An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives |
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Authors: | Thierry Levade Robert Salvayre Jean Sicre Louis Douste-Blazy |
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Institution: | 1. INSERM Unité 101, Biochimie des Lipides, Hôpital Purpan, 37 allées Jules Guesde, 31000 Toulouse, France;2. Laboratoire de Biochimie Médicale, Faculté de Médecine Purpan, 37 allées Jules Guesde, 31000 Toulouse, France |
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Abstract: | 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. |
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