TY - JOUR
T1 - Entrapment of alpha1-acid glycoprotein in high-performance affinity columns for drug-protein binding studies
AU - Bi, Cong
AU - Jackson, Abby
AU - Vargas-Badilla, John
AU - Li, Rong
AU - Rada, Giana
AU - Anguizola, Jeanethe
AU - Pfaunmiller, Erika
AU - Hage, David S.
PY - 2016/5/15
Y1 - 2016/5/15
N2 - A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37 °C for carbamazepine with AGP was found to be 1.0 (±0.5) × 105 M-1, which agreed with a previously reported value of 1.0 (±0.1) × 105 M-1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.
AB - A slurry-based method was developed for the entrapment of alpha1-acid glycoprotein (AGP) for use in high-performance affinity chromatography to study drug interactions with this serum protein. Entrapment was achieved based on the physical containment of AGP in hydrazide-activated porous silica supports and by using mildly oxidized glycogen as a capping agent. The conditions needed for this process were examined and optimized. When this type of AGP column was used in binding studies, the association equilibrium constant (Ka) measured by frontal analysis at pH 7.4 and 37 °C for carbamazepine with AGP was found to be 1.0 (±0.5) × 105 M-1, which agreed with a previously reported value of 1.0 (±0.1) × 105 M-1. Binding studies based on zonal elution were conducted for several other drugs with such columns, giving equilibrium constants that were consistent with literature values. An entrapped AGP column was also used in combination with a column containing entrapped HSA in a screening assay format to compare the binding of various drugs to AGP and HSA. These results also agreed with previous data that have been reported in literature for both of these proteins. The same entrapment method could be extended to other proteins and to the investigation of additional types of drug-protein interactions. Potential applications include the rapid quantitative analysis of biological interactions and the high-throughput screening of drug candidates for their binding to a given protein.
KW - Alpha-acid glycoprotein
KW - Drug-protein binding
KW - Entrapment
KW - High-performance affinity chromatography
KW - Human serum albumin
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U2 - 10.1016/j.jchromb.2015.11.021
DO - 10.1016/j.jchromb.2015.11.021
M3 - Article
C2 - 26627938
AN - SCOPUS:84949034151
VL - 1021
SP - 188
EP - 196
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
SN - 1570-0232
ER -