Assay for beta-ureidopropionase by high-performance liquid chromatography.
A sensitive assay for beta-ureidopropionase based on derivatization of the reaction product beta-alanine with phenylisothiocyanate has been developed. Purification of the resulting phenylthiocarbamoyl-beta-alanine is achieved on a LiChrospher 100 C18 reversed-phase high-performance liquid chromatography column using an isocratic elution system. Phenylthiocarbamoyl-beta-alanine is detected by its absorbance at 245 nm and quantitated by automatic peak integration referring to a calibration curve. This technique offers a high degree of sensitivity as beta-alanine quantities in the picomole range can be identified. N-Carbamoyl-beta-alanine, the natural substrate of beta-ureidopropionase, does not interfere with the described assay system. The enzymatic reaction is linear for an incubation time of 45 min with enzyme concentrations of 3.2 micrograms/ml.
Waldmann
G
G
Institute of Physiological Chemistry, University of Würzburg, Federal Republic of Germany.
Podschun
B
B
D000409:Alanine / Q000032:analysis; D000581:Amidohydrolases / Q000032:analysis*; D000641:Ammonia; D002851:Chromatography, High Pressure Liquid; D017879:Isothiocyanates; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013861:Thiocyanates; D015091:beta-Alanine / Q000031:analogs & derivatives / Q000032:analysis / Q000302:isolation & purification
D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate; D015091:beta-Alanine; C035122:N-carbamoyl-beta-alanine; D000641:Ammonia; D000581:Amidohydrolases; C021590:beta-ureidopropionase; D000409:Alanine