Preparative-scale amino acid separation by thermal parametric pumping on an ion-exchange resin

Thermal parametric pumping was experimentally investigated for the concentration and separation of amino acids. Previous theories of parametric pumping were improved by taking into account dissociation equilibria in the liquid phase. Experiments were carried out with a mixture of glutamic acid, aspartic acid, serine and threonine in a highly acidic solution (HCl). A multi-component equilibrium model was mainy used to simulate the experimental results and to investigate the effect of chloride concentration over a wide range. It is shown that it is always possible to concentrate the amino acids and to separate some of them under certain conditions.     

The separation and amino acid analysis of collagen crosslinks on an extended basic ion-exchange column

The major reducible crosslinks found in collagen were separated and analyzed on an extended basic amino acid analyzer column. Reaction with ninhydrin allows the direct analysis of collagen crosslinks, including hydroxyaldol-histidine, a naturally occurring, nonreducible crosslink. In addition to known crosslinks, direct amino acid analysis of tissue hydrolysates reveals the presence of an unknown, ninhydrin-reactive component, in both NaB³H₄-reduced and unreduced collagenous tissues. Initial fractionation of hydrolysates on a Bio-Gel P-2 gel filtration column provides partial separaton of amino acids and crosslinks and enables more direct analysis of the crosslinks present in the samples, as well as detecting potential new crosslinks. The results also show that, prior to NaB³H₄ reduction, substantial amounts of known crosslinks are normally present in bovine skin and bone.     

Tryptic cleavage of a peptide at modified aspartic acid

Conversion of the carboxylic acid side chain of aspartic acid in the peptide pyroGlu·Asp·Phe·amide to a carboxamidomethylamine side chain (structure $\text{l}$) results in the tryptic hydrolysis of the peptide at the Asp-Phe bond.     

Rapid purification of a recombinant protein using tandem radial flow ion-exchange column chromatography.

Tandem radial flow anion- and cation-exchange columns were used to partially purify and concentrate a dilute recombinant protein that had been refolded in vitro after production as insoluble inclusion bodies in E. coli. The refolded sample was first passed through a Q-Sepharose Fast Flow column in order to remove the majority of E. coli contaminating proteins and endotoxins, then purified on an S-Sepharose Fast Flow column connected to the outlet of the Q-Sepharose column. This tandem arrangement enabled the rapid processing of multiple preparations of refolded material during production method development.     

Chromatography of hen egg-white proteins on anion-exchange cellulose at high flow rates using a radial flow column.

The influence of column configuration on the separation of hen egg-white proteins using Whatman DE52 and QA52 anion-exchange cellulose has been investigated. Using a 100 ml volume axial flow column (6.6 cm x 4.4 cm i.d.) we achieved flow rates of up to 25 ml/min i.e. 15 bed volumes/h after which higher flow was restricted due to pressure constraints within the system. Under radial flow conditions using a 100 ml column flow rates of up to 150 ml/min i.e. 90 bed volumes/h were achieved using DE52 and QA52. While chromatographic resolution was superior under axial flow at the lower flow rates excellent resolution was maintained at up to 150 ml/min using the radial flow column. This is a consequence of the fast kinetics of adsorption/desorption exhibited by DE52 and QA52. The data indicate that it is the column configuration and not the cellulose matrix which influences flow performance.     

Resolution and productivity of conalbumin and lysozyme from fresh egg-white loaded at very high flow rate on a 250-mm length CM-HVFM column.

A 250 x 10 mm I.D. column of CM-HVFM, a novel carboxymethyl ion-exchange matrix, has been used as a preparative chromatographic column to separate fresh egg-white protein. When loading a diluted egg-white solution at pH 4.8 ovalbumin was not adsorbed and lysozyme was preferentially adsorbed compared to the conalbumin. As the column loading was increased from 24 to 450 kg m-3 column volume at the superficial velocity of 6.12 m h-1, the lysozyme continued to be absorbed eventually displacing conalbumin. A maximum lysozyme productivity at 16.7 kg m-3 h-1 was achieved at the highest loading. For conalbumin a maximum productivity of 8.8 kg m-3 h-1 occurred at the lower loading of 100 kg m-3. The purities of lysozyme and conalbumin were comparable at a column loading of 450 kg m-3 h-1. The performance of the column was not degraded, neither was the column blocked or channelled despite the high column loading at the high flow-rate.     

Rapid separation of gonadotropin-releasing hormone molecular forms by isocratic high-performance liquid chromatography on an ion-exchange column

The purpose of the present work was to develop a chromatographic system for the separation of five molecular forms of the gonadotropin-releasing hormone (GnRH); mammalian GnRH (mGnRH) (LHRH), salmon GnRH (sGnRH), chicken I GnRH (clGnRH), chicken II GnRH (cIIGnRH) and lamprey GnRH I (IGnRH-I). By using an ion-exchange HPLC column and isocratic elution, it was possible to separate properly the five peptides in approximately 20 min. The utility of the system in determining the GnRHs forms present in the brain of two species of vertebrates was examined.     

