An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives.
Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.
D002863:Chromogenic Compounds; D003124:Colorimetry; D004340:Drug Contamination; D005697:Galactosides; D005960:Glucosides; D006454:Hemoglobins / Q000302:isolation & purification*; D006867:Hydrolases / Q000302:isolation & purification*; D008247:Lysosomes / Q000201:enzymology*; D010767:Phosphorylcholine / Q000031:analogs & derivatives; D013108:Sphingomyelin Phosphodiesterase / Q000032:analysis; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D005697:Galactosides; D005960:Glucosides; D006454:Hemoglobins; D010767:Phosphorylcholine; C010733:2-hexadecanoylamino-4-nitrophenylphosphorylcholine; C013552:2-hexadecanoylamino-4-nitrophenyl-D-glucopyranoside; C014583:2-hexadecanoylamino-4-nitrophenyl-beta-D-galactopyranoside; D006867:Hydrolases; D013108:Sphingomyelin Phosphodiesterase