Adsorption Equilibrium and Kinetics of Egg-White Proteins on Immobilized Metal Ion Affinity Gels for Designing Fractionation

Designing an Immobilized Metal ion Affinity (IMA) chromatographic process on large scale demands a thorough understanding to be developed regarding the adsorption behaviour of proteins on metal loaded IMA (IMA-M(II)) gels and the characteristic adsorption parameters to be evaluated. This research investigation illustrates the significance of these aspects for the proposed fractionation of chicken egg-white proteins on these gels. Consequently, a systematic investigation of the adsorption characteristics of three chicken egg-white proteins viz., ovalbumin, conalbumin and lysozyme on Cu(II) and Ni(II) loaded IMA gels, iminodiacetate (IDA) and tris(2-aminoethyl)amine (TREN), has been undertaken. These gels differ in their selectivity towards the proteins of interest under the identical sets of experimental conditions. While TREN-Ni(II) was selective only for lysozyme, IDA-Cu(II), IDA-Ni(II) and TREN-Cu(II) showed varying affinities for all the three proteins. The equilibrium and kinetic data were analysed using various theoretical models and adsorption parameters were quantified. On the basis of these investigations, various strategies have been proposed for the efficient large-scale fractionation of chicken egg-white proteins on these gels.     

INDUCIBLE POSTREPLICATION REPAIR IS RESPONSIBLE FOR MINIMAL MEDIUM RECOVERY IN UV-IRRADIATED Escherichia coliK–12

Abstract— Ultraviolet (UV)-irradiated Escherichia coli K–12 uvrA cells showed higher survival if plated on minimal growth medium rather than on rich growth medium, i.e., they showed minimal medium recovery (MMR). A 2-hour treatment of UV-irradiated cells with rifampicin inhibited the subsequent expression of MMR, and produced a large reduction in survival. We have recently isolated a new mutant ( mmrA1 ) that does not show MMR. The mmrA mutation protected UV-irradiated uvrA cells from the effect of rich growth medium on survival, but not from the effect of rifampicin on survival. DNA daughter-strand gap (DSG) repair in UV-irradiated (4 J/m²) uvrA cells was inhibited to the same degree whether rich growth medium was added immediately after irradiation or after 10 min of postirradiation incubation in minimal growth medium. However, chloramphenicol added immediately after irradiation greatly reduced this repair; there was less reduction if it was added 10 min after UV irradiation. These findings suggest that MMR is an inducible repair phenomenon, and that rich growth medium inhibits this repair process itself rather than its induction.     

Temperature dependence of near-resonant vibration ↔ rotation energy transfer

V ? R,T deactivation of the ν₄ (243 cm^?1) level in BCl₃ by HCl has been measured over the range 220–340 K, with an infrared double resonance technique. The deactivation probabilities show an inverse temperature dependence below room temperature, indicating that long-range attractive forces are important in the resonant transfer process. The results are compared with the theory of Sharma and Brau.     

Solvation numbers and hydration constant for thorium(IV) in ethanol-water medium

The solvation number and hydration constant of Th⁴⁺ in ethanol-water medium were determined at 25°C using UV-spectral and electrochemical measurements. A solvate formation equilibrium is demonstrated and characterized. Three molecules of ethanol (S) can bond to the metal cation with strengths comparable to that for H₂O to form ThS₃(H₂O) ₃ ⁴⁺ Formation of thorium monochelate with lawsone (2-hydroxy-1,4-naphthoquinone) eliminates bonding with alcohol molecules. The dissociation constant of the chelating agent_sK_a and the formation contant of the monochelated metal ion_sK _f ^* that are essentially independent of the solution composition are evaluated. Hydration titrations involving thorium-lawsone monochelate are performed and the data obtained from the changes of pH with solvent composition are analyzed. The solution independent constant,_sK _f ^* for thorium-lawsone complex formation in mixed aqueous ethanol is given by $$log_s K_f * = vpK_a + log_s K_h - log[LH] - vpH + 3logv$$ where vpK_a is the dissociation constant of the chelating agent LH in the solvent system of ν volume fraction of water and_sK_h is the solution-independent hydration constant of thorium (IV) in the solvent system. Log-values for the constants_sK_h,_sK _f ^* and_sK _a ^* are found to be 7.8±0.02, 11.38±0.04 and ?0.753, respectively.     

