David B. Land, E. Jackim
Sep 1, 1966
Citations
0
Influential Citations
21
Citations
Journal
Analytical Biochemistry
Abstract
Abstract In the search for an ultrasensitive method for the determination of alkaline phosphatase to be incorporated in an automated system, the ammonium salt of flavone 3-diphosphate was synthesized and found to be a stable, versatile, and sensitive substrate for assaying both acid and alkaline phosphatase. Flavone 3-monophosphate was also synthesized, and exhibited comparable sensitivity to the diphosphate. The enzymic cleavage of the mono- and diphosphate groups liberates 3-hydroxyflavone, which can be assayed by direct absorption at 410 mμ or, with greater sensitivity, fluorometrically at 510 mμ. Ultrahigh sensitivity can be achieved by forming the fluorescent metallic chelate of 3-hydroxyflavone with an excess of aluminum ions at a slightly acidic pH value. After an incubation period of 5 min, approximately 10−11 gm of phosphatase can be assayed by the chelate method. The substrate appears to have excellent cytohistochemical fluorescent stain applications. The fluorogenic phosphatase substrate 3-O-methylfluorescein phosphate (1), although more sensitive, lacks the desired stability in solution for automation. The substrate, β-naphthyl phosphate (2–4), has been investigated and was found in our laboratory to be less sensitive than flavone 3-diphosphate.