Near-infrared spectroscopic determination of human serum albumin, γ-globulin, and glucose in a control serum solution with searching combination moving window partial least squares

Near-infrared (NIR) spectra in the region of 5000-4000 cm⁻¹ with a chemometric method called searching combination moving window partial least squares (SCMWPLS) were employed to determine the concentrations of human serum albumin (HSA), γ-globulin, and glucose contained in the control serum IIB (CS IIB) solutions with various concentrations. SCMWPLS is proposed to search for the optimized combinations of informative regions, which are spectral intervals, considered containing useful information for building partial least squares (PLS) models. The informative regions can easily be found by moving window partial least squares regression (MWPLSR) method. PLS calibration models using the regions obtained by SCMWPLS were developed for HSA, γ-globulin, and glucose. These models showed good prediction with the smallest root mean square error of predictions (RMSEP), the relatively small number of PLS factors, and the highest correlation coefficients among the results achieved by using whole region and MWPLSR methods. The RMSEP values of HSA, γ-globulin, and glucose yielded by SCMWPLS were 0.0303, 0.0327, and 0.0195 g/dl, respectively. These results prove that SCMWPLS can be successfully applied to determine simultaneously the concentrations of HSA, γ-globulin, and glucose in complicated biological fluids such as CS IIB solutions by using NIR spectroscopy.     

Determination of gamma-globulin at nanogram levels by its enhancement effect on the resonance light scattering of functionalized HgS nanoparticles

A novel assay of γ-globulin (γ-IgG) with a sensitivity at the nanogram level is proposed based on the measurement of enhanced resonance light-scattering (RLS) signals resulting from the interaction of functionalized nano-HgS with γ-globulin. At pH 5.03, the RLS signals of functionalized nano-HgS were greatly enhanced by γ-globulin in the region of 200-700 nm characterized by the peak around 362 nm. Linear relationship can be established between the enhanced RLS intensity and γ-globulin concentration in the range of 10-140 ng ml⁻¹. The limit of detection is 2.71 ng ml⁻¹. Based on this, a new direct quantitative determination method for γ-globulin in blood serum samples without separation of human serum albumin (HSA) is established. The contents of γ-IgG in blood serum samples were determined with recovery of 95.7-102.5% and R.S.D. of 1.6-2.4%. This method is proved to be very sensitive, rapid, simple and tolerance of most interfering substances.     

Determination of total basicity and available lysine in proteins by nonaqueous titrimetry

A simple method is presented for the determination of the available lysine residues of proteins. The sample is titrated directly with 0.05 or 0.1 M perchloric acid in anhydrous acetic or propionic acid to a potentiometric end-point. The titration is repeated after acylation of the sample. The difference between the two basicity values gives the available lysine content of the proteins. Results are provided for the available lysine contents of bovine serum albumin, human γ-globulin, β-casein, soya bean meals meat meal and milk protein; in most cases, they agree closely with literature data obtained by other methods. The standard error for the procedure is <3.9%.     

Langmuir Aggregation of Chromophore in Biomacromolecule and its Application: Interaction of Picramine CA (PCA) with Proteins

The microphase adsorption-spectral correction (MPASC) technique has been described. The formation of microelectrostatic fields in macromolecule is proposed and it causes the aggregation of a chromophore in protein. We have studied the interaction of picramine CA (PCA) with five proteins: bovine serum albumin (BSA), human n -globulin ( n -G), bovine hemoglobin (Hb), horse myoglobin (Mb) and ovalbumin (OVA). Results show that at pH 2.21, the adsorption ratios of PCA to BSA, n -G, Hb, Mb and OVA are 46, 54, 35, 15 and 21, respectively, their adsorption constants 3.17 ‐ 10 5 , 6.58 ‐ 10 4 , 1.40 ‐ 10 5 , 8.99 ‐ 10 4 and 1.68 ‐ 10 5 and their absorptivities 3.56 ‐ 10 5 , 3.06 ‐ 10 5 , 2.91 ‐ 10 5 , 1.10 ‐ 10 5 and 1.97 ‐ 10 5 l mol m 1 cm m 1 at 630 nm. The analysis of samples has given the satisfactory result.     

