The upper limit pH in the dye-binding method for the determination of serum protein via measurements of the absorbance increase produced by protein error.

In the dye-binding method for determining the albumin concentration, the absorbance increase due to the change of the color shade by protein error of a pH indicator can be measured by a spectrophotometer. This absorbance increase is observed only in a restricted pH region, but this pH region is not theoretically studied yet. Thus, the author investigated the upper limit pH (pHUL) at which the absorbance increase occurs by the theoretical calculation, and compared these results with those obtained experimentally using four pH indicators. The pHUL is not affected by the dye or protein concentrations, or by the formation constant of the dye-protein complex; but the value changes according to the acid-dissociation constant of the dye (KD) and the ratio of the molar absorptivities of the proton-dissociated dye anion (epsilonD) and the dye-protein complex (epsilonPD). The pHUL value can be calculated by the equation, found theoretically. The calculated pHUL values of BPB, BCG, BCP and BTB were 5.1, 4.8, 6.2 and 5.5, respectively. These values correlated with the experimental results of 4.5 for BPB, 4.7 for BCG, 5.9 for BCP and 5.2 for BTB, but were not associated with the pKD values of each dye. The pHUL of these dyes did not change significantly for various dye and protein concentrations, as was expected from the thoretical calculation.     

Influence of pH for the determination of serum albumin by a dye-binding method in the presence of a detergent

In the dye-binding method, the absorbance increase caused by a protein error of a pH indicator is observed only in a restricted pH range. However, this pH range in the presence of a detergent has not yet been examined. Thus, the author investigated the pH (pH(UL)) where the absorbance increase becomes zero by a calculation based on the chemical equilibrium of a protein error of a pH indicator, and by experiments using four sulfonephthalein dyes. The pH(UL) value changed only with the detergent concentration, but did not change at all due to the dye, buffer solution or protein concentrations. Although the pH(UL) value was different according to the kind of dye used, it correlated well with the pK(D) values (dissociation constant) of BPB, BCG, BCP and BTB. The characteristics of pH(UL) in the reactions of the four dyes indicated good agreement with that obtained by a calculation.     

Characteristics of a protein error in determination of serum protein in the presence of inorganic salt.

There is a possibility that the color development of the dye-binding method based on a protein error of a pH indicator is affected by the coexisting inorganic salt. Thus, the author theoretically and experimentally investigated the effect of the inorganic salt on the protein error. In a theoretical analysis, the anion of an inorganic salt, like the dissociated dye and buffer anions, was assumed to react with the protein, forming a colorless anion-protein complex. The calculated results were compared with those obtained by experiments using three pH indicators and various kinds of inorganic salts. The calculated results obtained are as follows: (1) The color development decreases with increasing the concentration of the inorganic salt and the equilibrium constant of the reaction between the inorganic salt and protein; (2) The rate of the absorbance decrease is larger for a lower concentration of the inorganic salt than for a higher one; (3) The larger is the equilibrium constant, the larger is the absorbance decrease. The absorbance decrease was caused by the anion, and was increased by increasing the anion concentration. The magnitude of the effect of the anion was iodide > bromide > chloride, which was associated with their ionic radius. The difference in the effect of the anion was thought to indicate that the equilibrium constant, in other words, the bonding strength of the anion to protein is iodide > bromide > chloride.     

Fluorimetric studies and noncovalent labeling of protein with the near-infrared dye HITCI for analysis by CE-LIF

1,1',3,3,3',3'-Hexamethylindotricarbocyanine iodide (HITCI) is a commercially available, positively charged, indocarbocyanine dye used typically as a laser dye in the near infrared (NIR). The absorbance and fluorescence properties of HITCI in a variety of solvent systems were determined. Results indicate that the fluorescence of HITCI is not significantly affected by the pH. Titration of HITCI with human serum albumin (HSA) and trypsinogen was carried out to investigate the interactions between this dye and proteins. These studies revealed that the absorbance and fluorescence properties of the dye change upon binding to protein in a wide range of solution pH's. The potential use of HITCI as a noncovalent protein labeling probe, therefore, was explored. Determination and separation of HITCI and HITCI-protein complexes was performed by capillary electrophoresis with diode-laser induced fluorescence detection (CE-LIF). Both pre-column and on-column noncovalent labeling methods are demonstrated.     

