Continuous assay for acid phosphatase using 1-naphthyl phosphate as a substrate.
The described continuous acid phosphatase assay is based on kinetics of the release of 1-naphthol in the course of the enzyme-catalyzed hydrolysis of 1-naphthyl phosphate, measured at 320 nm in aqueous solution and at 322 nm in sodium-bis(2-ethylhexyl)sulfosuccinate isooctane-water reverse micelles in a broad pH range (1.0-8.2). The method allows precise determination of the initial rate of the reaction and therefore may be used in the steady-state and pre-steady-state studies on the phosphatase-catalyzed reaction. The kinetic parameters (Km and kcat) for human prostatic acid phosphatase in aqueous solution and in reverse micelles, at pH 3.8, 4.5 and 5.7, by the proposed 1-naphthyl phosphate assay have been determined.
Luchter-Wasylewska
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Institute of Medical Biochemistry, Jagiellonian University, Collegium Medicum, Cracow, Poland. mbwasyle@cyf-kr.edu.pl
D000135:Acid Phosphatase / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008823:Micelles; D009281:Naphthalenes / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D009284:Naphthols; D009943:Organophosphorus Compounds / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D011467:Prostate / Q000201:enzymology; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D008823:Micelles; D009281:Naphthalenes; D009284:Naphthols; D009943:Organophosphorus Compounds; D012996:Solutions; C029350:1-naphthol; C014694:naphthyl phosphate; D000135:Acid Phosphatase