| | Re: for estimation of acid phosphatase
Acid phosphatase (AcP) hydrolyze disodium phenylphosphate in acidic medium (pH 4.9) to form phosphate and phenol. In fixed time, reaction is, by sodium arsenate, stopped and phenol liberated is condensed by 4-aminoanipyrine in alkaline (pH 7.9 ± 0.1 or 10.0) medium and in presence of potassium ferricyanide in a red complex that showed a absorption peak about 500 – 520 nm. This method, by me modified, is applicable in the phenol concentration range of 1 μg/L to 250 μg/L with a sensitivity of μg/L.
Substrate buffer (citrate buffer 0.2 M at pH 4.9 containing 10 mM of disodium phenylphosphate) : dissolve in 200 ml dH2O, 21.0 g di citric acid monohydrate, add 188 ml of 1 M NaOH and dilute to 500 ml with dH2O. Adjust the pH to 4.9, if necessary, with molar NaOH or molar HC1, then dissolve 10 mM of disodium phenylphosphate, 05 g/L NaN3 and keep at 4 °C, stable one year in opaque plastic bottle.
Blocking reagent : 30 mM 4-aminoantipyrine, 120 mM sodium arsenate dibasic heptahydrate (Fluka cod. 71625) in NH4OH/NH4Cl buffer at pH 10 ± 0.2 (dissolve 67.5 g of ammonium chloride in 570 ml of ammonium hydroxyde, and add freshly boiled and cooled water to make 1 liter), and 0.5 g/L NaN3. Keep at 4 °C, stable one year in opaque plastic bottle.
Color reagent : 75 mM potassium ferricyanide (III) anhydrous in phosphate buffer 0.2 M at pH 7.4 ± 0.2 (dissolve 4.415 g/L NaH2PO4, 22.5075 g/L Na2HPO4•Heptahydrate pH should be 7.4, if not adjust with 1.0 N NaOH or 1.0 N HCl) and 0.5 g/L NaN3. Filter if necessary and store in a brown glass bottle or in dark plastic bottle at 4 °C.
Mix 0.2 ml of serum or cell lysates, through twice cycle of frozen and thawed, with 0.8 ml of substrate buffer and incubate for 1 hr at 37 °C, then add 1 ml blocking reagent, mix and incubate for 10 min in the dark. In last, dispense 1 ml color reagent, mix again and incubate for 10 min. At the finish read Abs at 510 nm.
The inhibition of this activity was also measured after adding 0.05 ml of 1 M sodium L-tartrate to a sample
before its incubation for 1 h at 37 °C.
Acid phosphatase activity units were expressed as nM of phenol produced per minute per 106 cells