Rayleigh Light-Scattering Spectroscopy: Application to the Determination of Proteins Using Bromopyrogallol Red

Qi, Chun; Li, Ke An; Shen, Tong

doi:10.1246/bcsj.70.129

Cited by 45 publications

(10 citation statements)

References 16 publications

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“…As Table 3 shows, the present method is much more sensitive than the reported RLS methods using other organic dyes, including azo, triphenylmethane, and porphrins. [9][10][13][14][15][16][17][18][19][20][21][22][23][24] It was reported earlier [4][5][6] that enhanced RLS intensity mainly depends on the electrical properties of the individual chromophores, the strength of the electrical interaction between the chromophores and biomolecules, and the size of the thus-formed complex. Sometimes, the above-mentioned factors exert simultaneously effects, but sometimes one factor may display more significant than others.…”

Section: Calibration Curvesmentioning

confidence: 99%

A Resonance Light-Scattering Determination of Proteins with Fast Green FCF

Li¹,

Huang²,

Li³

2002

ANAL. SCI.

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Proteins assays are important in both analytical biochemistry and clinical tests. The common quantitative methods, including Biuret, 1 Bradford 2 and Lowry 3 assays, are spectrophotometric or spectrofluorometric tests through the binding of proteins with organic dyes, where color changes should be observed. However, most of these methods suffer from the disadvantages of low sensitivity and selectivity. Therefore, these dyes, whose interactions with proteins fail to display color changes, are limited for the spectrophotometric and spectrofluorometric assays of proteins.Recent studies have focused on resonance light scattering (RLS), since Pasternack et al. 4 showed that it is a powerful technique to detect chromophore aggregation. 5-10We have applied enhanced RLS signals for analytical purposes, having proved that this technique is exceedingly sensitive to determine trace amounts of nucleic acids and proteins. 8,9 Our former reports have shown that the enhanced RLS signals are associated with the size and the charge-couple of the formed dye-protein or dye-DNA complex. [8][9][10] If the size is the main factor responsible for the enhanced RLS signals, the enhanced RLS signals were found to be in proportion to the molecular weight of the proteins. 10The role of the charge-coupled chromophore, however, is much more complicate and it needs further studied. Besides the negative charges on the molecular structures, the pH and ionic strength of the medium that determines the positive charges of protein molecules, depending on the isoelectric points, have a strong effect on the enhanced RLS intensity. In this work, we studied the interaction of proteins with Fast Green FCF (FCF), and tried to elucidate the roles of the charge-couple played in the dye-protein interaction.FCF is an anionic triarylmethane dye (molecular structure in Fig. 1), acting as a fluorophore for the visualization of the acidophilic cell and tissue structures, 11 and for the determination of tamoxifen citrate.12 Herein we report on its interaction with proteins based on RLS measurements where color changes of the dye upon binding to proteins are scarcely observed. It was found that the enhanced RLS intensity of FCF by trace amounts of proteins at 279.0 nm is proportional to the concentration of proteins below 4.0 µg/ml. Although organic dyes, including azo, triphenylmethane and porphryins, have been reported as being used for the determination of proteins of nanogram, the present protein assay using FCF has a much higher sensitivity than that of the reported RLS methods. 9,10,13-24 Experimental ApparatusThe RLS spectra and intensities were recorded and measured with a Hitachi F-2500 spectrofluorometer (Tokyo, Japan), while the absorption spectra were obtained using a Tianmei 850 spectrophotometer (Hong Kong, China). An S-10A digital pH meter (Xiaoshan Scientific Instruments Plant, Zhejiang, China) was used to measure the pH values of aqueous solutions, and an MVS-1 vortex mixer (Beide Scientific Instrumental Ltd., Beijing, China) was used to blend th...

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Section: Calibration Curvesmentioning

confidence: 99%

A Resonance Light-Scattering Determination of Proteins with Fast Green FCF

Li¹,

Huang²,

Li³

2002

ANAL. SCI.

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“…[2][3][4][5][6][7][8] It has been proved that the enhanced RLS signals which resulted from the aggregation and assembly processes can be applied to highly sensitive quantifications of DNA, 9,11 proteins, [12][13][14] ions, 15 surfactants 16 and clinical medicines [17][18][19] by using a common spectrofluorometer. These measurements, however, depend on the measuring conditions, including the molecular absorption, instrument conditions, reflection and scattering of the inside and outside walls of the absorption cell.…”

mentioning

confidence: 99%

Determination of Proteins with Fast Red VR by a Corrected Resonance Light-Scattering Technique

Yang¹,

Li²,

Huang³

2003

ANAL. SCI.