The process\pi ^0 \to \lambda _\gamma \bar \lambda _\gamma as an alternative to\pi ^0 \to v\bar v

The process \(\pi ^0 \to \lambda _\gamma \bar \lambda _\gamma \) is investigated as an alternative to \(\pi ^0 \to v\bar v\) . It is shown that the experimental bound on the branching fraction for missing energy (in the form of \(\lambda _\gamma \bar \lambda _\gamma \) and/or \(v\bar v\) ) may be understood in terms of \(\pi ^0 \to \lambda _\gamma \bar \lambda _\gamma \) for the kinematically allowed photino mass, if the squark mass is >8 GeV.     

Can a single oxidant with two spin states masquerade as two different oxidants? A study of the sulfoxidation mechanism by cytochrome p450

Density functional calculations were performed on the sulfoxidation reaction by a model compound I (Cpd I) of cytochrome P450. By contrast to previous alkane hydroxylation studies, which exhibit a dominant low-spin (LS) pathway, the sulfoxidation follows a dominant high-spin (HS) reaction. Thus, competing hydroxylation and sulfoxidation processes as observed for instance by Jones et al. (Volz, T. J.; Rock, D. A.; Jones, J. P. J. Am. Chem. Soc. 2002, 124, 9724) are the result of a two-state reactivity scenario, whereby the hydroxylation originates from the LS pathway and the sulfoxidation from the HS pathway. In this manner, two spin states of a single oxidant (Cpd I) can be disguised as two different oxidants. The calculations rule out the possibility that a second oxidant (the ferric peroxide, Cpd 0 species) interferes in the observed results of Jones et al.     

Differentiation of Boc- alpha,beta- and beta,alpha-peptides and a pair of diastereomeric beta,alpha-dipeptides by positive and negative ion electrospray tandem mass spectrometry (ESI-MS/MS)

Positive and negative ion electrospray ionization (ESI) tandem mass spectral study of a new series of hybrid peptides, viz, BocN-alpha,beta-peptides and BocN-beta,alpha-peptides, synthesized from C-linked carbo-beta3-amino acids [Caa (S)] and L-Ala has been carried out. The alpha,beta-peptides have been differentiated from beta,alpha-peptides by the collision-induced dissociation (CID) of [M + H]+ and [M - H]- ions in positive and negative ion ESI-MS respectively. The fragment ion [M + H - C(CH3)3 + H]+ formed from [M + H]+ ions by the loss of 2-methyl-prop-2-ene in alpha,beta-peptides with L-Ala at the N-terminus is insignificant or totally absent for beta,alpha-peptides which have the Caa (S) at N-terminus. The fragment ion [M - H-C(CH3)3OH - HNCO]- formed from [M - H]- of beta,alpha-peptide acids is totally absent for alpha,beta-peptide acids. This has been attributed to the absence of the beta-methylene group in alpha,beta-peptides, and the participation of the beta-methylene group in the loss of HNCO in beta,alpha-peptide acids is confirmed by the deuteration experiments. The CID of [M + H-Boc + H]+ ions of these peptides also produce characteristic fragmentation. In the CID spectra of alpha,beta-peptides, the b(n)+ ions and the resulting y(n)+ ions occur at a mass difference of 243 and 71 Da corresponding to the successive losses of Caa and L-Ala, whereas a mass difference of 71 and 243 Da is observed for beta,alpha-peptides. In contrast to the CID of protonated peptides, the CID of [M - H]- ions of the alpha,beta- and beta,alpha-peptide acids do not give b(n)- ions and show abundant z(n) (-) ions. Further, a pair of diastereomeric dipeptide esters and acids have been distinguished by the CID of [M + H]+ ions. The loss of 2-methyl-prop-2-ene is more pronounced for Boc-NH-Caa(R)-D-Ala-OCH3 (21) and Boc-NH-Caa(R)-D-Ala-OH (23) with Caa (R) at the N-terminus, whereas it is totally absent for Boc-NH-Caa (S)-D-Ala-OCH3 (22) and Boc-NH-Caa(S)-D-Ala-OH (24) peptides, which have Caa (S) at the N-terminus. Thus, on the basis of our previous and present studies, we propose that the CID of [M + H]+ ions provides a simple and useful method for distinguishing the configuration of Caa (S or R) at the N-terminus of BocN-carbo beta,alpha- and beta,beta-dipeptides.     

