Comparative Thermodynamic Analysis of DNA−Protein Interactions Using Surface Plasmon Resonance and Fluorescence Correlation Spectroscopy

Schubert, Frank; Zettl, Heiko; Hafner, Wolfgang; Krauss, Gerhard; Krausch, Georg

doi:10.1021/bi034033d

Cited by 66 publications

(53 citation statements)

References 35 publications

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“…3,49 The molecular mass of the Antp homeodomain used by Seimiya and Kurosawa 44 is 8.5 kDa, i.e., almost 5-fold lighter than HRP used in these experiments. This feature can raise the association rate of lighter molecules.…”

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confidence: 90%

“…1,3,32 An SPR interaction plot generally contains three steps: the association, the dissociation and regeneration ones. The association and dissociation steps can be used to determine the rate constants and thereby the equilibrium constants.…”

Section: Spr Studies Of the Layers Formation On Gold Surfacementioning

confidence: 99%

“…These devices show great potential in predicting genotoxicity of drugs and environmental chemicals, thus future tools in chemical product development studies are very important. 18 Schubert et al 3 reported kinetic and thermodynamic characterizations of the ss-DNA and replication protein A using SPR and fluorescence correlation spectroscopy. However, no studies about the kinetics adsorption of horseradish peroxidase and ds-DNA were reported.…”

Section: Introductionmentioning

confidence: 99%

“…The main advantage of SPR is that no particular property of the interacting molecules is required; being possible to determine the true association and dissociation rates of the complex formation. [1][2][3]33 Thus, in the present study IES and SPR techniques were employed to systematically investigate the layer-by-layer formation in the kinetic and thermodynamic point of view. …”

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confidence: 99%

“…44 The dissociation constant of our system can be characterized as weak when compared to ss-DNA-RPA binding. 3,49 Even though the association constant is not so high, it is important to consider that the ration between them, k a e k d , is still favourable. The association rate data may imply certain dependency on the enzyme structure and the availability of the ligand structure.…”

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confidence: 99%

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Kinetic studies of HRP adsorption on ds-DNA immobilized on gold electrode surface by EIS and SPR

Ferreira

Kubota

2010

J. Braz. Chem. Soc.

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Este trabalho descreve a construção de um filme de multicomponentes via método layer-bylayer (LbL) e a análise cinética da interação da enzima peroxidase de raiz forte (HRP) sobre a camada de ds-DNA de timo de bezerro formada numa superfície de ouro modificada. As técnicas de ressonância de plásmon de superfície (SPR) e a espectroscopia de impedância eletroquímica (EIE) foram usadas para caracterizar a construção do filme sobre a superfície de ouro. A ressonância de plásmon de superfície forneceu informações essenciais para o estudo e caracterização da interação proteína e ácido nucléico. Este método não necessita de marcador e monitora as interações em tempo real. Os estudos cinéticos obtidos por SPR da formação do filme de HRP sobre o ds-DNA apresentaram valores de 24,7 L mol -1 s -1 e 1,2×10 -3 s -1 para k a e k d , respectivamente. A energia livre de Gibbs obtida para o sistema foi de -23,1 kJ mol -1. Os resultados obtidos mostram que a interação das moléculas da enzima sobre o ds-DNA é cinética e termodinamicamente favoráveis e a interação entre as camadas ocorre principalmente pela atração de cargas opostas. This paper describes the construction of a multicomponent film via layer-by-layer (LbL) method and the kinetic analysis of the interaction between horseradish peroxidase (HRP) enzyme on calf thymus double-stranded DNA layer on a modified gold surface. Surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the film on the gold surface. Surface plasmon resonance provided essential information for the study and characterization of protein and nucleic acid interaction and this method is label-free and monitors the interactions in real time. The kinetic studies determined by SPR of the horseradish peroxidase film formation on ds-DNA layer showed values of 24.7 L mol -1 s -1 and 1.2×10 -3 s -1 for k a and k d , respectively. The Gibbs free energy obtained for the system was -23.1 kJ mol -1. The results obtained show that the interaction of the enzymes molecules on ds-DNA is kinetically and thermodynamically favourable and the interaction among the layers probably occurs mainly by attraction of opposite charges.Keywords: SPR, EIS, kinetic adsorption, DNA, HRP, protein-DNA interaction IntroductionThe interaction of nucleic acids with protein has been a primary area of interest for therapeutic development and in the study of cellular processes.1-3 Development of devices based on this interaction presents application in the investigations of the toxicity pathways of nucleic acid. Recently, electrochemical sensors have been developed for the rapid detection of DNA damage. [4][5][6][7] Such devices are being successfully used in food industry, environmental fields and clinical chemistry. Biosensors for such analysis can be prepared with multicomponent films of polyions and other charged materials and to achieve high specificity, high sensitivity, rapid response and flexibility of use, it is clear that the research continues to focus ...

