Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection.
An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.
Thompson
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Chemistry Department, Oberlin College, Ohio 44074.
Barone
G C
GC
Halsall
H B
HB
Heineman
W R
WR
D000469:Alkaline Phosphatase / Q000032:analysis*; D000814:Aniline Compounds / Q000378:metabolism; D004563:Electrochemistry; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D013379:Substrate Specificity
D000814:Aniline Compounds; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C068798:4-aminophenylphosphate; D000469:Alkaline Phosphatase; C024836:4-nitrophenol