Quantitative determination of proteins by the Rayleigh light-scattering technique after optimization of the derivation reaction with Arsenazo-DBS

This paper researched the determination of proteins with 2-(2-arsonophehenylazo)-7-[(2,6-dibromo-4-sulfophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid (Arsenazo-DBS) by Rayleigh light-scattering (RLS). The reaction parameters, such as acidity, volume of buffer solution and concentration of Arsenazo-DBS, were examined by orthogonal array design (OAD). Under optimal conditions, the weak RLS of Arsenazo-DBS and BSA can be enhanced greatly and two enhanced RLS signals were produced at 340-350 and 400-420 nm. Based on this reaction, a new quantitative determination method for proteins has been developed. This method is proved to be very sensitive (the determination limits are 0.077 μg ml⁻¹ for bovine serum albumen (BSA) and 0.074 μg ml⁻¹ for human serum albumen (HSA)), rapid (<2 min), simple (one step) and tolerance of most interfering substances. The effects of different surfactants were also examined. The amount of proteins in human serum samples was determined and the maximum relative error was no more than 2% and the recovery was between 95 and 105%.     

Microdetermination of proteins by enhanced Rayleigh light scattering spectroscopy with morin

Under acidic conditions, the reaction between 3,5,7,2′,4′-pentahydroxyflavone (morin) and proteins enhances the weak light scattering of morin drastically. The enhanced light scattering intensity is proportional to the content of proteins. This fact is the basis of a new method for the quantitative analysis of proteins. The linear range is 0.45–7.15 and 0.46–11.14 μg/mL for BSA and HSA, respectively. The relative standard deviation is 3.76% (n = 12) for BSA in the middle of the linear range. The results of this assay for human serum samples were comparable with those from the traditional method (CBB method). There is almost no interfere from amino acids and most of the metal ions. The scattering spectrum of morin was also discussed. Received: 11 October 1998 / Revised: 18 February 1999 / Accepted: 24 February 1999     

Microdetermination of proteins by enhanced Rayleigh light scattering spectroscopy with morin

Under acidic conditions, the reaction between 3,5,7,2′,4′-pentahydroxyflavone (morin) and proteins enhances the weak light scattering of morin drastically. The enhanced light scattering intensity is proportional to the content of proteins. This fact is the basis of a new method for the quantitative analysis of proteins. The linear range is 0.45–7.15 and 0.46–11.14 μg/mL for BSA and HSA, respectively. The relative standard deviation is 3.76% (n = 12) for BSA in the middle of the linear range. The results of this assay for human serum samples were comparable with those from the traditional method (CBB method). There is almost no interfere from amino acids and most of the metal ions. The scattering spectrum of morin was also discussed.     

A sensitive and rapid method for the determination of protein by the resonance Rayleigh light-scattering technique with Pyrogallol Red

The resonance Rayleigh light-scattering (RRLS) technique was used to develop a simple, sensitive and selective method for the determination of proteins. The method is based on the interaction between proteins and Pyrogallol Red (PR) in the pH range 3.6-4.2, which causes a substantial enhancement of the resonance scattering signal of PR in the wavelength range 300-450 nm with the maximum scattering peak located at 347 nm. With this method, 0.25-13 microg ml(-1) of bovine serum albumin (BSA), 0.25-10 microg ml(-1) of human serum albumin (HSA) and 0.25-13 microg ml(-1) of human immunoglobulin G (IgG) can be determined, and the detection limits, calculated as three times the standard deviation of nine blank measurements, for BSA, HAS and IgG were 51, 48 and 57 microg l(-1), respectively. Moreover, the method shows almost no protein-to-protein variability and is free from interference from many amino acids and metal ions. The method, with high sensitivity, selectivity and reproducibility, was satisfactorily applied to the determination of the total protein in human serum and saliva samples. Mechanism studies indicated that PR can bind to BSA depending mainly on electrostatic forces, and this interaction can encourage the J-aggregation of PR, which results in enhanced Rayleigh light-scattering in the PR-protein system.     

Determination of nucleic acids with tetra-(N-hexadecylpyridiniumyl) porphyrin sensitized by cetyltrimethylammonium bromide (CTMAB) using a Rayleigh light-scattering technique

Using a common spectrofluorometer to measure the intensity of Rayleigh light-scattering (RLS), a method for determination of nucleic acids has been developed. At pH 10.24 and ionic strength 0.01 mol l-1 (NaCl), the Rayleigh light-scattering of the tetra-(N-hexadecylpyridiniumyl) porphyrin (TC16PyP) is greatly enhanced by nucleic acids in the presence of cetyltrimethylammonium bromide (CTMAB), with the scattering peak located at 311.8 nm. The enhanced RLS intensity is in proportion to the concentration of calf thymus DNA (ctDNA) in the range 0.2-6.0 microg ml-1 and to that of fish sperm DNA (fsDNA) in the range 0.05-3.0microg ml-1. The limits of detection are 0.016 microg ml-1 for calf thymus DNA and 0.023 microg ml-1 for fish sperm DNA when the concentration of TPP was chosen 2.0 x 10(-6) mol l-1. Four synthetic samples were determined satisfactorily.     

