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

  • 27 Citations

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.

LanguageEnglish (US)
Pages8085-8094
Number of pages10
JournalLangmuir
Volume16
Issue number21
DOIs
StatePublished - Oct 17 2000

Profile

liquid-liquid interfaces
Bovine Serum Albumin
albumins
serums
Labels
Labeling
Fluorescein-5-isothiocyanate
proteins
Proteins
Adsorption
Photobleaching
Kinetics
adsorption
marking
kinetics
Liquids
Recovery
Molecules
recovery
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

@article{486f9194e06040dab578c31a41b821d7,
title = "Effect of extrinsic fluorescent labels on diffusion and adsorption kinetics of proteins at the liquid-liquid interface",
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.",
author = "Arivalagan Gajraj and Ofoli, {Robert Y.}",
year = "2000",
month = "10",
day = "17",
doi = "10.1021/la000305r",
language = "English (US)",
volume = "16",
pages = "8085--8094",
journal = "Langmuir",
issn = "0743-7463",
publisher = "American Chemical Society",
number = "21",

}

TY - JOUR

T1 - Effect of extrinsic fluorescent labels on diffusion and adsorption kinetics of proteins at the liquid-liquid interface

AU - Gajraj,Arivalagan

AU - Ofoli,Robert Y.

PY - 2000/10/17

Y1 - 2000/10/17

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034300035&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034300035&partnerID=8YFLogxK

U2 - 10.1021/la000305r

DO - 10.1021/la000305r

M3 - Article

VL - 16

SP - 8085

EP - 8094

JO - Langmuir

T2 - Langmuir

JF - Langmuir

SN - 0743-7463

IS - 21

ER -