Abstract
Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of α-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against α-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 208-222 |
| Number of pages | 15 |
| Journal | Analytical Biochemistry |
| Volume | 315 |
| Issue number | 2 |
| DOIs | |
| State | Published - Apr 15 2003 |
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Keywords
- Cytochrome P450
- Esterase
- Fluorescent substrate
- High-throughput assay
- Human liver microsomes
- Native 2D gel electrophoresis
- Pyrethroid
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Evaluation of α-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes. / Wheelock, Craig E.; Wheelock, Åsa M.; Zhang, Rong; Stok, Jeanette E.; Morisseau, Christophe; Le Valley, Susanna E.; Green, Carol E.; Hammock, Bruce D.
In: Analytical Biochemistry, Vol. 315, No. 2, 15.04.2003, p. 208-222.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Evaluation of α-cyanoesters as fluorescent substrates for examining interindividual variation in general and pyrethroid-selective esterases in human liver microsomes
AU - Wheelock, Craig E.
AU - Wheelock, Åsa M.
AU - Zhang, Rong
AU - Stok, Jeanette E.
AU - Morisseau, Christophe
AU - Le Valley, Susanna E.
AU - Green, Carol E.
AU - Hammock, Bruce D.
PY - 2003/4/15
Y1 - 2003/4/15
N2 - Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of α-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against α-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.
AB - Carboxylesterases hydrolyze many pharmaceuticals and agrochemicals and have broad substrate selectivity, requiring a suite of substrates to measure hydrolytic profiles. To develop new esterase substrates, a series of α-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays. The use of these substrates as surrogates for Type II pyrethroid hydrolysis was tested. The results suggest that these novel analogs are appropriate for the development of high-throughput assays for pyrethroid hydrolase activity. A set of human liver microsomes was then used to determine the ability of these substrates to report esterase activity across a small population. Results were compared against standard esterase substrates. A number of the esterase substrates showed correlations, demonstrating the broad substrate selectivity of these enzymes. However, for several of the substrates, no correlations in hydrolysis rates were observed, suggesting that multiple carboxylesterase isozymes are responsible for the array of substrate hydrolytic activity. These new substrates were then compared against α-naphthyl acetate and 4-methylumbelliferyl acetate for their ability to detect hydrolytic activity in both one- and two-dimensional native electrophoresis gels. Cyano-2-naphthylmethyl butanoate was found to visualize more activity than either commercial substrate. These applications demonstrate the utility of these new substrates as both general and pyrethroid-selective reporters of esterase activity.
KW - Cytochrome P450
KW - Esterase
KW - Fluorescent substrate
KW - High-throughput assay
KW - Human liver microsomes
KW - Native 2D gel electrophoresis
KW - Pyrethroid
UR - http://www.scopus.com/inward/record.url?scp=0037446204&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0037446204&partnerID=8YFLogxK
U2 - 10.1016/S0003-2697(03)00002-2
DO - 10.1016/S0003-2697(03)00002-2
M3 - Article
C2 - 12689831
AN - SCOPUS:0037446204
VL - 315
SP - 208
EP - 222
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -