6M Urea Solutions Research Articles

Non-covalent assembly of poly(allylamine hydrochloride)/triethylamine microcapsules with ionic strength-responsiveness and auto-fluorescence

Ionic strength-responsive microcapsules with auto-fluorescence were fabricated by incubation of poly(allylamine hydrochloride) (PAH)-doped CaCO3 particles in triethylamine (Et3N), followed by core removal using HCl. Based on the combination of hydrophobic interaction and hydrogen bonding, PAH and Et3N formed a complex with a molar ratio of 3:1 (repeating unit of PAH: Et3N). The as-prepared capsules showed extraordinary stability against 1M HCl, 1M NaOH and 6M urea solutions, and could swell or shrink reversibly in response to the ionic strength. Furthermore, the capsules possessed auto-fluorescence, allowing easily tracking of capsules during applications. Such interaction may be expanded to formation of stimuli-responsive multilayer films and other colloidal particles.

Dating bloodstains with fluorescence lifetime measurements.

Dating bloodstains with fluorescence lifetime measurements.

Sequential recognition of antigenic markers of Toxoplasma gondii tachyzoite by pooled sera of mice with experimental toxoplasmosis

Accurate diagnosis of maternal toxoplasmosis can enhance the success of medical treatment and prevent congenital transmission. The current diagnostic methods have many limits, and they poorly differentiate between recent and latent infections. The present work was conducted to record the sequential recognition of antigenic markers of both Toxoplasma tachyzoites whole extract and glycosylinositolphospholipids (GIPLs)-enriched fraction by specific IgG and IgM, respectively, by immunoblotting analysis of the antigens against daily pooled serum samples from mice with experimentally induced recent and latent toxoplasmosis. IgG avidity immunoblotting was tested by using a wash with 6M urea solution as antigen-antibody disrupting agent. Band of 10kDa reacted exclusively with low-avidity IgG in pooled sera of mice with recent infection. Band of 39kDa was a good marker for the infection; reacting with both low-avidity IgG in recent infection and with high-avidity IgG in latent one. Bands of 15, 23, 30, 60, 66, and 97kDa reacted with variable avidity in both phases of infection. Two antigenic bands were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the GIPLs-enriched fraction of tachyzoite, the 14- and 30-kDa band. The 14-kDa band was recognized by IgM in pooled serum samples of recently infected mice only, while the 30-kDa band was recognized by serum samples of both recent and latent phases of infections. The study highlights the value of avidity immunoblotting assay to discriminate between recent and latent experimental toxoplasmosis. Further study must be carried on human to evaluate the values of the used technique.

Temperature and urea induced denaturation of the TRP-cage mini protein TC5b: A simulation study consistent with experimental observations.

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20-residue Trp-cage mini-protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force-field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR-NOE atom-atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40-90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force-field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side-chain side-chain contact (Trp6-Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.

Urea-induced conformational changes of chitosan molecules and the shift of break point of Mark-Houwink equation by increasing urea concentration

Chitosan solutions of the same 83% degree of deacetylation (DD) but different weight average molecular weights (Mws) (78–914 kDa) in 0.01M HCl containing different concentrations of urea (0–6M) were prepared. Intrinsic viscosity ([η]) and weight average molecular weight (Mw) of chitosan were measured with a capillary viscometer and light scattering, respectively. Mark–Houwink exponent a was used as the parameter of conformational index. The Mark–Houwink exponent a increased with increasing concentrations of urea. When solutions contained 0, 2, 3, 4, and 6M urea, the value of a increased from 0.715 to 0.839, 0.894, 1.000, and 1.060, respectively. This indicates the occurrence of urea-induced conformational transitions of chitosans. The break point shifted from 223 kDa in solutions containing no urea to 280 kDa in 2M urea solutions, to 362 kDa in 4M urea solutions and further to 481 kDa in 6M urea solutions. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 452–457, 2000

Heat capacities of transfer of some amino acids and peptides from water to aqueous urea solution

AbstractIntegral enthalpies of solution at low concentrations of several amino acids and peptides in 2 and 6M urea solutions have been determined at 25 and 35°C. These data have been used to derive the enthalpies of transfer (at 25 and 35°C) and heat capacities of transfer (at 30°C) of these amino acids and peptides from water to aqueous urea solutions. Furthermore, the enthalpies of transfer and heat capacities of transfer per CH2 group and per peptide group CONH have also been estimated. These results show that while the enthalpies and heat capacities of transfer per CH2 group are positive and negative, respectively, the reverse is true for CONH group. The implications of these results in the mechanism of the denaturation of proteins by urea are discussed.