D.C. and pulse polarographic studies of rhodium(III)-protein catalytic systems

The Rh(III) complexes of substituted ethylenediamines are observed to be catalytically active at the DME and studies of the effects of human serum proteins on this activity are reported. Rh(III) complex waves are shown to respond to six different proteins, albumin, α-, β-, γ-globulin, mercaptoalbumin, and glycoprotein at trace concentration levels. The effect gives linear calibration for all proteins in the range 0.1–0.8 μg ml^?1 with sensitivity in the order glycoprotein >albumin>γ-globulin > β-globulin > α-globulin > mercaptoalbumin at pH 8. Important factors controlling sensitivity are buffer composition and pH, and Rh complex structure and concentration, and at pH 9.2, almost equal response of proteins is obtained. The method for protein analysis is compared to the Brdicka method and found to have several advantages in terms of sensitivity, pH range, response of differing proteins and wave resolution.     

Direct quantification of γ-globulin in human blood serum by resonance light scattering techniques without separation of human serum albumin

We describe the preparation and characterization of functionalized nano-PbS. The functionalized nanoparticles are water-soluble. Reaction of mercaptoacetic acid functionalized nano-PbS with γ-globulin (γ-IgG) results in an enhanced resonance light scattering (RLS) at 385 nm. Based on this, a new direct quantitative method for γ-globulin in blood serum samples without separation of human serum albumin is established. Under optimal conditions, the enhanced RLS intensity is in proportion to the γ-IgG concentration in the range 10-500 ng ml⁻¹. The 3σ limit of detection is 2.75 ng ml⁻¹. The contents of γ-IgG in blood serum samples were determined with a recovery of 97-104% and R.S.D. of 1.5-2.1% (n=6). This method proved to be very sensitive, rapid, simple and tolerant of most interfering substances.     

批量法研究牛γ-球蛋白在尼龙亲和膜上的等温吸附行为

以尼龙膜为基质,L－色氨酸（Trp)为配基,合成了一种新的亲和介质用以吸附牛γ-球蛋白（BGG）。用批量法系统考察了温度、离子强度和pH以亲和等温吸附的影响。研究结果表明,BCG与氨基酸之间的亲和相互作用力主要是静电力和疏水相互作用力。在最适条件下吸附遵循Langmuir型吸附,并且亲和吸附量最大而非特异性吸附最小。偏离该条件则会发生在蛋白质在膜上的堆积,蛋白质构型变化及蛋白质与配基间的空间取向的变化,从而使吸附不再遵循Langmuir型吸附。     

Effect of polysaccharide structure on protein adsorption

Using X-ray photoelectron spectroscopy for quantification, the adsorption has been studied of chicken egg lysozyme, human serum albumin (HSA), bovine colostrum lactoferrin, and γ-globulin (IgG) from single solutions onto surface-immobilised polysaccharide coatings, which were produced by the covalent attachment of a series of carboxymethyldextrans (CMDs) onto aminated fluoropolymer surfaces. CMDs with differing degrees of carboxymethyl substitution were synthesized by the reaction of dextran with bromoacetic acid under different reactant ratios. Substantial amounts of protein adsorption onto these coatings were observed with the majority of the coating/protein combinations. On the most extensively substituted CMD (1 carboxyl group per 2 dextran units), lysozyme and lactoferrin adsorbed to approximately monolayer amounts whereas there was minimal adsorption of HSA, indicating the importance of electrostatic interfacial interactions. CMD 1:14 was similar whereas the least substituted, least dense coating, from CMD 1:30, adsorbed less lysozyme and lactoferrin but more HSA. Adsorption of the large multidomain protein IgG varied little with the coating. Grazing angle XPS data indicated that for the CMD 1:30 coating there occurred significant in-diffusion of the lower molecular weight proteins. The data suggest that elimination of adsorption of a broad spectrum of proteins is not straightforward with negatively charged polysaccharide coatings; elimination of protein accumulation onto/into such coatings may not be achievable solely with a balance of electrostatic and steric–entropic interfacial forces.     

Synchronous fluorescence determination of protein with functionalized CdS nanoparticles as a fluorescence probe

Nanometer-sized fluorescent particles have been successfully synthesized. A synchronous fluorescence method, with high sensitivity and selectivity, has been developed for rapid determination of protein with functionalized CdS as a fluorescence probe. When Δλ=260 nm, maximum synchronous fluorescence is produced at 274 nm at pH 7.0. Under optimal conditions, the calibration graphs are linear over the range 0.1-3.0 μg ml⁻¹ for bovine serum albumin (BSA), 0.1-11.0 μg ml⁻¹ for γ-globulin (γ-G) and 0.1-1.4 μg ml⁻¹ for human serum albumin (HSA), respectively. Limits of determination were 0.01 μg ml⁻¹ for BSA, 0.019 μg ml⁻¹ for γ-G and 0.021 μg ml⁻¹ for HSA, respectively. The relative standard deviations of seven replicate measurements were 1.8% for 1.0 μg ml⁻¹ BSA, 2.2% for 1.0 μg ml⁻¹ γ-G and 2.3% for 1.0 μg ml⁻¹ HSA.     