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A simple corrected resonance light-scattering (CRLS) technique was established to correct for any distortion of the resonance light scattering (RLS) spectra resulting from molecular absorption. By using an absorption cell holder to change the propagation direction of the incident light beam of a common spectrofluorometer, the molecular absorption was directly measured through a spectrofluorometer. With measurements of the CRLS signals of the interaction of Fast Red VR (FRV) and proteins, we proved that the present correction for the RLS spectra in terms of the molecular absorption of excitation and scattering radiation can improve the detection sensitivity by about two fold.

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“…Under the optimal conditions the decrease of peak current was proportional to the concentration of protein and further used to the determination of different kinds of proteins. The calibration curves for the determination of HSA, bovine serum albumin (BSA), ovalbumin (OVA), bovine hemoglobin (BHb), lipase were linear over the ranges of 4.0~28.0 mg L [6][7][8][9] In this paper electrochemical method was applied to investigate the interaction of orange G with proteins. Compared with other often-used methods such as spectrophotometry, fluorometry and chemiluminicence, electrochemical method is seldom used in studying the protein binding reaction.…”

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confidence: 99%

Voltammetric studies on the interaction of orange G with proteins: analytical applications

Sun¹,

Han²,

Yong³

et al. 2006

J. Braz. Chem. Soc.

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A interação do alaranjado G com proteína foi investigada pela técnica de voltametria. O alaranjado G, em solução tampão Britton-Robinson (B-R), pH 2,0, mostrou um pico voltamétrico irreversível de redução, a -0,17V (vs. SCE) utilizando mercúrio como eletrodo de trabalho. A adição de albumina de soro humano (HSA) à solução de alaranjado G, resultou no decréscimo do pico de corrente de redução, aparentemente sem variações dos portenciais de pico e sem o surgimento de novos picos de corrente. Os parâmetros eletroquímicos da solução de alaranjado G, na ausência e presença de HSA, foram calculados e comparados. Os resultados mostraram que não ocorreram variações significantes, indicando que o comportamento eletroquímico da solução reacional utilizando mercúrio como eletrodo de trabalho não variou e que foi formado um biocomplexo supramolecular, eletroquimicamente inativo. O mecanismo de interação foi devido à formação do campo microeletrostático na estrutura da albumina em solução aquosa, causando a interação com o alaranjado G, o que induziu o decréscimo da concentração de equilíbrio do alaranjado G na solução reacional, e o decréscimo do pico de corrente de redução. As condições de interação foram discutidas cuidadosamente. Sob condições ótimas, o decréscimo do pico de corrente foi proporcional à concentração da proteína e posteriormente, usado para a determinação de diferentes tipos de proteínas. As curvas de calibração para a determinação de HSA, albumina de soro bovino (BSA), ovalbumina (OVA), hemoglobina bovina (BHb) e lipase, foram lineares nos intervalos de 4,0~28,0 mg L The interaction of orange G with protein was investigated by voltammetric method in this paper. In pH 2.0 Britton-Robinson (B-R) buffer solution orange G displayed an irreversible voltammetric reduction peak at -0.17 V (vs. SCE) on mercury working electrode. The addition of human serum albumin (HSA) into the orange G solution resulted in the decrease of the reduction current peak apparently without the changes of peak potentials and no new peaks appeared. The electrochemical parameters of orange G solution in the absence and presence of HSA were calculated and compared. The results showed that there were no significant changes, which indicated that the electrochemical behaviors of reaction solution on the mercury working electrode showed no changes and a supramolecular electrochemical inactive biocomplex was formed. The interaction mechanism was due to the formation of the microelectrostatic field in albumin structure in aqueous solution and caused the interaction with orange G, which induced the decrease of the equilibrium concentration of orange G in the reaction solution, and the decrease of the reductive peak current. The interaction conditions were discussed carefully. Under the optimal conditions the decrease of peak current was proportional to the concentration of protein and further used to the determination of different kinds of proteins. The calibration curves for the determination of HSA, bovine serum albumin (BSA), ovalbumin (OVA)...

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Rayleigh Light-Scattering Spectroscopy: Application to the Determination of Proteins Using Bromopyrogallol Red

Cited by 45 publications

References 16 publications

A Resonance Light-Scattering Determination of Proteins with Fast Green FCF

A Resonance Light-Scattering Determination of Proteins with Fast Green FCF

Determination of Proteins with Fast Red VR by a Corrected Resonance Light-Scattering Technique

Voltammetric studies on the interaction of orange G with proteins: analytical applications

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