NMR Analysis of Thiodiglycol Oxidation by Mammalian Alcohol Dehydrogenases

Mark J. Novak1, Alan A. Brimfield2

1 Florida Institute of Technology, Melbourne, Florida, 2 United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving Ground, Maryland
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.20
DOI:  10.1002/0471140856.tx0420s29
Online Posting Date:  September, 2006
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Abstract

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for elucidating the metabolism of xenobiotics, as it allows for the least ambiguous assignment of chemical structure when compared to other forms of spectroscopy. In addition, it is a sensitive technique that can reveal the presence of transient species that otherwise would not be detected by utilizing either largeā€scale batch processes or other forms of spectroscopic analyses. The primary focus of this unit describes the use of NMR to identify metabolites arising from the oxidation of thiodiglycol by equine and human variants of alcohol dehydrogenase (ADH). Given that it is often risky to base metabolism studies on a single form of spectroscopy, a spectrophotometric method is also presented. In addition, incorporation of independent organic syntheses in conjunction with the spectroscopic studies to further solidify structural identification of the ADH metabolites is presented.

Keywords: alcohol dehydrogenase; NMR; spectroscopy; thiodiglycol; oxidative metabolism

     
 
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Table of Contents

  • Basic Protocol 1: UV/Visible Spectrophotometric Analysis of ADH Activity
  • Basic Protocol 2: 1H NMR Analysis of Thiodiglycol Oxidation by Alcohol Dehydrogenase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: UV/Visible Spectrophotometric Analysis of ADH Activity

  Materials
  • β‐nicotinamide adenine dinucleotide (NAD) solution, warmed to 30°C
  • Thiodiglycol (see recipe)
  • Equine and human alcohol dehydrogenases (ADH; see recipe for equine liver ADH), on ice
  • UV/visible spectrophotometer equipped with a constant temperature cuvette compartment (e.g., Beckman DU 70 or equivalent)
  • Standard 1.0‐cm quartz cuvettes
  • 30°C water bath
  • 2.0‐ml polypropylene microcetrifuge tubes (Eppendorf)

Basic Protocol 2: 1H NMR Analysis of Thiodiglycol Oxidation by Alcohol Dehydrogenase

  Materials
  • 0.1 M sodium phosphate buffer, pH 7.5/10% (v/v) D 2O (see recipe)
  • Lactate dehydrogenase (LDH from rabbit muscle; Sigma‐Aldrich)
  • β‐Nicotinamide adenine dinucleotide (NAD)
  • Sodium pyruvate
  • Thiodiglycol (Sigma‐Aldrich)
  • 1.6 U/mg equine or human alcohol dehydrogenase (ADH)
  • 2.0‐ and 5.0‐ml microcentrifuge tubes
  • 10.0‐ml centrifuge tubes
  • 5.0‐mm NMR tubes
  • 600‐MHz NMR spectrometer with variable temperature probe
NOTE: The individual steps in this protocol involve making and analyzing a multicomponent solution. Therefore, the solution recipes will not be repeated in Reagent and Solutions.
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Figures

Videos

Literature Cited

Literature Cited
   Abeles, R.H. and Lee, H.A. 1960. The dismutation of formaldehyde by alcohol dehydrogenase. J. Biol. Chem. 274:7106‐7107.
   Black, R.M., Brewster, K., Clark, R.J., Hambrook, J.L., Harrison, J.M., and Howells, D.J. 1993. Metabolism of thiodiglycol (2,2′‐thiobis‐ethanol): Isolation and identification of urinary metabolites following intraperitoneal administration to rat. Xenobiotica 23:473‐481.
   Brimfield, A.A., Zweig, L.M., Novak, M.J., and Maxwell, D.M. 1998. In vitro oxidation of the hydrolysis product of sulfur mustard, 2,2′‐thiobis‐ethanol, by mammalian alcohol dehydrogenases. J. Biochem. Mol. Toxicol. 12:361‐369.
   Corey, E.J. and Schmidt, G. 1975. Useful procedures for the oxidation of alcohols involving pyridinium dichromate in aprotic media. Tetrahedron Lett. 5:399‐402.
   Dudley, B.F., Brimfield, A.A., and Winston, G.W. 2000. Oxidation of 2,2′‐thiodiethanol by alcohol dehydrogenase: Comparison of human isozymes. J. Biochem. Mol. Toxicol. 14:244‐251.
   Henehan, G.T.M. and Oppenheimer, N.J. 1993. Horse liver alcohol dehydrogenase‐catalyzed oxidation of aldehydes: Dismutation precedes net production of reduced nicotinamide adenine dinucleotide. Biochemistry 32:735‐738.
   Hore, P.J. 1989. Nuclear magnetic resonance. Solvent suppression. Methods Enzymol. 176:64‐77.
   Silverstein, R.M., Webster, F.X., and Kiemle, D.J. 2005. Spectrometric Identification of Organic Compounds, 7th ed. John Wiley & Sons, Hoboken, N.J.
   Trivic, S. 1998. Influence of tris(hydroxymethyl) aminoethane on kinetic mechanism of yeast alcohol dehydrogenase. J. Enzy. Inhib. 13:57‐68.
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