DNA Quantification in Nanoliter Volumes

A novel method for the determination of proteins at nanogram levels was proposed based on the decrease of resonance light scattering (RLS) signal resulting from the interaction of dibromo-o-nitrophenylfluorone (DBONPF)-sodium lauroyl glutamate (SLG) with proteins. At pH 2.97, the decrease RLS intensity was proportional to the concentration of proteins in the range of nanogram levels with 3σ detection limits being 3.4 ng mL⁻¹ for bovine serum albumin (BSA), 1.7 ng mL⁻¹ for human serum albumin (HSA), 4.1 ng mL⁻¹ for γ-globulin (γ-IgG), 4.4 ng mL⁻¹ for egg albumin, 6.2 ng mL⁻¹ for pepsin (Pep) and 3.7 ng mL⁻¹ for α-chymotrypsin (Chy). The method is no protein-to-protein variability, simple, rapid, practical and relatively free from interference from coexisting substance, as well as much more sensitive than most of the reported methods. The proposed method was successfully applied to determine total protein in human serum samples.     

Evaluation of a potential prognostic parameter for the inflammatory status in COVID-19 patients: The inflammatory protein ratio

C-reactive protein (CRP), fibrinogen, and d -dimer are determined in the human plasma of 2745 hospitalized patients with and without coronavirus disease 2019 (COVID-19) by automated-latex enhanced immunoassay and immuno-turbidimetric assay. SARS-COV-2 RNA qualitative test, real time polymerase chain reaction (RT-PCR) based, is performed in nasopharyngeal swabs to confirm those with SARS-COV-2 positivity. Furthermore, serum proteins are separated and quantified in all the patients by serum protein electrophoresis (SPE). A new SPE parameter, inflammatory protein ratio (IPR), is elaborated for the first time by a mathematical equation that considers the albumin, α1-globulin, and α2-globulin. IPR normal reference range (10.7%–28.3%) is calculated considering the normal reference range of albumin, α1-globulin, and α2-globulin obtained for controls. Analysis of variance (ANOVA), Pearson's, Kruskal–Wallis, and Spearman's tests application show that IPR significantly correlates with direct proportionality with d -dimer, CRP, and fibrinogen. Significant (p < 0.001) increase of these parameters, IPR included, is detected in COVID-19 patients only. Our results show that IPR is more specific for monitoring inflammatory status thanks to its correlation with the only three serum proteins involved in inflammation: albumin, α1-globulin, and α2-globulin. Furthermore, IPR can simplify the interpretation of SPE results about inflammatory status, being of unique value compared to the six-serum protein classes separately presented in the typical SPE clinical reports.     

Microdetermination of Proteins with the 1,10-Phenanthroline–H2O2–Cetyltrimethylammonium Bromide–Cu(II) Chemiluminescence System

Proteins can enhance the chemiluminescence (CL) intensity of the 1,10-phenanthroline–H₂O₂–cetyltrimethylammonium bromide (CTMAB)–Cu(II) system because unsaturated complexes of Cu(II) with protein have a much stronger catalytic effect on the CL reaction than does Cu(II). On this basis, a new flow injection analysis method for detection of some proteins was established. The method gives linear responses over two orders of magnitude and detection limits at the 0.02–0.05 μg ml⁻¹level for bovine serum albumin, human serum albumin, γ-globulin, and egg albumin. The method was used for determination of proteins in human serum with satisfactory results.     