Structural modifications of aqueous ionic micelles in the presence of denaturants as studied by DLS and viscometry

Hydrophobic interactions control the morphologies of both surfactant aggregates and proteins. Globular proteins "denature" upon addition of excess amounts of denaturants such as urea. Understanding the microscopic basis of the urea effect on proteins or supramolecular aggregates such as micelles has always been a debated issue. Inspired by this need, the effect of urea (U), thiourea (TU), monomethylurea (MMU), dimethylurea(DMU), tetramethylurea (TMU), dimethylthiourea (DMTU), and tetramethylthiourea (TMTU) on the structural transition (spherical micelles to rod-shaped micelles, s --> r) in the sodium dodecylbenzenesulfonate (SDBS)-1-pentanol system has been investigated through dynamic light scattering(DLS) and viscosity measurements at 25 degrees C. 1-Pentanol, at 0.14 M, is found to promote s --> r in this system (0.2 M SDBS). The presence of the additives causes, in almost all cases, a decrease and increase in this 1-pentanol concentration depending upon the concentration and nature of the additive. These effects are explained in terms of an increased dielectric constant of the solvent medium due to the presence of additives and increased micellar hydration due to the repulsion of charged monomers caused by adsorption of the additives. Taken together, the data signal the exposure of biological assemblies to water at higher [additive], which causes a decrease in hydrophobic interactions responsible for compact structure formation (i.e., native protein).     

Physico-chemical studies on the chelation behaviour of biologically active 2-hydroxy-1-naphthaldehyde semicarbazone (HNAS) with some lanthanons

Summary The chelation behaviour of the complexes of La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Y³⁺, Tb³⁺, Dy³⁺, Ho³⁺ with biologically active 2-Hydroxy-1-naphthaldehyde semicarbazone (HNAS) has been studied potentiometrically in 75% (v/v) dioxane-water medium at various ionic strengths. The method of Bjerrum and Calvin, as modified by Irving and Rossotti has been used to find out the values of n andpL. The formation constants of metal chelates and the values ofS _min have been calculated. The order of formation constants of chelates was found to be: La³⁺3+3+3+3+3+3+3+3+3+3+.

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Physikochemische Untersuchungen zur Komplexierung von biologisch aktivem 2-Hydroxy-1-naphthaldehyd-Semicarbazon (HNAS) mit Lanthanoiden

Zusammenfassung Das Chelierungsverhalten von La³⁺, Ce³⁺, Pr³⁺, Nd³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Y³⁺, Tb³⁺, Dy³⁺ und Ho³⁺ mit 2-Hydroxy-1-naphthaldehydsemicarbazon (HNAS) wurde potentiometrisch in 75% (v/v) Dioxan-Wasser bei verschiedenen Ionenstärken untersucht. Die Werte für n undpL wurden nach der Methode von Bjerrum und Calvin in der Modifikation von Irving und Rossotti bestimmt. Die Komplexbildungskonstanten der Metallchelate und die WerteS _min wurden ermittelt. Die Reihung der Komplexbildungskonstanten war La³⁺3+3+3+3+3+3+3+3+33+.

     

Pervaporation from a dense membrane: roles of permeant-membrane interactions, Kelvin effect, and membrane swelling

Dense polymeric membranes with extremely small pores in the form of free volume are used widely in the pervaporative separation of liquid mixtures. The membrane permeation of a component followed by its vaporization on the opposite face is governed by the solubility and downstream pressure. We measured the evaporative flux of pure methanol and 2-propanol using dense membranes with different free volumes and different affinities (wettabilities and solubilities) for the permeant. Interestingly, the evaporative flux for different membranes vanished substantially (10-75%) below the equilibrium vapor pressure in the bulk. The discrepancy was larger for a smaller pore size and for more wettable membranes (higher positive spreading coefficients). This observation, which cannot be explained by the existing (mostly solution-diffusion type) models ofpervaporation, suggests an important role for the membrane-permeant interactions in nanopores that can lower the equilibrium vapor pressure. The pore sizes, as estimated from the positron annihilation, ranged from 0.2 to 0.6 nm for the dry membranes. Solubilities of methanol in different composite membranes were estimated from the Flory-Huggins theory. The interaction parameter was obtained from the surface properties measured by the contact angle goniometry in conjunction with the acid-base theory of polar surface interactions. For the membranes examined, the increase in the "wet" pore volume due to membrane swelling correlates almost linearly with the solubility of methanol in these membranes. Indeed, the observations are found to be consistent with the lowering of the equilibrium vapor pressure on the basis of the Kelvin equation. Thus, a higher solubility or selectivity of a membrane also implies stronger permeant-membrane interactions and a greater retention of the permeant by the membrane, thus decreasing its evaporative flux. This observation has important implications for the interpretation of existing experiments and in the separation of liquid mixtures by pervaporation.