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Anal Biochem. 2011 Nov 15;418(2):247-52. doi: 10.1016/j.ab.2011.07.021. Epub 2011 Jul 24.

Sensitive fluorogenic substrate for alkaline phosphatase.

Author information

1
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.

Abstract

Alkaline phosphatase serves both as a model enzyme for studies on the mechanism and kinetics of phosphomonoesterases and as a reporter in enzyme-linked immunosorbent assays (ELISAs) and other biochemical methods. The tight binding of the enzyme to its inorganic phosphate product leads to strong inhibition of catalysis and confounds measurements of alkaline phosphatase activity. We have developed an alkaline phosphatase substrate in which the fluorescence of rhodamine is triggered on P-O bond cleavage in a process mediated by a "trimethyl lock." Although this substrate requires a nonenzymatic second step to manifest fluorescence, we demonstrated that the enzymatic first step limits the rate of fluorogenesis. The substrate enables the catalytic activity of alkaline phosphatase to be measured with high sensitivity and accuracy. Its attributes are ideal for enzymatic assays of alkaline phosphatase for both basic research and biotechnological applications.

PMID:
21827735
PMCID:
PMC3172393
DOI:
10.1016/j.ab.2011.07.021
[Indexed for MEDLINE]
Free PMC Article

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