Spectrophotometric Determination of Total Proteins in the Serum with Water Soluble Aniline Blue
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It was found that water soluble aniline blue could react with proteins forming complexes in Britton-Robinson buffer at pH 2 36 and room temperature, which gives maximum absorption peak at 688 nm with 88 nm red shift in contrast with water soluble aniline blue. A molar absorptivitity of 4 25×105 L/(mol·cm) for BSA was determined. Four standard curves for proteins such as BSA, HAS, γ-G and casein were constructed under the optimum conditions. The analytical method was proposed to determine the content of total proteins in human serum samples with satisfactory results and in agreement with that of bromocresol green method.Keywords:
Bromocresol green
Bromocresol purple
Molar absorptivity
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Addition of aqueous protamine solution to Methyl Orange solution caused color change from bright orange to pale yellow at pH 6.80. A significant decrease in absorbance at 465 nm (λmax) and increase at 365 nm with increasing protamine concentration was observed with isosbestic point at 383 nm. This metachromasy was inhibited by the presence of ethanol. This phenomenon was used to determine ethanol content. The calibration curve was not a straight line. The applicable maximum range was 13( v/v %) of ethanol.
Absorbance
Metachromasia
Spectrophotometry
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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), bovine hemoglobin (BHb), lipase were linear over the ranges of 4.0~28.0 mg L-1, 4.0~30.0 mg L-1, 2.0~20.0 mg L-1, 2.0~25.0 mg L-1, 2.0~30.0 mg L-1, respectively. The detection limit was 3.0 mg L-1 for HSA, 3.5 mg L-1 for BSA, 1.0 mg L-1 for OVA, BHb and lipase. The new established electrochemical method was applied to determine the content of albumin in healthy human serum samples and the results were in good agreement with the traditional Coomassie Brilliant Blue (GBB G-250) spectrophotometric method.
Bovine serum albumin
Human serum albumin
Buffer solution
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In the present paper, the binding characteristics and spectral behavior of interaction of meso-tetra-(3, 5-dibromo-4-hydroxyphenyl) porphyrin [T(DBHP)P] as a new-style probe with protein were studied by the techniques of spectrophotometry. The experiment showed that Tween-80 microemulsion was efficiently used to enhance the sensibility and stability of the system at pH 4. 17(Britton-Robinson buffer solution). Under optimum conditions, the characteristics of absorption spectral of meso-tetra-(3,5-dibromo-4-hydroxyphenyl) porphyrin-protein were investigated, the maximum absorption was located at 425 nm, and the reducing value of absorbance A was in proportion to the concentration of proteins in the range 0.50-6.00 microg x mL(-1) for bovine serum albumin, 0.05-0.60 microg x mL(-1) for ovalbumin, and the limits of detection were 0.106 microg x mL(-1) for bovine serum albumin and 0.039 microg x mL(-1) for ovalbumin. The experiments indicated that the proposed method featured high sensitivity and good selectivity and stability, and was simple and relatively free from interference of coexistent substances. It has been applied to the determination of protein in milk samples with satisfactory. The recovery for the investigated protein from milk was 99.56%-100.2% and the relative standard deviations were less than 2.2%. The sensitive method for the quantitative determination of proteins was proposed and may be applicable to the determination of ultra amounts of protein in food analysis. The effect of ionic strength on the system was investigated, and the result indicated that the binding force between meso-tetra-(3, 5-dibromo-4-hydroxyphenyl) porphyrin and protein was judged as electrostatic force. The influence of protein denaturation was also studied, under higher temperature the structure of protein was destroyed, and thermodynamic movement of the molecular of protein was intensified as the heating time extended.
