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Measurement of free and bound fractions of extracellular ATP in biological solutions using bioluminescence
Author(s) -
Douillet Christelle D.,
Suy Sihong,
Zarzaur Ben L.,
Robinson William P.,
Milano Peter M.,
Boucher Richard C.,
Rich Preston B.
Publication year - 2005
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/bio.869
Subject(s) - extracellular , albumin , chemistry , adenosine triphosphate , chromatography , biochemistry , centrifugation
Measurement of extracellular ATP in biological solutions is complicated by protein‐binding and rapid enzymatic degradation. We hypothesized that the concentration of extracellular ATP could be determined luminometrically by limiting degradation and measuring the free and protein‐bound fractions. ATP was added (a) at constant concentration to solutions containing varying albumin concentrations; (b) at varying concentrations to a physiological albumin solution (4 gm/dL); (c) at varying concentrations to plasma. After centrifugation, a fraction of each supernatant was heated. ATP in heated and unheated samples was measured luminometrically. Blood was drawn into saline or an ATP‐stabilizing solution and endogenous plasma ATP measured. ATP‐albumin binding was a linear function of albumin concentration (3.5% ATP bound at 100 µmol/L to 33.2% ATP bound at 1000 µmol/L) but independent of ATP concentration (29.3%, 10–1000 nmol/L ATP in 602 µmol/L albumin). Heating released the majority of bound ATP from albumin‐containing solutions (94.8 ± 1.7%) and plasma (97.6 ± 5.1%). Total endogenous plasma ATP comprised 93 ± 27 nmol/L (free) and 150 ± 40 nmol/L (total fraction). Without stabilizing solution, degradation of free endogenous plasma ATP occurred. Within a physiological range (10–1000 nmol/L), ATP binds albumin independently of ATP concentration. Heating releases bound ATP, enabling accurate luminometric measurement of total extracellular ATP (free and bound) in biological samples. Copyright © 2005 John Wiley & Sons, Ltd.