A New Type of Ultrasensitive Bioluminogenic Enzyme Substrates. I. Enzyme Substrates with D-Luciferin as Leaving Group

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Abstract
Derivatives of D-luciferin, D-luciferin methyl ester, D-luciferin O-sulfate, D-luciferin O-phosphate, D-luciferyl-L-N.alpha.-arginine and D-luciferyl-L-phenylalaine were used as highly sensitive substrates for carboxylic esterase, arylsulfatase, alkaline phosphatase and carboxypeptidases A, B and N. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants have been determined for D-luciferin methyl ester and carboxylic esterase, for D-luciferin O-sulfate and arylsulfatase, for D-luciferin O-phosphate and alkaline phosphatase, for D-luciferyl-L-phenylalanine and carboxypeptidase A, and for carboxypeptidases B and N and D-luciferyl-L-N.alpha.-arginine. All compounds proved to be highly sensitive substrates for the respective enzymes, permitting a limit of detection for enzymes between 10 and 500 fg per assay.