Fractionation and amino acid composition of an aspartic acid-containing thermal proteinoid population

The heterogeneity of an aspartic acid-containing thermal polymer population has been studied by anion exchange chromatography, amino acid analysis of the resulting fractions and data processing by the principal components method. By using stepwise or continuous gradients of ionic strength both saw-toothed or bell-shaped elution profiles were obtained. This behaviour and the amino acid composition of analyzed fractions suggest a relatively high degree of heterogeneity in the polymer population although less than theoretically expected. This conclusion is compared with the findings reported in proteinoids made by similar procedures.     

A simple method for the separation of bile acid groups by ion-exchange chromatography

A simple ion-exchange chromatographic method for the separation of bile acid mixtures is described. The method employs Dowex 1 as anion exchanger and mixtures of aqueous ethanol and hydrochloric acid as eluants. The use of mixtures in which both the ethanol and acid concentrations are varied has permitted a clear separation of the major bile acid groups (nonconjugated, glycine conjugated, and taurine conjugated bile acids).     

Rapid separation of cobyrinic acid and its biosynthetic precursors by ion-exchange paper chromatography

Cobyrinic acid, a key intermediate in vitamin B₁₂ biosynthesis, can be rapidly separated from δ-aminolevulinic acid, S-adenosylmethionine, uroporphyrin III, and heptacar?ylic uroporphyrin III by car?ymethyl cellulose paper chromatography developed with 2% acetic acid.     

Mechanism of Ret activation by a mutation at aspartic acid 631 identified in sporadic pheochromocytoma

Mutations at aspartic acid 631 in Ret were reported in sporadic pheochromocytoma and medullary thyroid carcinoma. We replaced this aspartic acid with four other amino acids including tyrosine, glycine, asparagine, and alanine and investigated the transforming activity of these mutant cDNAs. Among them, RET cDNA with a mutation of aspartic acid to tyrosine (D631Y) that was reported in sporadic pheochromocytoma showed high transforming activity. The D631Y mutation activated Ret by inducing its disulfide-linked dimerization in the transfectant as observed for multiple endocrine neoplasia (MEN) 2A mutations at cysteine 609, 611, 618, 620, 630, or 634. Further mutation analysis suggested that cysteine 630 or 634 could be involved in the disulfide-linked Ret dimerization induced by the D631Y mutation.     

Study on separation of conalbumin and lysozyme from high concentration fresh egg white at high flow rates by a novel ion-exchanger

In this report, we show that it is possible to separate valuable proteins from egg-white using a Productiv(TM) CM ion-exchanger column operated at flow rates significantly higher than those than can be achieved using traditional particulate adsorbents. In the approach taken, sample pretreatment is restricted to a simple dilution of the egg-white, which can then be applied to the column at superficial velocities (V(s)) of up to 13.8 m/h. Under a loading of 220 mg total protein per milliliter of ion-exchanger, the resolution (R(s)) between the eluted conalbumin and lysozyme fractions was found to be almost constant during nine consecutive adsorption/desorption cycles. For all nine consecutive batches, the column average adsorption capacity was greater than 30 mg/mL, with 90% recovery of adsorbed protein being achieved in each run. The overall productivity achieved was 12.6 kg/m(3) h for lysozyme and 31.2 kg/m(3) h for conalbumin. (c) 1993 John Wiley & Sons, Inc.     

Site-specific pyrolysis-induced cleavage at aspartic acid residue in peptides and proteins

A simple and site-specific nonenzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220-250 degrees C in 10 s. Electrospray ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence-specific protein biomarkers.     

On-line monitoring of ethanol: in relation to the rate of carbon dioxide evolved

A simple method for rapid on-line measurement of ethanol, produced in a continuous bioreactor, in a steady state, is described. Soap film meter measures the rate of CO² evolved. This value is equated to the equimolar production of ethanol. A mean 3.3% error was observed, as compared to the gas chromatography method.     

Continuous Fermentation in high flow rate fermenter systems

In commercial batch processes the productivity of product formation is low. But a significant increase of productivity can be achieved in continuous fermentations. By using high flow rate fermenter systems characterized by a relatively long retention time of biomass in comparison with the retention time of the liquid we can realize a high-performance fermentation. The problem of holding back the biomass within the reactor could be solved by means of membranes being impenetrable to the cells, but permeable to the hydraulic phase. Such a process technology was successfully tested for its applicability in alcoholic and lactic acid fermentations. The maximum productivities obtained on this way were ? = 120 g/l. · h for ethanol production and ? = 51 g/l. h for lactic acid fermentation, respectively.