Modeling of immunoglobulin uptake by N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia particles under static and dynamic conditions

A matrix developed from N,N,N',N'-ethylenediaminetetramethylenephosphonic acid-modified zirconia beads (further referred to as r_PEZ); 25-38 microm in diameter and with a pore size of 22+/-3 nm, was utilized for the separation of immunoglobulins (Igs). r_PEZ has been shown to bind to various Igs originating from a wide variety of species. To understand the mechanisms controlling the uptake of Igs by r_PEZ, static protein uptake experiments were carried out. The protein uptake profiles were further modeled with a kinetic rate constant model. Individual studies were undertaken for human immunoglobulin A, G and M (HIgA, HIgG and HIgM). The kinetic rate constant model indicated that HIgG binding to r_PEZ was more favorable than its disassociation. The equilibrium rate constants were found to decrease with increasing concentration. The effect of continuous loading in a packed bed system utilizing r_PEZ matrix was evaluated by carrying out frontal studies, using different feed concentrations and linear velocities. The breakthrough profiles obtained for the uptake of HIgG were modeled with the pore diffusion model. The model was found to best describe the breakthrough profiles obtained at a feed concentration of 2.0 mg of HIgG per milliliter. The NTU for the packed bed was found to be equal to 2.     

Simulating the effects of N availability, straw particle size and location in soil on C and N mineralization

Predicting the C and N mineralization of straw added to soil is important for forecasting subsequent soil N availability during and between crop growth cycles. The decomposition module of the STICS model, parameterized under optimal conditions, was used to predict straw decomposition in sub-optimal conditions, i.e. when contact between soil and residue was poor (due to large size residues or surface placement) or when mineral N availability was restricted. The data used in the simulations were obtained from published studies of effects of residue size, location and N availability on C and N mineralization from straw under controlled laboratory conditions. We selected studies in which the dynamics of C and N mineralization were measured simultaneously. The dynamics of straw mineralization could be well predicted by the model under optimal conditions with standard parameter values as derived from measured C/N ratios of the residues, but not under sub-optimal conditions which required a new parameterization. A good fit could be obtained on these treatments by a marked reduction in the rate constants of residue and microbial biomass decomposition and a marked increase in the microbial biomass C/N ratio. Our results show the need to include in decomposition models routines for simulating effects of spatial heterogeneity of residue distribution, different particle sizes and limiting N availability.     

Characterization and optimization of a chromatographic process based on ethylenediamine-N,N,N',N'-tetra(methylphosphonic) acid-modified zirconia particles

The primary objective of work was to characterize, optimize and model a chromatographic process based on ethylenediamine-N,N,N',N'-tetra(methylphosphonic) acid (EDTPA)-modified zirconia particles. Zirconia particles were produced by spray-drying colloidal zirconia. Zirconia spheres produced were further classified, calcined and modified with EDTPA to yield a solid-phase support for use in bio-chromatography (r_PEZ). Specifically, the ability of r_PEZ to selectively bind and enrich IgG, IgA, and IgM from biological fluids was evaluated and demonstrated. To better understand the force of interaction between the IgG and the r_PEZ, the equilibrium disassociation constant (K(d)) was determined by static binding isotherms, as a function of temperature and by frontal analysis at different linear velocities. The maximum static binding capacity (Q(max)) was found to be in the range 55-65 mg IgG per ml of beads, and unaffected by temperature. The maximum dynamic binding capacity (Q(x)) was found to be in the range 20-12 mg IgG per ml of beads. The adsorption rate constant (k(a)) was determined by a split-peak approach to be between 982 and 3242 l mol(-1) s(-1) depending on the linear velocity. The standard enthalpy and entropy values were estimated for this interaction of IgG with this novel support.     

Size exclusion chromatography of cellulose in LiCl/N,N-dimethylacetamide

Size exclusion of cellulose in LiCl/N,N-dimethylacetamide has been used for the past 15 years, yet much of the current research is still devoted to fundamental studies, as many issues regarding column calibration, separation mechanisms and solution behavior have not been resolved yet. The paper reviews practical aspects of sample preparation and it is demonstrated that sample heating and several techniques to aid solvent exchange call for reevaluation. It is further shown that the use of internal standard may introduce minor improvements in repeatability. The commonly used column calibration procedures, chromatographic conditions and applications are also reviewed. Further research is needed to understand the mechanisms of separation, to optimize column calibration and to facilitate and optimize sample preparation.     

