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Acid Phosphatase


(Orhtophosphoric-monoester phosphohydrolase (acid optimum); EC 3.1.3.2)

Acid phosphatase is a general term associated with non-specific phosphomonoesterases with optimum activity in the pH range of 4-6. Following is an example of a reaction catalyzed by acid phosphatase:

ACID PHOSPHATASE
Orthophosphoric monoester + H2O Alcohol + H3PO4

   Acid phosphatase is widely distributed in nature in plants, animals and microorganisms. Potatoes, wheat germ, milk and bovine prostate gland are generally used as sources for commercial quantities of this enzyme. One of the most concentrated sources of acid phosphatase is the human prostate gland. Clinically, serum acid phosphatase levels are used for diagnosis of prostatic cancer (Kent, J.R., Hill, M. and Bischoff, A. Cancer, 25, 858, 1970).



1.Source: Human Prostate Gland
   Form: Freeze-dried powder 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: Stable when stored at -20C 
   Activity (approx.): 10-20 U/mg solid 
   Protein (approx.): 95% (biuret) 
   Catalog No.: 050A0020 
$12.00/U
2.Source: Wheat Germ
   Form: Freeze-dried powder 
   Solubility: Soluble in distilled water or dilute buffer 
   Stability: Stable when stored at -20C 
   Activity (approx.): 1 U/mg solid 
   Protein (approx.): 90% (biuret) 
   Catalog No.: 145A0001 
$15.00/g


The amount of enzyme which liberates one micromole p-nitrophenyl per minute at 37C and pH 4.8.



The rate of the reaction is determined by measuring the increase in absorbance at 405 nm resulting from the

release of p-nitrophenol from p-nitrophenyl phosphate.



1. 0.1 M NaOH.

2. Buffer/Substrate Solution (0.05 M sodium citrate buffer, pH 4.8, with 5.5 mM p-nitrophenyl phosphate).

3. Acid phosphatase (enzyme) solution. Dissolve in 0.05 M sodium citrate buffer, pH 4.8 to prepare a concentration of 1

mg/ml. Dilute as required. Must be prepared fresh prior to assay.



1. Set the water bath at 37C.

2. Into a two test tubes pipette the following reagents in the amounts indicated: TEST BLANK

        Buffer/Substrate solution                                          1.0 ml 1.0 ml

        Diluted enzyme                                                       0.2 ml ---------

        0.1 M NaOH                                                            --------- 1.0 ml

3. Mix and incubate test tube in water bath for 30 minutes at 37C.

4. Add the following to each test tube and mix:

         0.1 M NaOH                                                            1.0 ml ---------

         Diluted enzyme                                                        --------- 0.2 ml

5. Measure increase in absorbance at 405 nm using spectrophotometer against blank.

6. Calculate the ΔE405nm/min



Activity (U/mg) = (ΔE405nm/min)(Total Vol.)(Enz. Diln.)
(18.5)(Enz. Vol.)(mg Enz./ml)
 

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