Effect of buffer components and carrier solvents on in vitro activity of recombinant human carboxylesterases.
BACKGROUND
The effects of buffer and substrate solvent conditions on in vitro activity of carboxylesterases (CE) have not been previously described. Therefore, it is unknown if the many different assay conditions used by various laboratories have a substantial impact on the activity of CE enzymes.


METHODS
Three human CEs were expressed and purified, and the hydrolysis of 4-nitrophenyl butyrate was measured to assess enzyme activity. Four buffers (HEPES, potassium phosphate, sodium phosphate, and Tris) were evaluated for their effects on enzyme activity at concentrations ranging from 5 to 900 mM, as well as phosphate buffered saline. Five commonly used substrate-carrier solvents (acetone, acetonitrile, dimethyl sulfoxide, ethanol, and methanol) ranging from 0.25 to 6% were also assessed for their effect on enzyme activity.


RESULTS
The clearances for the CEs in HEPES, potassium phosphate, sodium phosphate, and Tris up to 100 mM were similar to the CE clearances obtained with phosphate buffered saline. Higher buffer concentrations resulted in differential activity of the CEs. All three CEs tolerated the substrate solvents up to 2% as indicated by little effect of solvent on catalytic activity. At substrate solvent concentrations above 2% the CE activities were found to gradually decrease. In general, CES3 displayed substantially lower activity than CES1 and CES2.


CONCLUSIONS
In conclusion, any of the buffers examined up to 100 mM resulted in clearance values similar to that of phosphate buffered saline for the hydrolysis of 4-nitrophenyl butyrate by the human CEs. With regard to the substrate solvents tested, acetone, acetonitrile, or dimethyl sulfoxide appear to be well tolerated by the CEs up to 2% of the total reaction volume.
Williams
Eric T
ET
Department of Drug Disposition, Lilly Research, Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, United States.
Ehsani
Mariam E
ME
Wang
Xiliang
X
Wang
He
H
Qian
Yue-Wei
YW
Wrighton
Steven A
SA
Perkins
Everett J
EJ
D000096:Acetone / Q000737:chemistry; D000097:Acetonitriles / Q000737:chemistry; D002021:Buffers; D002087:Butyrates / Q000737:chemistry; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry; D006531:HEPES / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D000432:Methanol / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012997:Solvents / Q000737:chemistry*; D014325:Tromethamine / Q000737:chemistry
D000097:Acetonitriles; D002021:Buffers; D002087:Butyrates; D010710:Phosphates; D017680:Potassium Compounds; D011994:Recombinant Proteins; D012997:Solvents; D014325:Tromethamine; D000096:Acetone; C033592:4-nitrophenyl butyrate; D000431:Ethanol; C013216:potassium phosphate; D002265:Carboxylic Ester Hydrolases; D006531:HEPES; C018279:sodium phosphate; D000432:Methanol; D004121:Dimethyl Sulfoxide; C032159:acetonitrile