Absorbance
Spectrophotometry
Bovine serum albumin
Tetra
Buffer solution
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Gold nanoparticles about 10 nm in size were prepared by improved trisodium citrate reduction procedure, and were used to label goat anti-human C3 to obtain a sensitive spectral probe for complement 3 (C3) in the condition of pH 7.5. The immune reaction between nanogold-labeled C3 antibody (anti-C3) and the antigen C3 took place to form the nanogold immune complex in pH 5.6 Na2 HPO4-C6H8O7 buffer solution and in the presence of polyethylene (PEG). The optimal immunoreaction conditions were pH 5.6-9.7 microg x mL(-1) nanogold-labeled anti-C3, 6.0% PEG 6000 and incubation time 15 min under ultrasonic irradiation. After centrifuging for 10 min at 12 000 r x min(-1), the excess nanogold-labeled anti-C3 in the upper solutions was obtained, and was used to catalyze the colored particle reaction between HAuCl4 and NH2 OH x HCl to produce gold particles with bigger size. The influence on the immunonanogold catalytic reaction was considered spectrophotometrically. A pH 2.97 Na3C6H5O7-HCl buffer solution, 53.33 microg x mL(-1) HAuCl4, 74.13 microg x mL(-1) NH2OH x HCl, and reaction time of 3 min at 37 degrees C water bath were chosen for use. Results demonstrated that with increasing C3, the concentration of gold labeled anti-C3 in the upper solution decreased, and the absorbance decreased linearly. Linear relationships between the decreased absorbance and the C3 concentration in the range of 0.025-0.60 ng x mL(-1) were obtained by spectrophotometry at 760 nm. The regress equation was deltaA760 nm = 0.276c+0.025 4, the correlation coefficient was 0.990 3, and the detection limit reached 0.007 2 ng x mL(-1) of C3. The influence of foreign substances such as HAS, BSA, and ammonia acid on the determination of 0.2 ng x mL(-1) of C3was examined. Results showed that this assay has high selectivity. The sensitive, rapid and highly specific assay was applied to the quantification of C3 in human sera, with satisfactory results.
Absorbance
Trisodium citrate
Spectrophotometry
Buffer solution
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ABTS
Peracetic acid
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The interaction of neutral red and heparin was studied by UV-Vis spectrophotometry in pH 3.0 Britton-Robinson (B-R) buffer solution. Neutral red has a strong absorbance at 523 nm and the addition of heparin into neutral red solution resulted in the decrease in the absorbance value at 523 nm without the appearance of new absorbance peak. The decrease in absorbance value was linear with the concentration of heparin. The conditions for the interaction were optimized and the interferences of coexistent substances were investigated. Under the optimal conditions a linear regression equation was obtained as deltaA = 0.044 + 0.076c (mg x L(-1)) (n = 12, r = 0.997) in the range of 0.10-15.0 mg x L(-1) with the detection limit of 0.073 mg x L(-1). The molar absorptivity of the method was calculated to be 2.037 x 10(6) L x mol(-1) x cm(-1). The method was applied to determine the heparin sodium injection solution with satisfactory results. The stoichiometry of heparin with neutral red was calculated as 1 : 3.
Absorbance
Molar absorptivity
Spectrophotometry
Buffer solution
Stoichiometry
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Molar absorptivity
Absorbance
Metachromasia
Bovine serum albumin
Bradford protein assay
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Spectrophotometric characters of protein interaction with chromazurol S(CAS), bromocresol green (BCG), bromopyrogallol red (BPR) and methylthymol blue (MTB) have been studied in acid and alkaline medium respectively. Protein can combine the above mentioned dye to reduce absorptivity of dye solution of CAS and BCG at pH 3.8 and pH 4.0, to add absorptivity of dye solution of BPR and MTB at pH 3.8 and pH 10.8 for the bathochromic shift of wave length of maximal absorption of them to add 10 nm and 20 nm respectively. The linear relationship holds between the reduced or added absorptivity of dye and the optimum concentration range of protein. The interaction mechanism of protein and dye has been discussed tentatively.
Molar absorptivity
Bathochromic shift
Bromocresol green
Bromocresol purple
Spectrophotometry
Acid dye
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To propose a new simple and sensitive voltammetric method for determination of proteins.Protein with sulfhydryl or disulfide bond in 0.5 mol x L(-1) NaOH, 1.5 x 10(-4) mol x L(-1) Pb2+ and 0.02% tetrabutylammonium iodide was heated in boiling water for 5 minutes. The reactive product gave a well defined reductive adsorption wave at -0.66 V (vs SCE) by means of single sweep polarography, and the height of derivative wave was proportional to the concentration of proteins.The peak height was linearly proportional to bovine serum albumin (BSA) or human serum albumin (HSA) concentration in range of 7.5 x 10(-10) -3.0 x 10(-7) mol x L(-1) (r(BSA) = 0.9995, and r(HSA) = 0.9990). The detection limit of BSA or HSA was 3.0 x 10(-10) mol x L(-1). For lysozyme (Lyso), the concentration range was from 1.4 x 10(-8) to 1.3 x 10(-6) mol x L(-10 (r(Lyso) = 0.9997) and the detection limit was 7.0 x 10(-9) mol x L(-1).The method is simple, rapid, sensitive and applicable to the assay of diluted human serum albumin samples.
Bovine serum albumin
Human serum albumin
Sodium hydroxide
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Bovine serum albumin
Buffer solution
Spectrophotometry
Blueshift
Absorption band
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