Isotope pattern vector based tandem mass spectral data calibration for improved peptide and protein identification

Tandem mass spectra contain noisy peaks which make peak picking for peptide identification difficult. Moreover, all spectral peaks can be shifted due to systematic measurement errors. In this paper, a novel use of an isotope pattern vector (IPV) is proposed for denoising and systematic measurement error prediction. By matching the experimental IPVs with the theoretical IPVs of candidate fragment ions, true ionic peaks can be identified. Furthermore, these identified experimental IPVs and their corresponding theoretical IPVs are used in an optimization process to predict the systematic measurement error associated with the target spectrum. In return, the subsequent spectral data calibration based on the predicted systematic measurement error enhances the data quality. We show that such an integrated denoising and calibration process leads to significantly improved peptide and protein identification. Different from the commonly employed chemical calibration methods, our IPV‐based method is a purely computational method for individual spectra analysis and globally optimizes the use of spectral data. Copyright © 2009 John Wiley & Sons, Ltd.     

葡萄糖氧化酶反应体系的动力学性质及在葡萄糖分析中的应用

本文运用紫外－可见光谱仪研究了葡萄糖氧化酶偶联辣根过氧化物酶－邻联茴香胺染料体系的催化动力学性质，通过控制酶和染料的含量，确定了反应呈假一级反应的葡萄糖浓度范围，在此反应体系中，葡萄糖浓度可以通过测定染料吸光度而获得，干扰小，回收率达９７％～１０３％。     

酶法测定乳酸

利用乳酸在氧化型烟酰胺腺嘌呤二核苷酸（NAD）存在的条件下,由乳酸脱氢酶（L-LDH）催化生成丙酮酸和还原型烟酰胺腺嘌呤二核苷酸（NADH）。通过测定NADH吸光度的变化率可得出乳酸的酶促反应速度,并制得标准曲线。样品中的乳酸可由标准曲线求得。讨论了试剂用量、pH、温度和共存离子对测定的影响。     

Spectrophotometric, difference spectroscopic, and high-performance liquid chromatographic methods for the determination of cefixime in pharmaceutical formulations

Three simple and sensitive spectrophotometric, difference spectroscopic, and liquid chromatographic (LC) methods are described for the determination of cefixime. The first method is based on the oxidative coupling reaction of cefixime with 3-methyl-2-benzothiazolinon hydrazone HCI in presence of ferric chloride. The absorbance of reaction product was measured at the maximum absorbance wavelength (wavelength(max)), 630 nm. The difference spectroscopic method is based on the measurement of absorbance of cefixime at the absorbance maximum, 268 nm, and minimum, 237 nm. The measured value was the amplitude of maxima and minima between 2 equimolar solutions of the analyte in different chemical forms, which exhibited different spectral characteristics. The conditions were optimized, and Beer's law was obeyed for cefixime at 1 to 16 microg/mL and 10 to 50 microg/mL, respectively. The third method, high-performance LC, was developed for the determination of cefixime using 50 mM potassium dihydrogen phosphate (pH 3.0)-methanol (78 + 22, v/v) as the mobile phase and measuring the response at wavelength(max) 286 nm. The analysis was performed on a Lichrospher RPC18 column. The calibration curve was obtained for cefixime at 5 to 250 microg/mL, and the mean recovery was 99.71 +/- 0.01%. The methods were validated according to the guidelines of the U.S. Pharmacopoeia and also assessed by applying the standard addition technique. The results obtained in the analysis of dosage forms agreed well with the contents stated on the labels.     

New insights in the spatially resolved dynamic pH measurement in macroscopic large absorbent particles by confocal laser scanning microscopy

Both, experimental investigation of protein adsorption processes and mathematical models describing such processes indicate, that the pH in the absorbent particle might be the key factor for an improved understanding of these chromatographic processes. Thus, a technique aiming at the spatially resolved pH measurement in macroscopic large absorbent particles is presented. The first application of this method, being based on confocal laser scanning microscopy (CLSM), revealed an apparent dependence of the pH calibration curve on the scanning depth. By a model-based approach, factors distorting the measurement signal are identified: The wavelength-dependent light scattering and the re-absorption of emitted light. The resulting consequences for further development and application of CLSM based techniques to measure pH in macroscopic large absorbent particles are illustrated and discussed.     

