Reactivity with Tris(hydroxymethyl)aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins
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- 作者：Philip C. Burcham ; Frank R. Fontaine ; Dennis R. Petersen ; and Simon M. Pyke
- 刊名：Chemical Research in Toxicology
- 出版年：2003
- 出版时间：October 2003
- 年：2003
- 卷：16
- 期：10
- 页码：1196 - 1201
- 全文大小：87K
- 年卷期：v.16,no.10(October 2003)
- ISSN：1520-5010
文摘
The toxic 
,
-unsaturated aldehyde acrolein readily attacks proteins, generating adducts atcysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised againstacrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linkedimmunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivityand specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysinebut not polyhistidine. Efforts to develop a Western blotting method for detecting adductedproteins in cell lysates were hampered by irreproducible outcomes, evidently due to adductinstability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model proteinto acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was moststriking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formedduring extended exposure to acrolein (
60 min) were completely stable to Tris. The timedependence of susceptibility raised the possibility that Tris interfered with specific steps inlysine modification, which involves stepwise Michael addition of two molecules of acrolein tothe same residue, followed by condensation and dehydration to form a heterocyclic adduct,N
-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michaeladducts may react with Tris by forming imines with the primary amine of the buffer. Consistentwith this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effecton acrolein-adducted protein. These effects of Tris may explain difficulties in the detection ofacrolein-adducted proteins during conventional Western blotting procedures.
,-unsaturated aldehyde acrolein readily attacks proteins, generating adducts atcysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised againstacrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linkedimmunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivityand specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysinebut not polyhistidine. Efforts to develop a Western blotting method for detecting adductedproteins in cell lysates were hampered by irreproducible outcomes, evidently due to adductinstability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model proteinto acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was moststriking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formedduring extended exposure to acrolein (60 min) were completely stable to Tris. The timedependence of susceptibility raised the possibility that Tris interfered with specific steps inlysine modification, which involves stepwise Michael addition of two molecules of acrolein tothe same residue, followed by condensation and dehydration to form a heterocyclic adduct,N-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michaeladducts may react with Tris by forming imines with the primary amine of the buffer. Consistentwith this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effecton acrolein-adducted protein. These effects of Tris may explain difficulties in the detection ofacrolein-adducted proteins during conventional Western blotting procedures.