Regime analysis of a Baeyer–Villiger bioconversion in a three-phase (air–water–ionic liquid) stirred tank bioreactor

The aim of this work was to conduct a regime analysis on a three-phase (air–water–ionic liquid) stirred tank bioreactor of the Baeyer–Villiger bioconversion process, using [MeBuPyrr][BTA] ionic liquid as the dispersed phase. The regime analysis based on characteristic times of the different mechanisms involved (mixing, mass transfer, reaction) can yield a quantitative estimate of bioreactor performance. The characteristic time obtained for oxygen uptake rate (54 s⁻¹) was among the characteristic times determined for oxygen transfer (13–129 s⁻¹) under different operating conditions, suggesting that the oxygen transfer rate under certain operating conditions could be a limiting step in the bioconversion process. Further enhancement of oxygen transfer rates requires proper selection of the bioreactor operational conditions, and improved design of the ionic liquid used as oxygen transfer vector.     

Aerobic purification of N5,N10-methylenetetrahydromethanopterin dehydrogenase, separated from N5,N10-methylenetetrahydromethanopterin cyclohydrolase, from Methanobacterium thermoautotrophicum strain Marburg

The N5,N10-methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum strain Marburg has been purified with reasonable yield and much higher specific activity than previously reported. For the first time it has been shown that both N5,N10-methylenetetrahydromethanopterin dehydrogenase and N5,N10-methenyltetrahydromethanopterin cyclohydrolase activities were stable under air and could be purified using aerobic operations. The dehydrogenase activity from Methanobacterium thermoautotrophicum Marburg was stable in phosphate buffer with or without glycerol or ammonium sulfate under both aerobic and anaerobic conditions. However, the presence of either 2-mercaptoethanol or dithiothreitol in the enzyme solution destroyed the enzyme activity during both aerobic and anaerobic incubations. Dehydrogenase was purified 62-fold using Phenyl-Sepharose and DEAE-Sephadex chromatography in succession under air. Both of these chromatographic methods separated dehydrogenase activity from N5,N10-methenyltetrahydromethanopterin cyclohydrolase; DEAE-Sephadex provided the best separation. Phenyl-Sepharose chromatography of the supernatant of cell extracts containing ammonium sulfate at 60% of saturation provided a 4.7-fold purification and 98% recovery of cyclohydrolase; this result established the air stability of N5,N10-methenyltetrahydromethanopterin cyclohydrolase from Methanobacterium thermoautotrophicum Marburg.     

Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases

We have used deuterium and 15N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid [Grimshaw, C.E., Cook, P.F., & Cleland, W.W. (1981) Biochemistry 20, 5655; Rife, J.E., & Cleland, W.W. (1980) Biochemistry 19, 2328]. These steps are almost equally rate limiting for V/Kammonia with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/Kammonia. Release of oxidized nucleotide is largely rate limiting for Vmax for both enzymes. When beta-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits Vmax, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of alpha-ketoglutarate by alpha-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with alpha-ketoglutarate but causes an 8-fold decrease in Vmax with alpha-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either alpha-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.     

Effects of ionic strength on lysozyme uptake rates in cation exchangers. I: Uptake in SP Sepharose FF

Fluorescence scanning confocal microscopy was used in parallel with batch uptake and breakthrough measurements of transport rates to study the effect of ionic strength on the uptake of lysozyme into SP Sepharose FF. In all cases the adsorption isotherms were near-rectangular. As described previously, the intraparticle profiles changed from slow-moving self-sharpening fronts at low salt concentration, to fast-moving diffuse profiles at high salt concentration, and batch uptake rates correspondingly increased with increasing salt concentration. Shrinking core and homogeneous diffusion frameworks were used successfully to obtain effective diffusivities for the low salt and high salt conditions, respectively. The prediction of column breakthrough was generally good using these frameworks, except for low-salt uptake results. In those cases, the compressibility of the stationary phase coupled with the shrinking core behavior appears to reduce the mass transfer rates at particle-particle contacts, leading to shallower breakthrough curves. In contrast, the fast uptake rates at high ionic strength appear to reduce the importance of mass transfer limitations at the particle contacts, but the confocal results do show a flow rate dependence on the uptake profiles, suggesting that external mass transfer becomes more limiting at high ionic strength. These results show that the complexity of behavior observable at the microscopic scale is directly manifested at the column scale and provides a phenomenological basis to interpret and predict column breakthrough. In addition, the results provide heuristics for the optimization of chromatographic conditions.     