The synthesis and application of 1-(o-nitrophenyl)-3-(2-thiazolyl)triazene for the determination of palladium(II) by the resonance enhanced Rayleigh light-scattering technique

The resonance Rayleigh light-scattering (RRLS) signal of a new triazene reagent; 1-(o-nitrophenyl)-3-(2-thiazolyl)triazene (o-NPTT), was firstly synthesized and characterized in this paper at 500-600 nm wavelength range and can be enhanced remarkably by Pd2+. According to this phenomenon, a new method was developed for the determination of Pd2+ by the RRLS technique in the presence of Tween-80. The calibration graph showed good linearity over a concentration range of 5.0-700 microg l(-1) with a detection limit of 1.0 microg l(-1). There are almost no foreign ions interfered in the determination at a more than fivefold concentration of Pd2+. The method is relatively sensitive, of good selectivity and has been successfully used for the determination of trace palladium in the slag of fertilizer factories and catalyst samples.     

Study of the reaction of proteins with Beryllon II-AlIII by the Rayleigh light scattering technique and its application

The determination of proteins with tetrasodium 2-(3,6-disulfo-8-hydroxynaphthylazo)-1,8-dihydroxynaphthalene-3,6-disulfonate (Beryllon II) by Rayleigh light scattering (RLS) was studied. The weak RLS of the Beryllon II-bovine serum albumin (BSA) complex can be greatly enhanced by the addition of Al3+ in the pH range 5.6-7.2; there was a maximum RLS platform at 400-420 nm. Based on the reaction between Beryllon II, Al3+ and proteins, a new method for the determination of proteins was developed. This method is very sensitive [0.20-41.42 micrograms ml-1 for BSA and 0.18-48.15 micrograms ml-1 for human serum albumen (HSA)], rapid (< 2 min), simple (one step) and tolerant towards most interfering substances. The effects of different surfactants were also examined. Four samples of protein in human serum were determined; the maximum relative error was no more than 5% and the recovery was 96-105%.     

铑-钨酸盐-罗丹明B缔合体系的共振光散射现象及其应用

在聚乙烯醇(PVA)存在下,Rh(Ⅲ)与钨酸盐及罗丹明B(RB)形成的离子缔合物在620nm处产生强烈的共振瑞利光散射现象,且相对于试剂空白,缔合物体系的散射光强度(ΔI)与Rh(Ⅲ)的质量浓度在0.004～0.30ng mL范围内具有良好的线性关系,据此建立的测定Rh(Ⅲ)的共振光散射法的检出限可达0 002ng mL,且大量存在的常见离子对Rh(Ⅲ)的测定不干扰。已用此方法测定了催化剂及工业产品中的铑,结果与标准方法(SnCl2法)测得值基本一致。     

A highly sensitive assay for protein using resonance light-scattering technique with dibromohydroxyphenylfluorone-molybdenum(VI) complex

At pH 2.8 and in the presence of 0.090% p-octylpolyethyleneglycol phenylether, the resonance light-scattering (RLS) spectrum of molybdenum(VI) complex with dibromohydroxyphenylfluorone (DBHPF) has a sharp peak at 586 nm. If the micro protein coexists with Mo(VI) and DBHPF, the RLS intensity of the complex at 586 nm is significantly enhanced by protein due to the binding interaction between protein and DBHPF-Mo(VI) complex. Based on this a new assay for protein is described. The dynamic ranges for bovine and human serum albumins are both 0.05-0.75 mg l-1 with detection limits of 13 and 15 ng ml-1, respectively. Besides high sensitivity, the method is characterized by good reproducibility, rapidity of reaction, good stability of chemical system, commonality of spectrofluorometer, few coexisting substances, especially detergents. The determinations of diluted human serum and urine by this method give the results very close to these by the Coomassie brilliant blue G-250 colorimetry, with relative standard deviations of five duplicates of 1.8-2.5%.     

Microdetermination of the amine oxide group in organic compounds by reduction with titanium(III)

A rapid and selective method for the determination of water-soluble boron in complex fertilizers is described. Boron is separated from the sample solution, which should be approximately 0.1 N in hydrochloric acid, by shaking twice with 20% solution of 2-ethyl-1,3-hexanediol in hexone. Following this extraction boron is back-extracted into the aqueous phase with 0.5 N sodium hydroxide. It is finally determined spectrophotometrically at 415 nm using azomethine H as reagent.     

