Langmuir Aggregation of Coomassie Brill Blue on Protein and Determination of Protein by MPASC

The microphase adsorption–spectral correction technique (MPASC) has been applied to investigate the aggregation of coormassie brill blue G250 (CBBG) on protein in which the microelectrostatic fields exist. The Langmuir adsorption of CBBG in protein was confirmed, and the interaction is sensitive at pH 3.78. Results show that the adsorption ratio of CBBG to five proteins, e.g., BSA, OVA, Hb, Mb and -G are 100, 65, 30, 98 and 210, respectively, their adsorption constants are 9.92 × 10⁴, 8.04 x 10⁴, 9.38 × 10⁴, 1.66 × 10⁵, and 2.75 × 10⁴, and their absorptivities are 2.33 × 10⁶, 1.56 × 10⁶, 1.98 × 10⁶, 5.61 × 10⁵, and 4.48 × 10⁶ Lmol^–1 at 670 nm. The study indicates that 1 mg of protein adsorbs about 1.54 mmol of CBBG. For the protein determination of samples, the recovery of protein is between 88.0 and 111% and the relative standard deviation is 2.6%.     

Identification of a Fluorescent Protein from Rhacostoma Atlantica

We have cloned a novel fluorescent protein from the jellyfish Rhacostoma atlantica. The closest known related fluorescent protein is the Phialidium yellow fluorescent protein, with only a 55% amino acid sequence identity. A somewhat unusual alanine–tyrosine–glycine amino acid sequence forms the presumed chromophore of the novel protein. The protein has an absorption peak at 466 nm and a fluorescence emission peak at 498 nm. The fluorescence quantum yield was measured to be 0.77 and the extinction coefficient is 58 200 M^?1 cm^?1. Several mutations were identified that shift the absorption peak to about 494 nm and the emission peak to between 512 and 514 nm.     

Construction of Protein Probe with a His-tag and an Electron-transfer Peptide for a Target Protein Sensing

A protein probe with an electron-transfer peptide and a His-tag was designed to electrochemically sense a target protein. We selected tyrosine-rich (Y₄C) and tryptophan-rich (W₄C) peptides for use as electron-transfer agents. The peak for oxidation was based on the oxidations of the phenolic hydroxy groups in Y₄C and on the indole rings in W₄C. Asialofetuin (ASF) with galactose residues was the protein probe, and a galactose recognition protein, soybean agglutinin (SBA), was the target protein. A protein probe composed of an amino acid and carbohydrate residue was expected to be biocompatible. When voltammetric measurements were performed using a glassy carbon electrode, the oxidation peaks of H₆Y₄C and ASF-H₆Y₄C appeared at the same potential. The peak current of ASF-H₆Y₄C was 4-fold that of H₆Y₄C because of the stronger adsorption of ASF-H₆Y₄C onto the electrode. The electrode response of ASF-H₆Y₄C with SBA was half that of ASF-H₆Y₄C alone. By contrast, the peak current of ASF-Y₄CH₆ was higher than that of ASF-H₆Y₄C, which was the result of a greater degree of contact between the Y₄C moieties and an electrode. On the other hand, the voltammetric behaviors of ASF with W₄C and a His-tag were similar to those with Y₄C and a His-tag. The sensitivity of SBA using ASF-Y₄CH₆ was at the 10⁻¹³ M level. To confirm the function of the sensing system, measurements were performed in human serum with SBA and ASF-Y₄CH₆. When SBA was added, the serum had a concentration that ranged between 5.0×10⁻¹³ and 4.0×10⁻¹² M, and the amount of SBA that could be recovered ranged from 97 to 101%. Consequently, this system could be applied to the detection of SBA in serum.     

Purification of Biliary Protein and Its Effect on Calcium Carbonate Mineralization

The biliary protein (BP) was isolated from pig bile by gel filtration. The interaction between Ca²⁺ and protein was measured by fluorescence spectra. The result showed that there was a strong coordination between biliary protein and Ca²⁺. The CaCO₃ crystals obtained in systems with and without BP were characterized by scanning electron microscopy, Fourier transform infrared spectrography and powder X‐ray diffractometry. The possible formation mechanism of CaCO₃ in biliary protein solution was discussed.     