Photophysics of a xanthenic derivative dye useful as an "on/off" fluorescence probe

The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total buffer concentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constant of the dye determined by absorbance is almost independent of the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis, yielding the following values of the rate constants describing excited-state dynamics: k01 = 1.29 x 10(10) s(-1), k02 = 4.21 x 10(8) s(-1), k21 approximately 3 x 10(6) M(-1) s(-1), k12B= 6.40 x 10(8) M(-1) s(-1), and k21B = 2.61 x 10(7) M(-1) s(-1). The decay rate constant values of k01, k21, k21B, along with the low molar absorption coefficient of the neutral form, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 M and any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye.     

Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution.

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.     

Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry

A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240 nm. Copper absorbance slowly increases from 240 to 190 nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226 nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu²⁺ ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu²⁺ ions at pH 10.3. To determine proteins, 1-1.5 ml of 1.8% KOH solution with 0-20 μg ml⁻¹ protein is treated with 25 mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226 nm in 1 cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20 μg ml⁻¹ protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.     

Photochemical reactivity of sulfamethoxazole and other sulfa compounds with photodiode array detection

The photochemical activities of six sulfa compounds [sulfacetamide (CET), sulfadiazine (DIA), sulfaguanidine (GUA), sulfamerazine (MER), sulfamethoxazole (SMX), and sulfamethizole (MET)] under different experimental conditions such as photolysis time, solvent and buffer pH are investigated by photodiode array (PDA) spectrophotometry. With no photolysis, the sulfa drugs CET and DIA show no absorbance at 332 nm and the other compounds only modest absorbance. Upon photolysis for 4 min, absorbance enhancements at 332 nm of three to four times for GUA and MET and 12-15 times for SMX and MER are observed. For CET and DIA after photolysis, the (absorbance) l/mg is now approximately 0.01-0.02 units. Although two pH optima of approximately 3-4 and 7 are noted, the optimum solvent for photolysis is ethanol without pH adjustment. For flow injection (FI) with on-line photolysis and PDA detection, a mobile phase of 100% ethanol with a step flow rate from 0.1 to 1 ml/min is used providing a 4-min reaction time. The FI detection limit for SMX with photolysis at 330 nm is 1 mg/l. The relative standard deviation data (n=4) of seven individual points in a calibration curve from 5 to 150 mg/l are 0-4%. The recovery of SMX from pharmaceutical tablets is 99.7% indicating no interference from trimethoprim which is not photochemically active.     

Line absorption of matrix elements as a background correction error in atomic absorption spectrometry

Accurate measurement of weak atomic absorption signals is only possible after exact background correction. If the accompanying elements in excess have their own resonance lines in the immediate vicinity of the resonance line of the element to be determined,and these fall within the spectral band transmitted by the monochromator, then the value the measured apparent background absorbance is higher than the true one. The error may usually be diminished by choosing an appropriate spectral bandwidth. Tables of such bandwidths for a large number of pairs of elements are given.     

高浓度F~--Al~(3+)体系的热力学分析及氟的测定

设定了Visual minteq软件F~--Al~(3+)体系的物种及平衡常数。模拟滴定和实验滴定结果的相似性证明了软件设定的合理。对F-(0.01/0.1 mol/L)-Al~(3+)(0.02 mol/L)-OH-体系的模拟和分析表明,当F与Al摩尔比相差大时,体系平衡物种分布不同,OH-掩蔽铝的机理也不同,但是在p H值11~12铝都被成功掩蔽;不同的反应机理导致F与Al摩尔比大的体系模拟和滴定曲线差别较大。进一步模拟得到了此p H值区间的最大可掩蔽铝浓度。考虑OH-对氟离子电极的影响,最终确定高氟(0.01~0.1 mol/L)高铝体系氟的测定条件:控制p H值为(11.5±0.2),不大于0.02 mol/L的铝。误差分析表明,当电势测定误差较大时,标准曲线法比标准加入法误差小。     

Comparison of tryptophan interactions to free and grafted BSA protein

The binding of d- and l-tryptophan molecules to bovine serum albumin (BSA) protein has been studied using liquid chromatography and ultrafiltration in the pH range from 7 to 11. A hydrophobic interaction between tryptophan and BSA has been observed at pH 7.0 on BSA grafted chromatographic column. However, this interaction is negligible at higher pH for which the interaction to the stereospecific site was predominant. For both grafted and free proteins, the complexation mechanism was a competitive binding of d- and l-enantiomers on a single site. The apparent complexation constants for both d- and l-tryptophan show a maximum in the pH range 9-10. The variations of the apparent complexation constants versus pH were the result of the protonation of both the amino acid and a single site of the protein assuming that the complexation occurs between the zwitter-ionic amino acid form and the unprotonated BSA site. The apparent pK(BSA) is slightly shifted from 8.3 for grafted BSA protein to 9.4 for free BSA protein. This shift is presumably as a result of the different protein conformation.     

