TY - JOUR
T1 - Capillary electrophoretic reactor for estimation of spontaneous dissociation rate of Trypsin–Aprotinin complex
AU - Sasaki, Yumiko
AU - Sato, Yosuke
AU - Takahashi, Toru
AU - Umetsu, Mitsuo
AU - Iki, Nobuhiko
N1 - Funding Information:
This research was supported by the Japan Society for the Promotion of Science (JSPS) through the Grants-in-Aid for Scientific Research (KAKENHI) (grant No. 17K05897 ).
Publisher Copyright:
© 2019 Elsevier Inc.
PY - 2019/11/15
Y1 - 2019/11/15
N2 - A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme–inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try–Apr complex to dissociate through the steady removal of free Try and Apr from the Try–Apr zone. By analyzing the dependence of peak height of Try–Apr on separation time, the dissociation rate kd H was obtained as 2.73 × 10−4 s−1 (298 K) at pH 2.46. The dependence of kd H on the proton concentration (pH = 2.09–3.12) revealed a first-order dependence of kd H on [H+]; kd H = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10−5 s−1, and k1 is the second-order rate constant and was 5.07 × 10−2 M−1 s−1. From the kd value, the half-life of the Try–Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO– of the Asp residue in Try, which breaks the salt bridge with the –NH3 + group of Lys in Apr.
AB - A capillary electrophoretic reactor was used to analyze the dissociation kinetics of an enzyme–inhibitor complex in a homogeneous solution without immobilization. The complex consisting of trypsin (Try) and aprotinin (Apr) was used as the model. Capillary electrophoresis provided a reaction field for Try–Apr complex to dissociate through the steady removal of free Try and Apr from the Try–Apr zone. By analyzing the dependence of peak height of Try–Apr on separation time, the dissociation rate kd H was obtained as 2.73 × 10−4 s−1 (298 K) at pH 2.46. The dependence of kd H on the proton concentration (pH = 2.09–3.12) revealed a first-order dependence of kd H on [H+]; kd H = kd + k1[H+], where kd is the spontaneous dissociation rate and was 5.65 × 10−5 s−1, and k1 is the second-order rate constant and was 5.07 × 10−2 M−1 s−1. From the kd value, the half-life of the Try–Apr complex at physiological pH was determined as 3.4 h. The presence of the proton-assisted dissociation can be explained by the protonation of -COO– of the Asp residue in Try, which breaks the salt bridge with the –NH3 + group of Lys in Apr.
KW - Aprotinin
KW - Biomolecular complex
KW - Capillary electrophoresis
KW - Dissociation kinetics
KW - Enzyme
KW - Inhibitor
KW - Trypsin
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U2 - 10.1016/j.ab.2019.113406
DO - 10.1016/j.ab.2019.113406
M3 - Article
C2 - 31445899
AN - SCOPUS:85071135524
VL - 585
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
M1 - 113406
ER -