Acid ceramidase assay

From LipidomicsWiki

Determination of acid ceramidase activity with a micellar assay system

Reaction principle: pre-column derivatization of reaction product, separation, and fluorimetric detection  

1. Incubation mixture and assay conditions
The incubation mixture contains in a final volume of 100 µL:
a) 10-50 µL undiluted enzyme solution
b) 17 nmol N-lauroyl-sphingosine, dissolved in 0.5% (w/v) Triton X-100, 0.2% (w/v) Tween 20, 0.2% (w/v) NP-40, and 0.8% (w/v) sodium cholate, 250 mM sodium acetate buffer (pH 4.0)
c) 5 mM EDTA.
Incubation time: 30 min at 37°C.

2. Termination and recovery of the released reaction product
The enzyme reaction is terminated by adding 800 µL HPLC-grade (2:1, v/v) chloroform/methanol and 200 µL of a 100 mM ammonium hydrogen carbonate solution. D-erythro-1,3-dihydroxy-2-aminotetradecane (C₁₄-sphinganine) and D-erythro-1,3-dihydroxy-2-aminohexadecane (C₁₆--sphinganine), solubilized in HPLC-grade methanol, are added to the incubation mixture as internal standards in an amount of 500 pmol each. Samples are mixed for 10 min and then centrifuged for 5 min at 10,000 x g. The (lower) chloroform phase containing the sphingosine liberated in the enzyme-catalyzed reaction together with the internal standards is carefully removed and collected in a new vial. For quantitative recovery of sphingosine and the internal standards, the extraction is repeated by adding 600 µL (2:1 v/v) chloroform/methanol to each incubation vial, followed by mixing, centrifugation, and collection of the chloroform phase as described above. The combined organic phases are evaporated to dryness in a stream of nitrogen.The dried enzyme assays are solubilized in 50 µL HPLC-grade ethanol and mixed for 10 min at 50°C.

3. HPLC analysis of sphingoid bases after pre-column derivatization
The samples are cooled down to room temperature, and 10 µL o-phthaldialdehyde reagent solution complete (Sigma, contains beta-mercaptoethanol) is added to each incubation vial. Samples are mixed for 5 min at room temperature, and are then diluted with 960 µL HPLC buffer (acetonitril/2 mM potassium phosphate, pH 7.0, 9/1 v/v ) to a final volume of 1 mL. An aliquot of each sample (100-200 µL) is injected onto a Purosphere™ RP18-e column (125 x 4 mm, Merck), equilibrated in HPLC buffer. The derivatized sphinganine and sphingosine bases are isocratically eluted (flow rate: 1 mL/min, Shimadzu LC-10 AT solvent delivery system) and detected by a fluorescence detector (Shimadzu RF-10 AX, excitation wavelength: 355 nm, emission wavelength: 435 nm).
Quantification of liberated sphingosine is performed with a Windows-based standard liquid chromatography software (Class CR-10, Shimadzu).

4. Source: Modified from: <ref>Bernardo K, Hurwitz R, Zenk T, Desnick RJ, Ferlinz K, Schuchman EH, Sandhoff K, Purification, characterization, and biosynthesis of human acid ceramidase. J Biol Chem. 1995, 270, 11098-102</ref> according to H. Schulze, Dissertation Universität Bonn 2007.