sensitivity may be achieved.
The greatly improved fluorometric instrumentation becoming commercially available for automated analyses has further contributed to the increasing utility of fluorometry in such diverse applications as environmental pollution, clinical chemistry, biology and metallurgy. Criteria for the choice of a set of reaction conditions for use in fluorometry include: rapid rates, fluorescent product stability, lack of internal quenching, and a highly fluorescent product
The utilization of enzymes in assay procedures is predicated on the availablity of enzymes of known characteristics with respect to substrate association constants (Km), inhibition constants (K1), pH-activity relationships, etc. Commercially available enzymes are limited. However, microorganisms offer an untapped enzyme source. By a process of enzyme induction in adaptable microorganisms, the required enzymes may be obtained. The inducible microorganisms are propagated obligatorily in a synthetic growth medium containing the inducing substate as a sole source of carbon or nitrogen. The surviving cells are harvested and propagated further. The cells are processed to isolate the enzymes responsible for the metabolism of the inducing substrate. A variety of enzymes are thus obtained, including esterases, dehydrogenases, oxygenases—specific for the inducing substrate.
With this background, a fluorometric enzyme assay was devised for atropine, a compound of considerable medicinal interest. II isatoic anhydrida Pseudomonas putida, indigenously growing about Atropa belladonna rhizomes were isolated and propagated in synthetic media containing atropine or tropic acid as the sole carbon source. The induced enzymes in the surviving organisms were isolated and found to display a potent atropinase (an esterase that splits atropine to tropic acid and tropine), tropic acid dehydrogenase (an enzyme that dehydrogenates tropic acid in the presence of DPN to yield phenylmalonic and phenylacetic acids) and uncharacterized oxygenases. By coupling the atropinase and tropic acid dehydrogenase, atropine, and tropic acid could be determined spectrofluorometrically by the following sequence
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