Effect of extrinsic fluorescent labels on diffusion and adsorption kinetics of proteins at the liquid-liquid interface

Arivalagan Gajraj, Robert Y. Ofoli

    Research output: Contribution to journalArticle

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    Abstract

    We present experimental results of the effect of fluorescent labels on the adsorption kinetics and diffusion of bovine serum albumin (BSA) at the oil-water interface. We performed comparative studies on BSA labeled with exactly 1, an average of 1.7, and exactly 2 fluorescein-5-isothiocyanate (FITC) molecules. We used total internal reflection fluorescence microscopy along with fluorescence photobleaching recovery as an in-situ, noninvasive measure of diffusion and adsorption of proteins at the interface. We used ion-exchange chromatography to exploit the difference in electronegativity of proteins with different labeling ratios to effect the separation required to prepare the monodisperse (single- and double-labeled) samples. Absorbance spectroscopy measurements at 278 nm (BSA) and 490 nm (FITC) were used to calibrate the eluant from the chromatography column and determine the labeling ratio. The results showed that the attachment of an extrinsic label has a pronounced effect on both adsorption and diffusion of proteins. For instance, the apparent diffusion coefficient of a BSA molecule conjugated with 2 FITC molecules was estimated to be 40% greater than that of BSA, to which only a single label had been attached. The effects of concentration quenching on the fluorescence recovery after photobleaching were examined, and the recovery curves were shown to be free of quenching effects, even at a labeling ratio of 2.

    Original languageEnglish (US)
    Pages (from-to)8085-8094
    Number of pages10
    JournalLangmuir
    Volume16
    Issue number21
    DOIs
    StatePublished - Oct 17 2000

    Profile

    Feline Sarcoma Viruses
    albumins
    serums
    Labels
    Adsorption
    Proteins
    proteins
    adsorption
    Mastication
    Muscle Contraction
    Molecules
    Labeling
    Recovery
    marking
    recovery
    fluorescence
    molecules
    Birth Certificates
    Hyperkalemia
    Quenching

    ASJC Scopus subject areas

    • Colloid and Surface Chemistry
    • Physical and Theoretical Chemistry

    Cite this

    Effect of extrinsic fluorescent labels on diffusion and adsorption kinetics of proteins at the liquid-liquid interface. / Gajraj, Arivalagan; Ofoli, Robert Y.

    In: Langmuir, Vol. 16, No. 21, 17.10.2000, p. 8085-8094.

    Research output: Contribution to journalArticle

    Gajraj, Arivalagan; Ofoli, Robert Y. / Effect of extrinsic fluorescent labels on diffusion and adsorption kinetics of proteins at the liquid-liquid interface.

    In: Langmuir, Vol. 16, No. 21, 17.10.2000, p. 8085-8094.

    Research output: Contribution to journalArticle

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