Effect of the acyl group and the dodecylsulfonate chain on the inclusion of pyrene inside the cavity of β-cyclodextrin

The solubilization of pyrene in aqueous solution of β-cyclodextrin (β-CD) or its derivatives such as β-CD-hexanoyl, β-CD-benzoyl and β-CD-dodecylsulfonate was investigated by spectrophotometry. Linear and non-linear regression methods were used to estimate the association constants (K₁). A 1:1 stoichiometric ratio and different effects of the hexanoyl, benzoyl and dodecylsulfonate groups on the association constant were observed for the binary inclusion complex between pyrene and β-CD. The formation constant was shown to decrease when β-CD was modified by a dodecylsulfonate chain. The value of K₁ was 190 ± 10 L mol⁻¹ for the [pyrene/β-CD] complex and 145 L mol⁻¹ for the [pyrene/β-CD-dodecylsulfonate] complex. Partitioning of the pyrene molecules between the dodecylsulfonate chains and cyclodextrin cavities can explain the decrease in the association constant value. In the cases of β-CD-hexanoyl and β-CD-benzoyl derivatives, no association constants were detected. Results suggest that the high hydrophobicity of the hexanoyl and benzoyl groups prevents the inclusion of pyrene molecules inside the cyclodextrin cavity.     

Adduct Formation Between γ-Cyclodextrin and the Bifluorophore 1,3-Bis(1-pyrenyl)propane

The complexation of the bifluorophore 1,3-bis(pyrenyl)propane with γ-cyclodextrin in water has been studied by means of steady state and time-resolved fluorescence spectroscopy. It was found that in the association with γ-cyclodextrin the propane chain of 1,3-bis(pyrenyl)propane folds and the two pyrene units enter the same cyclodextrin cavity where they form weakly bound ground state dimers, which upon excitation emit excimer fluorescence. In addition to this 1:1 excimer emitting complex, two more complexes were detected, which emit monomer pyrene fluorescence. One has 1:1 stoichiometry, i.e. isomeric to the previous complex, and the other, with 2:1 stoichiometry, is comprised of two γ-cyclodextrin units and one 1,3-bis(pyrenyl)propane.     

On the factors affecting the enhanced resonance light scattering signals of the interactions between proteins and multiply negatively charged chromophores using water blue as an example

Electrostatic interactions of proteins, including bovine serum albumin (BSA), human serum albumin (HSA), γ-globulin (γ-IgG), α-chymotrypsin (Chy), lysozyme (Lys) and cellulase (Cel), with multiply negatively charged chromophores were investigated based on the measurements of the enhanced resonance light scattering (RLS) signals. Using triply negatively charged water blue (WB) as an example, the factors were discussed that affect the enhanced resonance light scattering signals of the interactions between proteins and the negatively charged chromophores. It was found that the enhanced RLS signals with the maximum light scattering peak at 346.0 nm in these interacting systems are strongly dependent on the isoelectric points of proteins and show adverse linear relationships with increasing ionic strength depending on the positive charges of the inorganic metal ions used to control the ionic strength of the medium, sufficiently disclosing that the electrostatic attraction performs an important role in the combination of proteins with WB. Linear responses were discovered between the enhanced RLS signals and the protein molecular weights (M_w), displaying the dimensions of scattered particles formed by proteins and WB make a key contribution to the RLS enhancements. An empirical equation is proposed which possibly displays the factors affecting the enhanced RLS signals of the interactions between proteins with negatively charged chromophores.     

Evidence of neutral radical induced analyte ion transformations in APPI and Near-VUV APLI

We report on the reactions of neutral radical species [OH, Cl, O(³P), H], generated in a typical atmospheric pressure ionization (API) source upon irradiation of the sample gases with either 193 nm laser radiation or 124 nm VUV light, the latter commonly used in atmospheric pressure photoionization (APPI). The present investigations focus on the polycyclic aromatic hydrocarbon pyrene as representative of the aromatic compound class. Experimental results are supported by computational methods: simple kinetic models are used to estimate the temporal evolution of the concentrations of reactants, intermediates, and final products, whereas density functional theory (DFT) energy calculations are carried out to further elucidate the proposed reaction pathways. The neutral radicals are generated upon photolysis of background water and oxygen always present in appreciable mixing ratios in typical API sources. Substantial amounts of oxygenated analyte product ions are observed using both techniques. In contrast, upon atmospheric pressure laser ionization (APLI) with 248 nm radiation, oxygenated products are virtually absent. In addition, kinetic data evaluation yielded a bimolecular rate constant of k=(1.9±0.9)×10⁻⁹ cm³ molecule⁻¹ s⁻¹ for the reaction of the pyrene radical cation with OH radicals.     