Microdetermination of Proteins by Enhanced Rayleigh Light Scattering Spectroscopy with Thorin

ＩｎｔｒｏｄｕｃｔｉｏｎＰｒｏｔｅｉｎｄｅｔｅｒｍｉｎａｔｉｏｎｉｓａｌｗａｙｓｉｍｐｏｒｔａｎｔｉｎｃｌｉｎｉｃａｌａｐｐｌｉｃａｔｉｏｎｓ .Ｎｅｗｍｅｔｈｏｄｓｉｎｃｌｕｄｉｎｇｓｐｃｅｔｒｏｐｈｏｔｏｍｅｔｒｙ ,1ｆｌｕｏｒｏｍｅｔｒｙ2 ａｎｄｃｈｅｍｉｌｕｍｉｎｉｃｅｎｃｅ3 ,4 ａｒｅｃｏｎｔｉｎｕｏｕｓｌｙｂｅ ｉｎｇｄｅｖｅｌｏｐｅｄ .Ｐａｓｔｅｒｎａｃｋｅｔａｌ.5,6ｄｅｖｅｌｏｐｅｄａｔｅｃｈ ｎｉｑｕｅｔｏｍｅａｓｕｒｅｔｈｅｉｎｔｅｎｓｉｔｙｏｆｓｃａｔｔｅｒｉｎｇｌｉｇｈｔｕｓｉ…     

A novel method for study of the aggregation of protein induced by metal ion aluminum(III) using resonance Rayleigh scattering technique

We present a novel method for the study of the aggregation of protein induced by metal ion aluminum(III) using resonance Rayleigh scattering (RRS) technique. In neutral Tris-HCl medium, the effect of this aggregation of protein results in the enhancement of RRS intensity and the relationship between the enhancement of the RRS signal and the Al concentration is nonlinear. On this basis, we established a new method for the determination of the critical induced-aggregation concentrations (C(CIAC)) of metal ion Al(III) inducing the protein aggregation. Our results show that many factors, such as, pH value, anions, salts, temperature and solvents have obvious effects. We also studied the extent of aggregation and structural changes using ultra-violet spectrometry, protein intrinsic fluorescence and circular dichroism to further understand the exact mechanisms of the aggregation characteristics of proteins induced by metal ion Al(III) at the molecular level, to help us to develop effective methods to investigate the toxicity of metal ion Al, and to provide theoretical and quantitative evidences for the development of appropriate treatments for neurodementia such as Parkinson's disease, Alzheimer's disease and dementia related to dialysis.     

Determination of proteins with Fast Red VR by a corrected resonance light-scattering technique.

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.     

Determination of proteins with Alizarin Red S by Rayleigh light scattering technique

A new protein determination method by enhanced Rayleigh light scattering (RLS) technique has been developed. In acid condition (pH=3.60), RLS of 1,2-dihydroxyanthraquinone-3-sulfonate (Alizarin Red S) can be greatly enhanced by addition of proteins, resulting in two characteristic peaks, 360 and 505 nm, respectively. The new protein assay is based on the RLS enhancement and spectrum change. The optimum condition for the reaction was investigated. The linear range is 0.20-24.9 μg ml⁻¹ for BSA and 0.20-15.5 μg ml⁻¹ for HSA. The detection limits (S/N=3) are 9.59 ng ml⁻¹ for BSA and 9.51 ng ml⁻¹ for HSA. The results of determination for human serum samples were comparable to those obtained by Bradford method. The binding stoichiometry was determined.     

Microdetermination of arsenic(III) and osmium(VIII) through osmium-thiourea reaction

Osmium-thioureau reaction is slow under conditions described in earlier methods. In the present study it has been observed that arsenic(III) catalyzed the reaction. Based on this, qualitative and quantitative methods are described for the determination of both osmium and arsenic. Interference study of 30 ions showed they do not interfere.     

Microdetermination of gold(III), silver(I) and ruthenium (III) by ring colorimetry after separation by thin-layer chromatography (TLC)

Summary Clean and speedy analytical separation ofg quantities of Au(III), Ru(III) and Ag(I) from their mixed solution is accomplished by ascending thin-layer chromatography. For the evaluation of these metal ions, the spots are scooped out with the help of a Mottier gadget and the collected material transferred to the paper set on the ring oven. Separate rings were obtained for individual metal ions and the determination is carried out by ring colorimetry, using PTC as a fixing and complexing agent for washing to the ring.