Radiation Chemical Studies of Protein Reactions: Effect of Irradiation Liquids Containing Aromatic Hydrocarbons and pH on the Breaking of Secondary Bonding in Protein

When protein in various liquids containing aromatic hydrocarbons, such as benzene, naphthalene, and phenanthrene, is irradiated by γ-rays from a ⁶⁰Co source, the breaking of secondary bonding in the protein molecule varies with the irradiation liquids containing aromatic hydrocarbons. Protein irradiated by γ-rays from a ⁶⁰Co source in air showed the effect of pH on the breaking of secondary bonding in the protein molecule. In both cases an empirical equation for the viscosity change was obtained, and the phenomena were explained on the basis of the molecular mechanism.     

Determination of Deuterated Phenylalanine and Tyrosine in Egg Protein by GCQ

In order to study protein digestibility by means of noninvasive tracer techniques (stable isotopes), a representative oral tracer, i.e. a stable isotope labeled protein, is needed. Therefore, egg white containing L-[ring-²H₅]phenylalanine and L-[ring-²H₄]tyrosine was prepared. The aim of this study was to measure the isotopic enrichment of the labeled amino acids in the egg white. The use of a standard GC-MS, based on ion trap technology was found to be a reliable technique. The enrichment of L-[ring-²H₅]phenylalanine and L-[ring-²H₄]tyrosine, expressed in Molar Percent (MP) amounted to 23.2 MP and 2.8 MP respectively.     

Probing Membrane Protein Insertion into Lipid Bilayers by Solid-State NMR

Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton-detected solid-state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic-angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site-specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid-protein transfer rate of 6 s⁻¹ was determined for residues protected from exchange. Transfer within the protein, as tracked in the ¹⁵N-¹H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s⁻¹ for several resolved residues, explaining the site specificity.     

Mg2+与胆汁蛋白质相互作用研究

胆汁是一中性溶液,在胆汁固形物中,胆汁酸盐、胆固醇、磷脂、蛋白质、胆红素分别占总质量的65%、3%、20%、5%及1%。蛋白质是胆汁固形物中第四多成分,在胆汁的生理功能的发挥和胆结石的形成中起到重要的作用。研究表明,胆汁蛋白质的主要成分是糖蛋白,主要由胆囊粘膜上皮细胞产生     

CZE with On-line Micellar Sample Stacking for Determination of Protein Concentration of Biopharmaceuticals

A capillary zone electrophoresis total protein assay was developed and validated in polyethylene oxide (PEO) dynamically coated capillaries. On-line large-volume sample stacking was employed. Protein samples were denatured using SDS and then injected into PEO-filled capillaries. Such treatment enabled injection of a sample volume of ??8% of the total capillary volume and stacking of protein-SDS molecules at the interface between the sample plug and the PEO plug. Results showed that SDS enhanced the sensitivity not only by protein denaturation but also by forming micelles, in which protein-SDS partitioned. Sensitivity of the method was further enhanced through using capillaries with (tenfold) extended detection pathlength. Such strategies resulted in a limit of detection of 0.26 ??g mL^?1 (3.64 nM BSA). A linear relationship between protein concentration and integrated peak area was obtained over a wide concentration range (8.49?C135.87 ??g mL^?1??R ² = 0.995). The method is particularly useful for determination of total protein concentration in chromatography fractions. It overcomes low UV absorptivity of proteins, presence of UV absorbing additives and high salt content. Contrary to conventional methods for determination of protein concentration, this method does not involve an interaction with a dye. Thus, variations due to differences in surface properties among proteins or due to differences in posttranslational modifications of the same protein are eliminated. The protocol was successfully applied for the determination of the concentration of a biopharmaceutical protein rhMBP in chromatography fractions. This protein has been previously produced in milk of transgenic cows and several charge isoforms were detected.     

Interaction of Trypan Blue with Protein and Application

The microphase adsorption ‐ spectral correction (MPASC) technique has been described and applied to the aggregation of trypan blue (TB) in proteins. The formation of the microelectrostatic field in protein causes the Langmuir monolayer aggregation of TB. The adsorption ratio of TB to bovine serum albumin (BSA), ovalbumin (OVA), hemoglobin (Hb) and human γ‐globulin (γ‐G) was determined to be 14.8, 8.4, 2.8 and 27.6, respectively, and the adsorption constant of the aggregates to be 7.17 × 10⁵, 4.88 × 10⁶, 4.85 × 10⁶ and 2.99 × 10⁶. The adsorption ratio of TB to proteins interestingly indicates almost no relation to the array sequence of amino acid residues. The interaction of TB with proteins is sensitive at pH 3.29, and the reaction was applied to the determination of protein trace with satisfactory results.     

