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A sensitive detection method for peptide using 4‐fluoro‐7‐nitrobenozo‐2‐oxa‐1,3‐diazole and its application to measure prolyl endopeptidase activity
Author(s) -
Yoshinaga Koji,
Kobayashi Naomi,
Nagatani Yasunori,
Tanaka Yoshiaki,
Ikeda Yugo
Publication year - 1994
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130080610
Subject(s) - chemistry , chromatography , detection limit , peptide , derivatization , calibration curve , endopeptidase , reagent , standard curve , high performance liquid chromatography , substrate (aquarium) , reversed phase chromatography , biochemistry , enzyme , organic chemistry , oceanography , geology
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‐NH 2 , which was released from Arg‐vasopressin by PEP. Arg‐Gly‐NH 2 was reacted with NBDF at 65°C for 5 min at pH 7.6 and the reaction mixture was analysed by HPLC on a reverse‐phase column by monitoring the gluorescence 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 × 10 −3 unit of PEP activity was detectable. This method was also applicable to the measurement of PEP activity Using subsytance P as a substrate by detecting the derivative of its fragment peptide (Arg‐Pro‐Lys‐Pro).