Spectrophotometric comparison of several reactions used for cerebrospinal fluid protein determinations

Three total protein procedures were analyzed by using spectral comparisons. Biuret reagent, Folin-Ciocalteau reagent, and Coomassie brilliant blue dye utilize different reactions to produce their final measured chromophores. Implicit in this statement is that there is no biuret component measured in the Folin-Ciocalteau color that is generated, for it is a reduced mixed heteropoly complex rather than a complex of copper with peptide bonds that is measured. The major conclusion of this study deals with the reliability of these three methods to effectively assay total protein mixtures in low-concentration solutions such as cerebrospinal fluid. Our results indicate that the biuret reaction is the least sensitive and the most linear while providing almost equal reactions on a weight per unit volume basis for albumin and gamma globulin as models. The Folin-Ciocalteau and Coomassie brilliant blue reactions are much more sensitive in absorbance signal generated, but they are nonlinear in calibration as well as unequal in their reactivities toward albumin and globulin. All of these factors must be carefully considered in making a choice of any of the three reactions for the analytical purposes intended.     

Indirect ultrasonication for protein quantification and peptide mass mapping through mass spectrometry-based techniques

We report in this work a fast protocol for protein quantification and for peptide mass mapping that rely on ¹⁸O isotopic labeling through the decoupling procedure. It is demonstrated that the purity and source of trypsin do not compromise the labeling degree and efficiency of the decoupled labeling reaction, and that the pH of the labeling reaction is a critical factor to obtain a significant ¹⁸O double labeling. We also show that the same calibration curve can be used for MALDI protein quantification during several days maintaining a reasonable accuracy, thus simplifying the handling of the quantification process. In addition we demonstrate that ¹⁸O isotopic labeling through the decoupling procedure can be successfully used to elaborate peptide mass maps. BSA was successfully quantified using the same calibration curve in different days and plasma from a freshwater fish, Cyprinus carpio, was used to elaborate the peptide mass maps.     

The Spectrophotometric Determination of gallium (III) Using O-Hydroxyhydroquinonephathalein in the Presence of Surfactant Micellar

Abstract

The color reaction systems between various metal ions and o-hydroxyhydroquinonephthalein(Qnph) as a xanthene dye, in the presence of various water soluble surfactants(cationic. anionic, non-ionic surfactants) alone or in combination, were systematically investigated at various pH areas. The coexistence of cationic and non-ionic surfactants, such as Zephiramine (Zp) and Brij 35, was most effective for the color reaction systems between Qnph and gallium(III), as a metal ion, at weakly acidic media. By using the color reaction between Qnph and gallium(III) in the coexistence of Zp and Brij 35, an improved and sensitive spectrophotometric determination of gallium(III) was proposed as method 1, and the calibration curve was rectilinear in the range of 0～7.0 μg of gallium(III) in a final solution of 10ml at pH 6.4. The apparent molar absorptivity was 1.5 × 10⁵ 1 mol^?1 cm^?1 at 560 nm, and the interference of foreign ions was decreased by ½～ ¼-fold in comparison with other methods; method 3—in the presence of Zp alone at pH 6.4, method 2—in the presence of Tween or Brij 35 alone, without Zp, at pH 8. Thus, the use of Qnph as a xanthene dye and the combination of cationic and non-ionic surfactants, such as Zp and Brij 35(perhaps, on the mixed micellar media), was most effective and its color reaction was used for the separative assay of gallium(III).     

Spectrophotometric reaction rate method for the determination of osmium by its catalytic effect on the oxidation of gallocyanine by bromate

A simple kinetic spectrophotometric method was developed for the determination of osmium. The method is based on the catalytic effect of osmium as osmium tetroxide on the oxidation of gallocyanine by bromate at pH 7. The reaction is monitored spectrophotometrically by measuring the decreasing absorbance of gallocyanine at 620 nm by the fixed-time method. A detection limit of 0.01 ng/ml and linear calibration curve from 0.1 to 100 and from 100 to 1200 ng/ml Os(VIII) is reported. The relative standard deviation for 0.0100 microg/ml Os(VIII) is 0.8% (N = 10). The method is free from most interferences. Osmium in synthetic samples is determined by this method, with satisfactory results.