Thermodynamic Characterization of Two Nearly Simultaneous Protein Denaturations

Near-UV circular dichroism and differential scanning calorimetry were used to analyse the thermal denaturation of bovine α-lactalbumin at pH 7.5 and various Ca²⁺ concentrations. Both analyses revealed that the denaturated protein consists of two fractions, a Ca²⁺-loaded and a Ca²⁺-free fraction. Despite this agreement, the two methods afforded significantly different values for the thermodynamic parameters of the denaturations. It was demonstrated that the ellipticity changes were more appropriate than the excess heat capacities for analysis of the two types of denaturation. The values of the thermodynamic parameters obtained with the former method are therefore more reliable.     

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D ¹H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (D_M) of 3.3. The 2D ¹H-¹³C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for ¹H, 15 ppb for ¹³C and 98% peaks with equivalent peak height. Finally, 2D ¹H-¹⁵N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.     

Radiation Chemical Studies of Protein Reactions: Effect of Post-irradiation on Optical Rotation

When protein was irradiated by γ-rays from a ⁶⁰Co source, a post-irradiation effect of protein reaction was caused. An empirical equation for the optical rotation was obtained, and the phenomena were explained on the basis of the molecular mechanism. The general equation for the optical rotation is given by [α] f= a ? b log t, where [α] f is the final specific rotation of the solution, t is time after γ irradiation, and a and b are adjustable constants.     

Studies on the Spectral Behavior between Protein with Poniacyl Carmine 2B and its Analytical Application

In this paper, the spectral behavior of protein and Poniacyl Carmine 2B (PC 2B) has been studied by spectrophotometric method. The conditional constants, apparent combination constant K and maximum binding number n, were used to express the combination ability of the reactions between PC 2B and protein under a set of given conditions. The Sandell index s was used to express the sensitivity of the determination of protein. The factors, acidity, PC 2B concentration and the ionic strength, were discussed by the change of apparent combination constant and maximum binding number. It was found that acidity of the solution, PC 2B concentration and ionic strength had a significant effect on the sensitivity of the assay of protein. Under the optimal conditions, the apparent combination constant K and the maximum binding number n were 2.36 × 10⁶ L mol^?1 and 95, respectively. With further investigation, it was found that the Scatchard model was suitable in treating the data obtained in the experiments. In the buffer medium of HCl‐KCl at 1.87, the addition of protein made the maximum absorption of the system move from 527 nm to 513 nm. Its apparent molar absorptivity is 4.46 × 10⁵ Lmol^?1 cm^?1 at 513 nm. Beer's law is obeyed in the range of 0 ? 55 μg mL^?1. The system developed in this paper has been used for the determination of protein in milk powder successfully.     

Native Chemical Ligation to Minimize Aspartimide Formation during Chemical Synthesis of Small LDLa Protein

The inhibition of the G protein‐coupled receptor, relaxin family peptide receptor 1 (RXFP1), by a small LDLa protein may be a potential approach for prostate cancer treatment. However, it is a significant challenge to chemically produce the 41‐residue and three‐disulfide cross‐bridged LDLa module which is highly prone to aspartimide formation due to the presence of several aspartic acid residues. Known palliative measures, including addition of HOBt to piperidine for N^α‐deprotection, failed to completely overcome this side reaction. For this reason, an elegant native chemical ligation approach was employed in which two segments were assembled for generating the linear LDLa protein. Acquisition of correct folding was achieved by using either a regioselective disulfide bond formation or global oxidation strategies. The final synthetic LDLa protein obtained was characterized by NMR spectroscopic structural analysis after chelation with a Ca²⁺ ion and confirmed to be equivalent to the same protein obtained by recombinant DNA production.     

The Thermal Diastereomerization of the Tryptophane-Derived Green Fluorescent Protein Chromophore

Summary. Two model compounds for the tryptophane variant of the green fluorescent protein chromophore containing a 3-indolyl and 2-pyrrolyl moiety were prepared. For the first one the (Z)-diastereomer was found to be more stable than the (E)-diastereomer by 5.7 kJ mol⁻¹. It could be photo-diastereomerized and its thermal equilibration was studied, whereas the second one underwent photo-destruction. From an Arrhenius plot an activation barrier for the (E) to (Z) diastereomerization of 85.6 kJ mol⁻¹ could be determined. Thus, it could be demonstrated that in contrast to the corresponding phenyl derivative studied recently the tyrosine- and tryptophane-derived chromophores of the green fluorescent protein are amenable to fast thermal diastereomerization, which is of fundamental importance for the fluorescence and photoswitching processes in the corresponding proteins.     