Experimental and theoretical evidence for convective nutrient transport in an immobilized cell support.

Even though immobilized-cell reactors possess several engineering advantages over free-cell reactors, their full potential has not been realized because mass transfer often limits the rate of nutrient supply and product removal from immobilized cell supports. We studied the interaction between mass transfer and reaction kinetics in the anaerobic conversion of glucose to CO2 and ethanol by yeast immobilized in a porous rotating disk on the agitator shaft of a conventional CSTR. A Sherwood number correlation was used to show that external mass-transfer resistances were negligible under typical operating conditions. The modulus of Weisz based on observable reaction parameters was used to gauge the importance of pore diffusion limitations. Under conditions for which significant pore diffusion effects and hence low effectiveness factors (eta = ca. 0.1) would be predicted, the observed reaction rates were much higher than expected (eta = ca. 1), suggesting that pore diffusion limitations were at least partially relieved by convective transport of glucose into the support. Two possible mechanisms of convective transport are discussed. We hypothesize that gas evolution was responsible for the convective enhancement of glucose supply.     

Gross mineralization and plant N uptake from animal manures under non-N limiting conditions, measured using 15N isotope dilution techniques

To utilise wisely the manure resource, a better understanding of the processes that control the breakdown of organic N to inorganic N (mineralization) is required. ¹⁵N isotope dilution techniques should allow estimates of plant N uptake and gross mineralization from organic manures under non-N limiting conditions to be made. In natural systems the study of organic nitrogen breakdown to inorganic nitrogen, mineralization, is confounded by the processes of nitrification, nitrate leaching, gaseous N losses and plant N uptake. The ¹⁵N isotope dilution approach allows measurement of gross mineralization independently of these processes. Greenhouse experiments were conducted to determine plant N uptake from organic manures under non-N limiting conditions using the soil pre-labelling isotope dilution approach. The soil was pre-labelled with ¹⁵N and maize plants were then grown on the control treatments (no organic amendment) or on the manure treatments. The principle is thus that the control crop has a ¹⁵N abundance which reflects the ¹⁵N status of the soil and the treatment crop has a ¹⁵N enrichment diluted by the contribution of mineralized unlabelled manure N. Using this technique, it was estimated that maize plants derived 17 and 34% of their N from sewage sludge and turkey manure, respectively. The soil pre-labelling isotope dilution approach allowed yield-independent estimation of nitrogen derived from manures under non-N limiting conditions. Estimates of gross N mineralization were made to determine the breakdown of manure under field conditions. Results suggested that there was a rapid mineralization of turkey manure N in the initial weeks after application, in the order of 50 kg N ha^?1, which tailed off in the following weeks. The technique suggested that the soil used in the study had an extremely low basal mineralization rate, and a high nitrification rate.     

The use of confocal laser scanning microscopy to study the transport of biomacromolecules in a macroporous support

Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 microm in diameter and have 3.5% solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites throughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.     

Root and hyphal interactions influence N transfer by arbuscular mycorrhizal fungi in soybean/maize intercropping systems

In maize-soybean intercropping systems, the transfer of N from soybean to maize gives the intercropping system the advantage of improved N utilization and higher yields. Mycorrhiza acts as an important pathway for N transfer, providing a constant supply of N to sustain the growth and development of maize in its early stages. However, it is not clear how arbuscular mycorrhizal fungi (AMF) drive the transfer of N from soybean to maize in the intercropping system. Therefore, we quantified the amount of N transferred from soybean to maize under low and high N levels in the intercropping system, and the abundance and diversity of AMF involved in N transfer (¹⁵N-AMF) under different conditions by ¹⁵N leaf marker and DNA-SIP technology. We found that the interaction between roots and reducing the application of N fertilizer increased the amount of N transfer from soybean to maize. Compared with plastic plate separation (PS), no separation (NS) and mesh separation (MS) significantly increased the N fixation rate (from 14.33% to 39.09%), and the amount of N transfer under NS was 1.95–3.48 times that under MS. N transfer from soybean to maize ranged from 9.7 to 43.42 mg per pot in the no N treatment, while the addition of N fertilizer reduced N transfer by 14.12–66.28%. This is due to root interaction and reduced N fertilization increased the abundance and diversity of the ¹⁵N-AMF community, thereby promoting AMF colonization of maize and soybean roots. AMF colonization in soybean and maize roots under NS treatment was 6.47–17.24% higher than under MS treatment in all three levels of N addition. The increase of mycorrhiza in root system increased the N transfer from soybean to maize significantly. These results suggest that reduced N fertilizer in maize-soybean intercropping systems can increase N transfer by the mycorrhizal pathway, meeting maize N requirements and reducing chemical N fertilizer, which is important for sustainable agricultural development.     

