A critical reexamination of the continuous spectrophotometric assay for adenosine deaminase. Export

@article{citeulike:5485777, abstract = {The continuous spectrophotometric assay for adenosine deaminase has been reinvestigated, using both adenosine and 9-β- -arabinofuranosyladenine as substrates. This assay is based on the reported decrease in absorbance at or near 265 nm between the adenine nucleoside substrate and the hypoxanthine nucleoside product. In the substrate concentration range 1,5 – 8.0 × 10−4 , the progress of the reaction is associated with an anomalous sigmoidal dependence of absorbance on time, and the overall change in absorbance decreases with increasing substrate concentration. Near 8 × 10−4 substrate, the deamination proceeds with no change in absorbance, while at higher concentrations, small increases in absorbance are observed. These effects, if ignored, generate initial ” rate” data exhibiting an apparent substrate inhibition whieh, however, is completely an artifact induced by the spectral anomalies. Over the entire concentration range 5 × 10−6–1 × 10−3 , alternative assay methods (e.g., discontinuous detection of the product, ammonia) yeld normal Michaelis-Menten kineties. The anomalous behavior manifested in the continuous spectrophotometric assay is due to large negative deviations from Beer's law. These deviations are observed for all four of the nucleosides tested, viz., adenosine, 9-β- -arabinofuranosyladenine, inosine, and 9-β- -arabinofuranosylhypoxanthine. The departure from Beer's law is detectable anywhere in the concentration range 5 × 10−6–1 × 10−3 , but is most marked at concentrations above 1 × 10−4 . Thus, the continuous spectrophotometric assay for adenosine deaminase should be utilized withextreme caution, and should not be employed at concentrations exceeding 1 × 10−4 , irrespective of the Km value for the substrate. Specific recommendations are given for future assays.}, author = {Murphy, J. and Baker, D. C. and Behling, C. and Turner, R. A.}, citeulike-article-id = {5485777}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/7114454}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=7114454}, citeulike-linkout-2 = {http://dx.doi.org/10.1016/0003-2697(82)90291-3}, day = {15}, doi = {10.1016/0003-2697(82)90291-3}, issn = {0003-2697}, journal = {Analytical biochemistry}, keywords = {amp, assay, deaminase, spectrophotometry}, month = {May}, number = {2}, pages = {328--337}, posted-at = {2009-08-19 10:14:20}, priority = {2}, title = {A critical reexamination of the continuous spectrophotometric assay for adenosine deaminase.}, url = {http://dx.doi.org/10.1016/0003-2697(82)90291-3}, volume = {122}, year = {1982} }

TY - JOUR ID - citeulike:5485777 L3 - citeulike-article-id:5485777 N2 - The continuous spectrophotometric assay for adenosine deaminase has been reinvestigated, using both adenosine and 9-β- -arabinofuranosyladenine as substrates. This assay is based on the reported decrease in absorbance at or near 265 nm between the adenine nucleoside substrate and the hypoxanthine nucleoside product. In the substrate concentration range 1,5 – 8.0 × 10−4 , the progress of the reaction is associated with an anomalous sigmoidal dependence of absorbance on time, and the overall change in absorbance decreases with increasing substrate concentration. Near 8 × 10−4 substrate, the deamination proceeds with no change in absorbance, while at higher concentrations, small increases in absorbance are observed. These effects, if ignored, generate initial “rate” data exhibiting an apparent substrate inhibition whieh, however, is completely an artifact induced by the spectral anomalies. Over the entire concentration range 5 × 10−6–1 × 10−3 , alternative assay methods (e.g., discontinuous detection of the product, ammonia) yeld normal Michaelis-Menten kineties. The anomalous behavior manifested in the continuous spectrophotometric assay is due to large negative deviations from Beer's law. These deviations are observed for all four of the nucleosides tested, viz., adenosine, 9-β- -arabinofuranosyladenine, inosine, and 9-β- -arabinofuranosylhypoxanthine. The departure from Beer's law is detectable anywhere in the concentration range 5 × 10−6–1 × 10−3 , but is most marked at concentrations above 1 × 10−4 . Thus, the continuous spectrophotometric assay for adenosine deaminase should be utilized withextreme caution, and should not be employed at concentrations exceeding 1 × 10−4 , irrespective of the Km value for the substrate. Specific recommendations are given for future assays. IS - 2 JF - Analytical biochemistry SN - 0003-2697 EP - 337 TI - A critical reexamination of the continuous spectrophotometric assay for adenosine deaminase. VL - 122 SP - 328 KW - amp KW - assay KW - deaminase KW - spectrophotometry AU - Murphy, J AU - Baker, DC AU - Behling, C AU - Turner, RA PY - 1982/05/15/ UR - http://dx.doi.org/10.1016/0003-2697(82)90291-3 ER -