Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography

Alexander Kurosky, Brian T. Miller, Susan L. Knock

    Research output: Contribution to journalArticle

    11 Citations (Scopus)

    Abstract

    A procedure employing C18 reversed-phase high-performance liquid chromatography (HPLC) is described for evaluating the kinetics of biotinylation of specific residues of peptides after reaction with N-hydroxysuccinimide esters of biotin. Utilizing this HPLC method we determined the observed pseudo-first-order reaction rate constant (k′1) of biotinylation of lysyl residues in two model peptides [biotinyl-Ser108]ProA-egg laying hormone (108-121) and pGlu-Lys-Trp-Ala-Pro, to be 1.22 · 10-2 s-1 and 1.08 · 10-2 s-1, respectively in 0.05 M sodium phosphate buffer, pH 8.2, at 25°C. The respective reaction half-lives of the two peptides were 57 s and 64 s. In addition, HPLC analytical methods were established for determining the time-course of hydrolysis of biotinylating reagents at acidic alkaline pH and for evaluating biotin reagent homogeneity.

    Original languageEnglish (US)
    Pages (from-to)281-287
    Number of pages7
    JournalJournal of Chromatography A
    Volume631
    Issue number1-2
    DOIs
    StatePublished - Feb 12 1993

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    Biotinylation
    High performance liquid chromatography
    High Pressure Liquid Chromatography
    Biotin
    Peptides
    Kinetics
    Reverse-Phase Chromatography
    Reaction rates
    Ovum
    Hydrolysis
    Rate constants
    Buffers
    Esters
    Hormones

    ASJC Scopus subject areas

    • Analytical Chemistry
    • Clinical Biochemistry
    • Molecular Medicine

    Cite this

    Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography. / Kurosky, Alexander; Miller, Brian T.; Knock, Susan L.

    In: Journal of Chromatography A, Vol. 631, No. 1-2, 12.02.1993, p. 281-287.

    Research output: Contribution to journalArticle

    Kurosky, Alexander ; Miller, Brian T. ; Knock, Susan L. / Kinetic analysis of biotinylation of specific residues of peptides by high-performance liquid chromatography. In: Journal of Chromatography A. 1993 ; Vol. 631, No. 1-2. pp. 281-287.
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