Methods for Determining Rates of Protein Synthesis via Dark CO2 Fixation by Marine Prokaryote

Methods were developed to determine rates of particulate and dissolved protein synthesis via dark CO₂ fixation by marine prokaryotic assemblages. The methods are based on incorporation of [¹⁴C]-bicarbonate and separation of proteins as TCA-insoluble materials. Results indicated that particulate protein constitutes a significant fraction (～67%) of cellular organic matter produced via prokaryotic dark CO₂ fixation. The microcentrifuge method allowed us to estimate the rate of total protein synthesis via CO₂ fixation. Time-series data proved useful for determining the optimum incubation period, and for estimating the rate of protein synthesis by regression analysis. The total prokaryotic dark CO₂ fixation rate (10.3 ng C l^?1 h^?1) estimated by these methods was 2.7-times the rate of particulate CO₂ fixation, which was comparable to the CO₂ fixation rate estimated by methods used in previous studies. Thus, total prokaryotic dark CO₂ fixation appears to be more important in the marine carbon cycle than previously thought.     

Dendrimeric Bisphosphonates for Multivalent Protein Surface Binding

A single weak‐binding event is multiplied into an efficient receptor site for protein surfaces (<10^?1 to >10⁶ M ^?1 in buffered aqueous solution) in a biomimetic fashion. This has hitherto been done with natural host/guest pairs, but not with artificial receptors. The organic reaction presented is one of very few that enable chemists to fuse multiple ionic building blocks covalently in highly polar solution; this one‐pot reaction proceeds with virtually quantitative yield. According to this concept, other building blocks with aldehyde groups can likewise be multiplied into monodisperse functional dendrimers. Small basic proteins are bound by octameric dendrimers in 1:1 or 1:2 complexes with millimolar to submicromolar affinities. The complexation event is studied independently in buffered aqueous solution by three different spectroscopic methods (PFG‐LED, UV/Vis, and fluorescence). Potential new applications include recombinant protein purification through Arg tags on immobilized dendrimers and on/off switching of protein function by reversible active‐site capping of enzymes.     

Allosterically Activated Protein Self‐Assembly for the Construction of Helical Microfilaments with Tunable Helicity

Protein allostery, a chemical‐to‐mechanical effect that can precisely regulate protein structure, exists in many proteins. Herein, we demonstrate that protein allostery can be used to drive self‐assembly for the construction of tunable protein architectures. Calmodulin (CaM) was chosen as a model allosteric protein. Ca²⁺‐mediated contraction of CaM to a closed state can activate CaM and its ligand to self‐assemble into a 1D protein helical microfilament. Conversely, relaxation of CaM to the open state can unwind and further dissociate the helical assemblies. Fine regulation of the protein conformation by tuning the external Ca²⁺ level allows us to obtain various protein helical nanostructures with tunable helicity. This study offers a new approach toward chemomechanically controlled protein self‐assembly.     

Development and Characterization of Novel Composite Films Based on Soy Protein Isolate and Oilseed Flours

The possibility of using oilseed flours as a waste source for film-forming materials with a combination of soy protein isolate in preparation of edible films was evaluated. Physical, mechanical and barrier properties were determined as a function of the oilseed type: hemp, evening primrose, flax, pumpkin, sesame and sunflower. It was observed that the addition of oilseed flours increased the refraction and thus the opacity of the obtained films from 1.27 to 9.57 A mm⁻¹. Depending on the type of flours used, the edible films took on various colors. Lightness (L*) was lowest for the evening primrose film (L* = 34.91) and highest for the soy protein film (L* = 91.84). Parameter a* was lowest for the sunflower film (a* = −5.13) and highest for the flax film (a* = 13.62). Edible films made of pumpkin seed flour had the highest value of the b* color parameter (b* = 34.40), while films made of evening primrose flour had the lowest value (b* = 1.35). All analyzed films had relatively low mechanical resistance, with tensile strength from 0.60 to 3.09 MPa. Films made of flour containing the highest amount of protein, pumpkin and sesame, had the highest water vapor permeability, 2.41 and 2.70 × 10⁻⁹ g·m⁻¹ s⁻¹ Pa⁻¹, respectively. All the edible films obtained had high water swelling values from 131.10 to 362.16%, and the microstructure of the films changed after adding the flour, from homogeneous and smooth to rough. All blended soy protein isolate–oilseed flour films showed lower thermal stability which was better observed at the first and second stages of thermogravimetric analysis when degradation occurred at lower temperatures. The oilseed flours blended with soy protein isolate show the possibility of using them in the development of biodegradable films which can find practical application in the food industry.