A sensitive detection method for peptide using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to measure prolyl endopeptidase activity.
A measuring method sensitive to prolyl endopeptidase (EC 3.4.21.26, PEP) activity using native peptides (Arg-vasopressin or substance P) as substrates was established. The investigation of three different derivatization reagents, which had been developed for an amino acid analysis, demonstrated that 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (NBDF) was the most suitable for the detection of Arg-Gly-NH2, which was released from Arg-vasopressin by PEP. Arg-Gly-NH2 was reacted with NBDF at 65 degrees C for 5 min at pH 7.6 and the reaction mixture was analysed by HPLC on a reverse-phase column by monitoring the fluorescence intensity. The detection limit was 1 picomol per injection and the linear standard calibration curve could be constructed in the range of 1 to 100 picomol per injection with a 3.0% relative standard deviation. This sensitive detection method for peptide was applied to the measurement of PEP activity using Arg-vasopressin as a substrate and 1 x 10(-3) unit of PEP activity was detectable. This method was also applicable to the measurement of PEP activity using substance P as a substrate by detecting the derivative of its fragment peptide (Arg-Pro-Lys-Pro).
Yoshinaga
K
K
Central Research Laboratories, Zeria Pharmaceutical Co. Ltd., Saitama, Japan.
Kobayashi
N
N
Nagatani
Y
Y
Tanaka
Y
Y
Ikeda
Y
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives*; D000595:Amino Acid Sequence; D000818:Animals; D001127:Arginine Vasopressin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides; D001127:Arginine Vasopressin; C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D013373:Substance P; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D009327:4-Chloro-7-nitrobenzofurazan