Simple and sensitive binding assay for measurement of adenosine using reduced S-adenosylhomocysteine hydrolase

Background Adenosine has been suggested to play an important role in the re gulation of renal function. We developed a simple and sensitive binding ass ay for the detection of adenosine based on the displacement of [H-3]adenosi ne from S-adenosylhomocysteine (SAH) hydrolase in its reduced form. Methods: SAH hydrolase was purified to apparent homogeneity from bovine kid ney by standard chromatographic methods. SAH hydrolase was converted in its reduced form, which had the advantage that the SAH hydrolase is enzymatica lly inactive. This reduced enzyme retains its ability to bind adenosine wit h high affinity. To determine adenosine in urine or tissues, samples must b e deproteinized (e.g., with 10 g/L sulfosalicylic acid or 0.6 mol/L perchlo ric acid). Results: The reduced SAH hydrolase bound adenosine with a dissociation cons tant of 33.0 +/- 2 nmol/L. Displacement of adenosine binding by the adenine 5'-nucleotides, adenine and hypoxanthine, required >1000-fold higher conce ntrations than adenosine itself. The intra- and interassay imprecision (CV) was <3.9% and 7.8%, respectively, and the values obtained showed acceptabl e correlation with those by HPLC. Conclusions: The highly sensitive adenosine-binding protein assay is a simp le test that allows detection of adenosine in samples with small volumes wi thout purification, and is in this respect superior to HPLC. (C) 2000 Ameri can Association for Clinical Chemistry.