Reactivity with Tris(hydroxymethyl) aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins

Philip Burcham, F.R. Fontaine, D.R. Petersen, S.M. Pyke

    Research output: Contribution to journalArticle

    12 Citations (Scopus)

    Abstract

    The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (greater than or equal to60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.
    Original languageEnglish
    Pages (from-to)1196-1201
    JournalChemical Research in Toxicology
    Volume16
    Issue number10
    DOIs
    Publication statusPublished - 2003

    Fingerprint

    Acrolein
    Tromethamine
    Proteins
    Lysine
    Buffers
    Imines
    Amines
    Immune Sera
    Western Blotting
    Immunosorbents
    Polylysine
    Poisons
    Electrophoresis
    Dehydration
    Histidine
    Aldehydes
    Cysteine
    Polyacrylamide Gel Electrophoresis
    Condensation
    Assays

    Cite this

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    title = "Reactivity with Tris(hydroxymethyl) aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins",
    abstract = "The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (greater than or equal to60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.",
    author = "Philip Burcham and F.R. Fontaine and D.R. Petersen and S.M. Pyke",
    year = "2003",
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    Reactivity with Tris(hydroxymethyl) aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins. / Burcham, Philip; Fontaine, F.R.; Petersen, D.R.; Pyke, S.M.

    In: Chemical Research in Toxicology, Vol. 16, No. 10, 2003, p. 1196-1201.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Reactivity with Tris(hydroxymethyl) aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins

    AU - Burcham, Philip

    AU - Fontaine, F.R.

    AU - Petersen, D.R.

    AU - Pyke, S.M.

    PY - 2003

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    N2 - The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (greater than or equal to60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.

    AB - The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (greater than or equal to60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N-epsilon-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.

    U2 - 10.1021/tx0341106

    DO - 10.1021/tx0341106

    M3 - Article

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    EP - 1201

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    JF - Chemical Research in Toxicology

    SN - 0893-228X

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