A kinetic, chromatographic method for studying protein hydrodynamic behavior

A chromatographic method based on "split-peak" behavior was described for the determination of the coefficient of mass transfer of proteins on small reversed-phase columns. The coefficient of mass transfer was found to be a linear function of the protein translational diffusion coefficient and inversely proportional to the square of the support particle diameter, as predicted by chromatographic theory. As an example of the practical application of this method for the measurement of protein diffusion coefficients, the denaturation of bovine serum albumin with decreasing solution pH was followed by measuring the change in the coefficient of mass transfer. A major advantage of this method was that the results were not affected by the interaction of the protein with the stationary phase.     

Modelling of the chromatographic separation of xylose-mannose in ion-exchange resin columns

A mathematical model was proposed for the chromatographic separation of xylose and mannose on an ion-exchange resin in the Pb form: dispersion in the mobile phase, external mass transfer around the particles and internal diffusion were taken into account. Small-scale experiments provided an evaluation of the different parameters. Dispersion in the mobile phase was found to be the predominant phenomenon. The Peclet numbers were calculated by identification in the Laplace domain of the elution profiles. Influence of temperature and initial concentration of the sample were studied.     

A mechanism of microbial cell growth

Presently empirical expressions, especially the Monod equation, are used to quantitatively relate microbial growth rate to limiting substrate concentration in the solution. In this paper microbial growth is postulated to occur by a mechanism involving a mass transfer or assimilation process. The assimilation process is assumed to be substrate mass transfer limited and hence proportional to the limiting substrate concentration. The ingestion is assumed independent of limiting substrate concentration and only dependent upon internal reaction rates. The quantitative relationship between limiting substrate and microbial growth rate resulting from this mechanism is developed. Under certain limiting conditions this expression is shown to reduce to the Monod equation and under other conditions it reduces to the Lotka-Volterra relationship. This mechanism is applied to batch and continuous cultures and the results obtained are compared quantitatively with experiment.     

Amine inversion in proteins. A 13C-NMR study of proton exchange and nitrogen inversion rates in N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine,N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine methyl ester, and reductively methylated concanavalin A

Exchange rates were calculated as a function of pH from line widths of methylamine resonances in 13C-NMR spectra of N epsilon,N epsilon,N alpha,N alpha-[13C]tetramethyllysine (TML) and N epsilon,N epsilon,N alpha,N alpha-tetramethyllysine methyl ester (TMLME). The pH dependence of the dimethyl alpha-amine exchange rate could be adequately described by assuming base-catalyzed chemical exchange between two diastereotopic methyl populations related by nitrogen inversion. Deprotonation of the alpha-amine was assumed to occur by proton transfer to (1) OH-, (2) water, (3) a deprotonated amine or (4) RCO2-. Microscopic rate constants characterizing each of these transfer processes (k1, k2, k3 and k4, respectively) were determined by fitting the rates calculated from line width analysis to a steady-state kinetic model. Using this procedure it was determined that for both TML and TMLME k2 approximately equal to 1-10 M-1 s-1, k3 approximately equal to 10(6) M-1 s-1 and ki, the rate constant for nitrogen inversion was about 10(8)-10(9) s-1. Upper limits of 10(12) and 10(3) M-1 s-1 could be determined for k1 and k4, respectively. A similar kinetic analysis was used to explain pH-dependent line-broadening effects observed for the N-terminal dimethylalanyl resonance in 13C-NMR spectra of concanavalin A, reductively methylated using 90% [13C]formaldehyde. From exchange data below pH 4 it could be determined that amine inversion was limited by the proton transfer rate to the solvent, with a rate constant estimated at 20 M-1 s-1. Above pH 4, exchange was limited by proton transfer to other titrating groups in the protein structure. Based upon their proximity, the carboxylate side chains of Asp-2 and Asp-218 appear to be likely candidates. The apparent first-order microscopic rate constant characterizing proton transfer to these groups was estimated to be about 1 X 10(4) s-1. Rate constants characterizing nitrogen inversion (ki), proton transfer to OH- (k1) and proton transfer to the solvent (k2) were estimated to be of the same order of magnitude as those determined for the model compounds. On the basis of our results, it is proposed that chemical exchange processes associated with base-catalyzed nitrogen inversion may contribute to 15N or 13C spin-lattice relaxation times in reductively methylated peptides or proteins.     

