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Cholesterol Esterase (CE)

(Sterol-ester hydrolase; EC

Cholesterol esterase (CE) is also known as cholesterol ester hydrolase. This enzyme catalyzes the following reaction:

Sterol Ester --------> Sterol + Fatty Acid 
Cholesterol esterase activity has been demonstrated in pancreas, intestine, liver and kidney. The enzyme is inactivated by proteolytic enzymes but stabilized by proteolytic enzyme inhibitors and by bile salts. CE from rat pancreas has a molecular weight of 65,000-69,000. In the presence of bile salts, it aggregates to a hexamer which is possibly the active form of the enzyme (Hyun, J., Steinberg, M., Treadwell, C.R., and G.V. Vahouny, Biochem. Biophys. Res. Comm., 44, 819, 1971).

Cholesterol esterase is widely used in clinical medicine for determination of serum cholesterol (Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and P.C. Fu, Clin Chem, 20, 470, 1974).

1.Source: Candida Rugosa
   Form: Freeze-dried powder 
   Solubility: Distilled water or dilute buffer 
   Stability: Store at -20 C (-4 F) 
   Activity (approx.): 25-100 U/mg 
   Protein (approx.): 90% (biuret) 
   Catalog No.: 134A0025 

That amount of enzyme which will catalyze the hydrolysis of one micromole of cholesterol ester, in the presence of sodium cholate, per minute at 37C and pH 6.7.

Cholesterol esterase (CE) activity (please inquire with Calzyme for the assay method for cholesterol esterase from Candida rugosa) is assayed in a coupled reaction sequence where the free cholesterol formed by CE is oxidized by cholesterol oxidase (ChO) to cholest-4-en-3-one with the simultaneous production of hydrogen peroxide. Finally, in the presence of peroxidase, the H2O2 oxidatively couples with 4-aminoantipyrine to produce a chromogen which has an absorbance maximum at 500 nm (Allain, C.C.; Poon, L.S.; Chan, C.S.G.; Richmond, W. and P.C. Fu, Clin Chem., 20, 470, 1974).

  1. 0.1 M Potassium phosphate buffer, pH 6.7.
  2. Combined Reagent - 100 ml. Prepare by dissolving the following in 100 ml 0.1M potassium phosphate buffer, pH 6.7. Prepare fresh immediately prior to assay

    Sodium cholate 129 mg
    4-aminoantipyrine 16 mg
    Phenol 132 mg
    Polyethylene glycol-6000 102 mg
    Peroxidase 200 Units
    Cholesterol oxidase 25 Units

  3. Substrate - Serachol (General Diagnostics) Specs: >375 mg/dl esters.
  4. Cholesterol esterase (CE) solution (0.1-0.2 U/ml). Dissolve 10 mg of enzyme in 10 ml 0.1 M phosphate buffer. Dilute further, in the same buffer, to yield a final concentration of 0.1-0.2 U/ml. Must be prepared fresh immediately prior to assay.

  1. Set spectrophotometer (equipped with a strip chart recorder and temperature control) at 500 nm and 37C.
  2. Into a cuvette pipette the following:

    Combined Reagent 3.00 ml
    Substrate 0.05 ml

  3. Incubate cuvette in spectrophotometer at 37C for 10 min. to attain temperature equilibration.
  4. Record blank rate at 500 nm.
  5. Add 0.1 ml enzyme solution to the cuvette. Mix and record the increase in absorbance at 500 nm for 8-10 min.
  6. Calculate the (delta)E500 nm/min from the linear portion of the curve.

Activity (U/mg) = (ΔE500nm/min)(Total Vol.)(Enz. Diln.)
(5.33)(Enz. Vol.)(mg Enz./ml)

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