Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolases and Phosphoric Triester Hydrolases

Douglas D. Anspaugh1, R. Michael Roe1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.10
DOI:  10.1002/0471140856.tx0410s13
Online Posting Date:  November, 2002
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Abstract

Assays for the Classification of Two Types of Esterases: Carboxylic Ester Hydrolase and Phosphoric Triester Hydrolase (Douglas D. Anspaugh and Michael Roe, North Carolina State University, Raleigh, North Carolina). This unit describes assays that quantitate two types of esterase the carboxylic ester hydrolases and the phosphoric triester hydrolases. Carboxylic ester hydrolases include the B‐esterases, which are inhibited by organophosphorus compounds. Among the phosphoric triester hydrolases is aryldialkylphosphatase, which has been called A‐esterase or paraoxonase due to its ability to oxidize paraoxon and other organophosphates. These assays are colorimetric and miniaturized for rapid simultaneous testing of multiple, small‐volume samples in a microtiter plate format. There is also a discussion of the history of esterase nomenclature and the reasons why this large group of enzymes is so difficult to classify.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of Carboxylic Ester Hydrolase (CEH) Activity
  • Support Protocol 1: CEH Specific Activity Calculations
  • Basic Protocol 2: Measurement of Phosphoric Triester Hydrolase (PTEH) Activity
  • Support Protocol 2: PTEH Specific Activity Calculations
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of Carboxylic Ester Hydrolase (CEH) Activity

  Materials
  • 100 µmol/ml 1‐naphthol (see recipe)
  • 200 mM 1‐naphthyl acetate (see recipe)
  • 0.3% (w/v) Fast Blue B Salt solution containing 3.4% (w/v) SDS (see recipe)
  • 100 mM methyl paraoxon (see recipe)
  • Enzyme sample (purified protein or clarified homogenate)
  • 100 mM sodium phosphate assay buffer, pH 7.4 (see recipe)
  • Spectrophotometer for microtiter plates with temperature control
  • Plastic 96‐well, flat‐bottom microtiter plates

Support Protocol 1: CEH Specific Activity Calculations

  Materials
  • 100 µmol/ml p‐nitrophenol (see recipe)
  • 100 mM methyl paraoxon (see recipe)
  • Enzyme sample (purified or clarified homogenate)
  • 100 mM Tris⋅Cl assay buffer, pH 8.0 (see recipe)
  • Spectrophotometer for microtiter plates with temperature control
  • Plastic 96‐well, flat‐bottom microtiter plates
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Figures

Videos

Literature Cited

Literature Cited
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   Aldridge, W.N. 1953. Serum Esterases 1. Two types of esterase (A and B) hydrolysing p‐nitrophenyl acetate, propionate, and butyrate, and a method for their determination. Biochem. J. 53:110‐117.
   Aldridge, W.N. 1989. A‐esterases and B‐esterases in perspective. In Enymes Hydrolyzing Organophosphorus Compounds (E. Reiner, W.N. Aldridge, and F.C.G. Hoskin, eds.) pp. 1‐14. John Wiley and Sons, New York.
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   Gomori, G. 1953. Human esterases. J. Lab. Clin. Med. 42:445‐453.
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   Mackness, M.I. 1989. Possible medical significance of human serum ‘A’‐esterases. In Enzymes Hydrolyzing Organophosphorus Compounds (E. Reiner, W.N. Aldridge, and F.C.G. Hoskin, eds.) pp.202‐213. John Wiley and Sons, New York.
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   Roe, R.M., Jesudason, P., Venkatesh, K., Kallapur, V.L., Anspaugh, D.D., Majumder, C., Linderman, R.J., and Graves, D.M. 1993. Developmental role of juvenile hormone metabolism in Lepidoptera. Amer. Zool. 33:375‐383.
   Shirai, K., Ohsawa, J., Saito, Y., and Yoshida, S. 1988. Effects of phospholipids on hydrolysis of trioleoylglycerol by human serum carboxylesterase. Biochim. Biophys. Acta 962:377‐383.
   Shishido, T. and Fukami, J. 1972. Enzymatic hydrolysis of diazoxon in rat tissue homogenates. Pestic. Biochem. Physiol. 2:39‐50.
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   van Asperen, K. 1962. A study of housefly esterases by means of a sensitive colorimetric method. J. Insect Physiol. 8:401‐416.
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   Walker, C.H. 1989. The development of an improved system of nomenclature and classification of esterases. In Enymes Hydrolyzing Organophosphorus Compounds (E. Reiner, W.N. Aldridge, and F.C.G. Hoskin, eds.) pp.236‐245. John Wiley and Sons, New York.
Key References
   Abdel‐Aal et al., 1990. See above.
  Describes, in detail, the carboxylic ester hydrolase assay (called 1‐naphthyl acetate esterase assay in that publication) that was modified and miniaturized for microtiter plate format. A modification of this procedure is presented in .
   Aldridge, 1953. See above.
  Original description of A‐ and B‐esterase distinction.
   Devorshak and Roe, 2001. See above.
  Describes, in detail, the phosphoric triester hydrolase assay that was miniaturized for microtiter plate format. This procedure was modified (see ) to provide endpoint absorbance data.
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