Evaluation of kinetic and mass transfer parameters for adsorption of clavulanic acid into natural and synthetic zeolites

This work investigates the adsorption of clavulanic acid using natural six cationic forms (Na⁺, Ca⁺², Ba⁺², Sr²⁺, K⁺, and Mg²⁺) of the X and NZ zeolites in a stirred tank reactor since the separation is an important step of the biomolecule production. A mathematical model was proposed taking into account the transport of CA molecules from the liquid phase to the surface of the adsorbent and after diffusion into the particles. The estimated kinetic and mass transfer parameters were used to evaluate adsorption rates and mass transfer resistances involved in the separation of clavulanic acid from the broth. It has been shown that mass-transfer phenomena were a limiting step in the clavulanic acid adsorption process and that the adsorption rate should be considered to evaluate the system. Amongst the materials, the synthetic zeolite NaX was selected as the most appropriate material to separate clavulanic acid because this material presented the highest values for the observed reaction rate, compensating for the external mass transfer resistance. Modeling and simulation of clavulanic acid purification using the zeolite NaX showed a satisfactory fitting of experimental data. The model was used to simulate the process and it was evaluated for its technical and economical viability by comparisons considering the influence of the solid:liquid ratio on the adsorption equilibrium time and on the hydrolysed mass of biomolecule.     

PROCAL: A Set of 40 Peptide Standards for Retention Time Indexing,Column Performance Monitoring,and Collision Energy Calibration

Beyond specific applications, such as the relative or absolute quantification of peptides in targeted proteomic experiments, synthetic spike‐in peptides are not yet systematically used as internal standards in bottom‐up proteomics. A number of retention time standards have been reported that enable chromatographic aligning of multiple LC–MS/MS experiments. However, only few peptides are typically included in such sets limiting the analytical parameters that can be monitored. Here, we describe PROCAL (ProteomeTools Calibration Standard), a set of 40 synthetic peptides that span the entire hydrophobicity range of tryptic digests, enabling not only accurate determination of retention time indices but also monitoring of chromatographic separation performance over time. The fragmentation characteristics of the peptides can also be used to calibrate and compare collision energies between mass spectrometers. The sequences of all selected peptides do not occur in any natural protein, thus eliminating the need for stable isotope labeling. We anticipate that this set of peptides will be useful for multiple purposes in individual laboratories but also aiding the transfer of data acquisition and analysis methods between laboratories, notably the use of spectral libraries.     

Modeling of protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) is commonly used to separate protein monomer and aggregate species in the purification of protein therapeutics. Despite being used frequently, the HIC separation mechanism is quite complex and not well understood. In this paper, we examined the separation of a monomer and aggregate protein mixture using Phenyl Sepharose FF. The mechanisms of protein adsorption, desorption, and diffusion of the two species were evaluated using several experimental approaches to determine which processes controlled the separation. A chromatography model, which used homogeneous diffusion (to describe mass transfer) and a competitive Langmuir binary isotherm (to describe protein adsorption and desorption), was formulated and used to predict the separation of the monomer and aggregate species. The experimental studies showed a fraction of the aggregate species bound irreversibly to the adsorbent, which was a major factor governing the separation of the species. The model predictions showed inclusion of irreversible binding in the adsorption mechanism greatly improved the model predictions over a range of operating conditions. The model successfully predicted the separation performance of the adsorbent with the examined feed.     

Determination of diffusion coefficients of proteins in stationary phases by frontal chromatography

A strategy to determine effective diffusion coefficients of proteins in chromatographic gels is presented in this article. An experimental methodology based on frontal liquid chromatography was combined with a numerical methodology based on a mathematical model describing the chromatographic process including the extra-column dispersion, the dispersion due to the packed bed, the external mass transfer from the bulk phase to the stationary phase, and the diffusive transport within the stationary phase. The methodology has several advantages compared to previously reported methods to determine diffusion coefficients in that no other equipment than an HPLC is required, any class of stationary phases can be investigated as long as the experiments are performed under non-binding conditions, and no modification, e.g., moulding of slabs or membranes, to the stationary phase is required. To show the applicability of the methodology, the effective diffusion coefficients of lysozyme, bovine serum albumin, and immunoglobulin gamma in Sepharose CL-4B were determined and shown to be comparable with those determined with other methods.