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confidence: 90%

Section: Spr Studies Of the Layers Formation On Gold Surfacementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Kinetic studies of HRP adsorption on ds-DNA immobilized on gold electrode surface by EIS and SPR

Ferreira

Kubota

2010

J. Braz. Chem. Soc.

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Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

2008

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in Wiley InterScience (www.interscience.wiley.com).Peptides constructed from a-helical subunits of the Lac repressor protein (LacI) were designed then tailored to achieve particular binding kinetics and dissociation constants for plasmid DNA purification and detection. Surface plasmon resonance was employed for quantification and characterization of the binding of double stranded Escherichia coli plasmid DNA (pUC19) via the lac operon (lacO) to ''biomimics'' of the DNA binding domain of LacI. Equilibrium dissociation constants (K D ), association (k a ), and dissociation rates (k d ) for the interaction between a suite of peptide sequences and pUC19 were determined. K D values measured for the binding of pUC19 to the 47mer, 27mer, 16mer, and 14mer peptides were 8.8 6 1.3 3 10 210 M, 7.2 6 0.6 3 10 210 M, 4.5 6 0.5 3 10 28 M, and 6.2 6 0.9 3 10 26 M, respectively. These findings show that affinity peptides, composed of subunits from a naturally occurring operonrepressor interaction, can be designed to achieve binding characteristics suitable for affinity chromatography and biosensor devices.

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Study of Diffusion of Organic Dyes in a Triblock Copolymer Micelle and Gel by Fluorescence Correlation Spectroscopy

Ghosh

Mandal

Adhikari

et al. 2009

Chemistry An Asian Journal

118

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Fluorescence correlation spectroscopy (FCS) has been used to study translational diffusion of three fluorescent dyes in a micelle and a gel. It was demonstrated that a highly hydrophobic dye, DCM, remains confined to a particular micelle during the passage of the micellar aggregation through the confocal volume. As a result, DCM exhibits slow diffusion of the large micellar aggregate with a diffusion coefficient (D(t)) approximately 25 times slower compared with that of water. In contrast, a hydrophilic probe (C343 or C480) occasionally diffuses out of the micelle into bulk water and displays a large D(t) (twofold smaller in F127 and approximately six times smaller in the P123 micelle compared with that in bulk water). In a gel, diffusion of the individual micelles is completely arrested and hence, the autocorrelation in FCS arises solely from the diffusion of the dye in the gel. In this case, all the three dyes exhibit extremely slow diffusion (300, 45, and 20 times slower than that in water for DCM, C480, and C343 in F127 gel, respectively). In a P123 and F127 gel, diffusion of DCM is respectively, seven and 29 times slower compared with that of the ionic probe C343. The relatively small value of red-edge excitation shift (REES) of the emission maximum, suggests that DCM is confined within the core of the triblock copolymer micelles and gels. The hydrophilic probes (C343 or C480) exhibit fast diffusion in the micelles and gels. However, their REES is very different. The large REES of C480 suggests that it is distributed over a large region of the micelle, whereas the low REES of C343 indicates that it is located primarily in the peripheral corona region.

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Comparative Thermodynamic Analysis of DNA−Protein Interactions Using Surface Plasmon Resonance and Fluorescence Correlation Spectroscopy

Cited by 66 publications

References 35 publications

Kinetic studies of HRP adsorption on ds-DNA immobilized on gold electrode surface by EIS and SPR

Kinetic studies of HRP adsorption on ds-DNA immobilized on gold electrode surface by EIS and SPR

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

Study of Diffusion of Organic Dyes in a Triblock Copolymer Micelle and Gel by Fluorescence Correlation Spectroscopy

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