Mechanism and kinetics of secretion degradation in aqueous solutions.
In order to clarify the mechanism of secretin degradation in aqueous solutions, the formation of degradation products from secretin, aspartoyl3 secretin and beta-aspartyl3 secretin was investigated; the stabilities of these three peptides were investigated as well. Aspartoyl3 secretion and beta-aspartyl3 secretin, degradation peptides produced during the storage of secretin in aqueous solutions, were isolated by preparative reversed-phase HPLC (RP-HPLC). The amounts of secretin and its two degradation peptides resulting from storage of secretin in various buffer solutions (pH 2.3 to 10.0, mu = 0.5 M, 60 degrees C) were determined by analytical RP-HPLC. Secretin and the isolated degradation peptides were stored separately in various aqueous buffer solutions resulting in the degradation of each peptide. A mixture of secretin and its degradation or cleavage peptides was formed in each solution. The observed degradation rates for each peptide approximately followed first-order kinetics. The pH-rate profiles for conversion of secretin and beta-aspartyl3 secretin were similar, while that for aspartoyl3 secretin was different from these two. Aspartoyl3 secretin was more stable than the others at pH 2.3 to 4.0, but it was easily degraded between pH 5.0 and 10.0. Investigation of aspartoyl3 secretin degradation showed that its degradation was related to the pH value of the solution, and that hydroxide ion catalyzes the ring opening of the aspartoyl peptide. Secretin was most stable in pH 7.0 buffer solution and more stable in acidic solutions than in alkaline solutions. Secretion was mainly degraded through the following pathways: cleavage peptides reversible secretin in equilibrium aspartoyl peptide in equilibrium beta-aspartyl peptide vector cleavage peptides.
Tsuda
T
T
Research and Development Division, Eisai Company, Ltd., Ibaraki, Japan.
Uchiyama
M
M
Sato
T
T
Yoshino
H
H
Tsuchiya
Y
Y
Ishikawa
S
S
Ohmae
M
M
Watanabe
S
S
Miyake
Y
Y
D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D012633:Secretin / Q000031:analogs & derivatives / Q000032:analysis* / Q000493:pharmacokinetics; D012996:Solutions
D002021:Buffers; D007202:Indicators and Reagents; D012996:Solutions; D012633:Secretin; C064168:secretin, beta-Asp(3)-; C034421:secretin, Asp(3)-