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Zusammenfassung Mikrogrammengen Au(III), Ru(III) und Ag(I) lassen sich voneinander aus Lösungsgemischen sauber und rasch durch aufsteigende Dünnschichtchromatographie trennen. Für die Auswertung der Flecken werden diese abgekratzt, mit Hilfe des vonMottier beschriebenen Sauggerätes gesammelt und auf dem Ringofen auf Papier übertragen. Für die einzelnen Metallionen erhält man getrennte Ringe, die kolorimetrisch vermessen werden, wobei Kaliumthiocarbonat zur Fixierung und Komplexbildung dient.

     

Determination of proteins by flow injection analysis coupled with the Rayleigh light scattering technique

A novel flow injection analysis (FIA) method with Rayleigh light scattering (RLS) detection was developed for the determination of protein concentrations. This method is based on the weak intensity of RLS of p-nitrohenzene-azo-3,6 disulfo-1-amino-8-naphthol-7-azo-benzene disodium salt (Amide Black-10B) which can be enhanced by addition of protein in weakly acidic solution. It has proved that the application of this method to quantify the proteins by using human serum albumin was available in real samples. In addition, this method is very sensitive (the determination limits are 0.11 μg/mL for human serum albumen (HSA) and 0.85 μg/mL for bovine serum albumen, BSA), simple, rapid and tolerance of most interfering substances. The FIA-RLS method was more stabile than the general RLS method and the average R.S.D. value of FIA-RLS less than general RLS. The effects of different interfering substances will be also examined. The amount of proteins in human serum sample was determined and the maximum relative error was no more than 3.00% as well as the recovery was between 94.9 and 105.9%.     

An investigation of a lipopolysaccharide-protein complex fromYersinia pseudotuberculosis by the light-scattering method

Summary An estimate has been made of the molecular weights and characteristic viscosities of aggregates of the LPPC fromYersinia pseudotuberculosis in water, and also in aqueous solutions of sodium chloride, urea, guanidine hydrochloride, and sodium dodecyl sulfate. It has been shown that a high ionic strength of the solution and the presence of disaggregating agents lead to a considerable decrease in the molecular weight of the LPPC.Pacific Ocean Institute of Bioorganic Chemistry of the Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 51–53, January–February, 1979.     

Quantitative determination of proteins at nanogram levels by the resonance light-scattering technique with macromolecules nanoparticles of PS-AA

The polystyrene-acrylic acid (PS-AA) nanoparticles have been prepared by ultrasonic polymerization, characterized by FT-IR and TEM. It is the first report on the determination of proteins with macromolecules nanoparticles of PS-AA by resonance light-scattering (RLS). At pH 6.9, the RLS of macromolecules nanoparticles of PS-AA can be enhanced by proteins. Based on this, a novel quantitative assay of proteins at the nanogram levels has been proposed. At pH 6.9, the RLS signals of PS-AA were greatly enhanced by proteins in the region of 250-700 nm characterized by the peak at 342 nm. Under optimal conditions, the linear ranges of the calibration curves were 0.02-11.0 microgml-1, 0.04-10.0 microgml-1 and 0.03-10.0 microgml-1 for gamma-globulin (gamma-IgG), bovine serum albumin (BSA) and human serum albumin (HSA), respectively. The detection limits were 16.0 ngml-1, 19.0 ngml-1, and 15.0 ngml-1 for gamma-IgG, BSA and HSA, respectively. The method has been applied to the analysis of total proteins in human serum samples collected from the hospital and the results were in good agreement with those reported by the hospital, which indicates that the method presented here is not only sensitive, simple, but also reliable and suitable for practical application.     

Microdetermination of ruthenium(III) in presence of other metals

Summary Ruthenium(III) reacts with thiovioluric acid in aqueous medium at p_H 4.45–6.00 to yield pinkish-violet complex with absorption maximum at 540 nm. Full colour development requires heating for about 30 minutes on a boiling water bath. The system obeys Beer's law in the range 0–5.6 ppm of ruthenium. The sensitivity of the reaction is 0.0046g/cm² (for log 10/I=0.001) at 540 nm. The composition of the complex is 12, as shown by Job's and Bent and French methods. The effect of various foreign ions in the determination of ruthenium has also been investigated.

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Zusammenfassung Ruthenium(III) reagiert mit Thioviolursäure in wäßriger Lösung bei p_H 4,45–6,00 unter Bildung einer rosa-violetten Komplexverbindung mit dem Absorptionsmaximum bei 540 nm. Die volle Farbentwicklung tritt beim Erwärmen auf dem siedenden Wasserbad in etwa 30 min. ein. Das Beer'sche Gesetz ist bis 5,6 ppm Ru erfüllt. Die Empfindlichkeit der Reaktion beträgt 0,0046g/cm² bei 540 nm. Die Zusammensetzung des Komplexes entspricht dem Molverhältnis 12. Der Einfluß verschiedener Fremdionen wurde untersucht.