Changes in Isoelectric Focusing Patterns of Milk Proteins from Storage in Aqueous Urea-Mercapthoethanol Solution

Patterns of isoelectric focusing of β-lactoglobulin, α-lactalbumin, and bovine serum albumin changed when these proteins were stored in aqueous 6M urea solutions containing mercaptoethanol. These changes, which were not detected by electrophoresis, involved progressive loss of some bands observed on focusing of freshly prepared solutions. They occurred rapidly (less than 15min) at pH 7 and 9 but required 2 to 3 days at pH 5.7. Isoelectric focusing patterns were not altered on storage at pH 5.0 or 2.0, or if the urea concentration was reduced substantially below 6M, or in the absence of mercaptoethanol. The half cystine residues in the proteins were implicated in changes in isoelectric focusing pattern as the changes were not evident on storage of αS- or β-casein (free of cystine residues) and as reduction and blockage of these residues in β-lactoglobulin rendered the patterns stable on storage.These changes were not accompanied to any considerable extent by aggregation. It is likely that these changes were the result of unfolding of the proteins under the influence of the 6M urea, followed by -SS/-SH interchange involving mercaptoethanol.

Determination of the number of cross-links in a protein gel from its mechanical and swelling properties.

AbstractThe relation between the chemical structure of a protein and the physical properties of a heat‐set gel of that protein has been investigated. The physical properties of the gel are determined by means of mechanical experiments in which the viscoelastic properties of the gel are determined in terms of the storage shear modulus, the loss modulus and the stress‐strain curve. The storage shear modulus defined the solid (elastic) character of the gel. The chemical structure of the protein and the nature of the solvent determine the nature and number of cross‐links in the gel. The cross‐links in gels formed by heating concentrated solutions of ovalbumin in 6M urea solutions were found to be disulfide bridges and the mechanical properties of these ovalbumin/urea gels approximated those of an ideal rubber. The latter finding enables one to calculate the number of cross‐links per ovalbumin molecule from the value of the storage modulus, using the classical theory of rubber elasticity. This theory, together with the Flory‐Huggins lattice model, can also be used to calculate the number of cros‐links per ovalbumin molecule from the swelling behavior of ovalbumin/urea gels. The number of cross‐links per ovalbumin molecule calculated from these two types of experiments are in mutual agreement and correspond with the number of thiol groups in ovalbumin. We conclude, thereforee, that theories of polymer physics can be used to relate the chemical structure of a protein to the physical properties of its gel.

Chemical studies on the cysteine and terminal peptides in tryptic digests of actin.

1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide ;maps' of tryptic digests of actin treated with thiol reagents. 3. The three peptides, each containing one residue of CMCys, were isolated from tryptic digests of carboxymethylated actin by ion-exchange chromatography. 4. One of these peptides was possibly the N-terminal peptide and contained about 17-18 residues; another was CMCys-Asp-Ile-Asp-Ile-Arg; the other, CMCys-Phe, was the C-terminal tryptic peptide. 5. The chemical evidence suggests that the actin molecule consists of a single polypeptide chain of molecular weight about 44000.

Studies of Casein. III. The Molecular Size of α-, β-, and γ-Casein

α-, β-, and x-casein are aggregated at neutral pH and room temperature in salt solution. They may be disaggregated at high pH or at neutral pH in concentrated urea solution. At neutral pH in salt solution, α-casein forms aggregates centring around a preferred size. As the pH is increased the size decreases until at pH 11 (I 0.20) it is disaggregated completely. Measurements of the molecular weight by sedimentation and diffusion at pH 11 give a value of 24,800�1000. Approach to sedimentation-equilibrium measurements at pH 12, using the Archibald method, gives a value of 25,500�1000. In 6M urea solution at pH 7.3 sedimentation and diffusion give a value of 27,600�1000. At room temperature and neutral pH, β-casein is present as single molecules in equilibrium with an aggregate of very high molecular weight. At pH 11, it is disaggregated with a molecular weight of 17,300�800 (by sedimentation-diffusion). At pH 7 in 631 urea a value of 19,800�1000 is obtained for the molecular weight. Preliminary measurements by the Archibald method at pH 12 give a value of 26,000�3000 for the molecular weight of x-casein. These results are discussed in relation to those of other workers.