Characterization of an opioid peptide-containing protein and of bovine α-lactalbumin by electrospray ionization and liquid secondary ion mass spectrometry

Mass spectrometry methods have been used to characterize two proteins: an opioid peptide-containing protein extracted from bovine pituitary, and bovine α-lactalbumin (BAL). A protein that contains β-endorphin was found in bovine pituitary, and that protein was characterized with electrospray ionization mass spectrometry (ESIMS), gel permeation chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), radioimmunoassay, trypsinolysis, and liquid secondary ion mass spectrometry (LSIMS). BAL is a protein that was used as a model to develop analytical methods to study opioid peptide-containing proteins. Commercial BAL was purified by RP-HPLC, and its molecular weight (M.W.) was determined by ESIMS. The shift in mass observed following dithiothreitol (DTT) reduction estimated the number of disulfide bonds. For all of the data obtained for BAL with or without RP-HPLC separation, ESIMS determined the M.W. of the peptides produced by trypsin treatment of BAL, and LSIMS selected a precursor ion, the protonated molecule ion [M + H]⁺, of a tryptic peptide, which was analyzed by tandem mass spectrometry. Following DTT reduction, ESIMS and LSIMS detected each peptide that contained disulfide bonds in that mixture of tryptic peptides.     

Identification of Highly Active Flocculant Proteins in Bovine Blood

Synthetic polymeric flocculants are used extensively for wastewater remediation, soil stabilization, and reduction in water leakage from unlined canals. Sources of highly active, inexpensive, renewable flocculants are needed to replace synthetic flocculants. High kaolin flocculant activity was documented for bovine blood (BB) and blood plasma with several anticoagulant treatments. BB serum also had high flocculant activity. To address the hypothesis that some blood proteins have strong flocculating activity, the BB proteins were separated by SEC. Then, the major proteins of the flocculant-active fractions were separated by SDS-PAGE. Identity of the major protein components was determined by tryptic digestion and peptide analysis by MALDI TOF MS. The sequence of selected peptides was confirmed using TOF/TOF-MS/MS fragmentation. Hemoglobin dimer (subunits α and β) was identified as the major protein component of the active fraction in BB; its high flocculation activity was confirmed by testing a commercial sample of hemoglobin. In the same manner, three proteins from blood plasma (fibrinogen, γ-globulin, α-2-macroglobulin) were found to be highly active flocculants, but bovine serum albumin, α-globulin, and β-globulin were not flocculants. On a mass basis, hemoglobin, γ-globulin, α-2-macroglobulin were as effective as anionic polyacrylamide (PAM), a widely used synthetic flocculant. The blood proteins acted faster than PAM, and unlike PAM, the blood proteins flocculants did not require calcium salts for their activity.     

Effects of low-dose ionizing radiation on α,β-globulins solutions studied by DSC

An attempt has been made to detect the effect of a small dose of ionizing radiation on the course of α,β-globulin thermal denaturation in aqueous solutions. Doses of 0.1 and 1.8 Gy have been delivered using γ-rays emitted by ⁶⁰Co isotope while doses of 10 and 100 Gy have been supplied by X-rays produced by linear accelerator. The highest dose has visibly changed DSC curve of protein solution while the changes due to lower doses are hardly detectable. Although very weak, the irradiation effect found has been dose dependent. The results suggest that the influence of ionizing radiation on globulins solution is bigger when the dose rate is lower at given dose. The opposite direction of differences between irradiated and control samples for fresh and stored protein solutions suggests various characters of changes in initial and later period of sample aging. This may be an important reason for difficulties in an investigation of the effect of ionizing radiation on protein solution, especially for low doses delivered very slowly.     

Preparation of composite monoliths of quaternized chitosan and diatom earth for protein separation

In this study, composite monoliths with porous structures were prepared using quaternized chitosan and diatom earth for protein separation. Quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride) dissolved in water was mixed with diatom earth and crosslinked with glutaraldehyde under low-temperature conditions to form a cryogel. Interconnected porous monoliths were obtained after removing ice crystals from the cryogel. The monoliths adsorbed bovine serum albumin selectively from the solution mixture of bovine serum albumin and bovine ɤ-globulin, and bovine ɤ-globulin was recovered in the flow-through fraction. The adsorption selectivity was enhanced by changing the solution pH from 6.8 to 5.5. The adsorption of bovine serum albumin by the monolith was replicated at least five times following its washing with a buffer containing 400 mM NaCl and subsequent regeneration with a 10 mM acetate buffer. The composited monolith is a promising adsorbent for the removal of acidic proteins, such as serum albumin contamination in neutral proteins, for example, ɤ-globulins, in bioproduction processes.