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A multienzyme bioluminescent time-resolved pyrophosphate assay.

Ye Sun¹, K Bruce Jacobson, Val Golovlev

¹Sci-Tec, Inc., 10425 Cogdill Road, Suite 300, Knoxville, TN 37932, USA.

Analytical Biochemistry

|June 2, 2007

Summary

We developed a sensitive assay for measuring inorganic pyrophosphate (PPi) in samples with adenosine 5'-triphosphate (ATP). This method uses luminescence kinetics and enzymatic reactions for accurate PPi detection, crucial for RNA expression analysis.

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Area of Science:

Biochemistry
Analytical Chemistry
Molecular Biology

Background:

Accurate measurement of inorganic pyrophosphate (PPi) is challenging in samples contaminated with adenosine 5'-triphosphate (ATP).
Existing methods may lack the sensitivity or specificity required for certain biological applications.

Purpose of the Study:

To develop a high-sensitivity assay for quantifying PPi in ATP-contaminated samples.
To establish a theoretical model for optimizing luminescence kinetics and assay performance.
To create algorithms for reliable PPi measurement from luminescence data.

Main Methods:

Development of a luminescence-based assay utilizing enzymatic conversion of PPi to ATP.
Time-resolved measurement of luminescence kinetics.
Implementation of two distinct algorithms for analyzing short (<1 min) and long (3-5 min) kinetic data.
Theoretical modeling of luminescence kinetics to optimize assay conditions.

Main Results:

The developed assay demonstrates high sensitivity, detecting PPi down to 15 pM (7 pg/ml) in ATP-contaminated samples.
A theoretical model accurately predicted experimental luminescence kinetics.
Two algorithms were validated for PPi measurement, with sensitivities of 15 pM and 20 pM for long and short kinetics, respectively.
Enzyme activity was controlled during experiments to ensure assay reliability.

Conclusions:

The novel assay provides a sensitive and reliable method for PPi detection in challenging samples.
The assay is suitable for RNA expression analysis and other applications requiring precise PPi quantification.
The developed algorithms offer flexibility for different experimental formats, including microplate assays.