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Related Experiment Videos

N-(1-pyrene)maleimide: a fluorescent cross-linking reagent

C W Wu, L R Yarbrough

    Biochemistry
    |June 29, 1976
    PubMed
    Summary

    N-(1-Pyrene)maleimide is a fluorescent probe that can label sulfhydryl groups. It also acts as a fluorescent cross-linking reagent, revealing the proximity of amino and sulfhydryl groups in proteins.

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    Area of Science:

    • Biochemistry
    • Chemical Biology
    • Protein Chemistry

    Background:

    • N-(1-Pyrene)maleimide (NPM) is a nonfluorescent reagent in aqueous solution.
    • It forms fluorescent adducts with sulfhydryl groups, enabling detection and monitoring of conjugation reactions via fluorescence intensity.
    • The presence of primary amino groups can lead to spectral shifts in these adducts.

    Purpose of the Study:

    • To investigate the spectral shifts observed in N-(1-Pyrene)maleimide adducts.
    • To elucidate the mechanism behind these spectral shifts and their implications for protein structure.
    • To establish N-(1-Pyrene)maleimide as a fluorescent cross-linking reagent for assessing spatial proximity of functional groups in proteins.

    Main Methods:

    • Conjugation reactions of N-(1-Pyrene)maleimide with model compounds (L-cysteine, cysteamine, N-acetyl-L-cysteine, beta-mercaptoethanol) and proteins (bovine serum albumin).
    • Spectroscopic analysis (fluorescence emission spectra) to monitor spectral shifts.
    • Chemical analysis and nuclear magnetic resonance (NMR) studies to confirm reaction mechanisms.
    • Use of denaturing reagents (e.g., sodium dodecyl sulfate) to probe cross-linking dynamics.

    Main Results:

    • N-(1-Pyrene)maleimide forms fluorescent adducts with sulfhydryl groups, with conjugation rate monitored by fluorescence increase.
    • A red shift in emission spectra, characterized by the disappearance of peaks at 376, 396, 416 nm and appearance of peaks at 386, 405 nm, was observed in the presence of primary amino groups.
    • Model studies indicated this spectral shift results from intramolecular aminolysis of the succinimido ring.
    • N-(1-Pyrene)maleimide cross-links the N-terminus and cysteine residue in bovine serum albumin, confirmed by spectral shift and blocked alpha-amino group.
    • The spectral shift is dependent on the proximity of sulfhydryl and amino groups, as demonstrated by prevention with denaturants and failure with PM-glutathione.

    Conclusions:

    • N-(1-Pyrene)maleimide adducts undergo intramolecular aminolysis in the presence of primary amino groups, causing characteristic spectral shifts.
    • N-(1-Pyrene)maleimide functions as a fluorescent cross-linking reagent, providing insights into the spatial arrangement of sulfhydryl and amino groups within proteins.
    • The reagent's ability to detect proximity is critical for understanding protein structure and function.

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