Separation of proteins by high-performance anion-exchange chromatography

The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.     

Separation of proteins by high-speed pressure liquid chromatography

Two chromatographic systems for separation of proteins by high-speed pressure liquid chromatography are described. Molecular size exclusion chromatography was achieved by the use of porous silica deactivated by Carbowax-20M to prevent protein adsorption. Protein separations were successful provided the salt concentration in the eluting buffer was relatively high.The second system described is adsorption chromatography of proteins on deactivated Porasil. This technique involves elution of the proteins from the gel by means of a salt and pH gradient. In both systems the total time required for the chromatography is less than 1 hr.     

Separation of proteins by ion-exchange chromatography using gradient elution

Chromatographic separation of proteins by the gradient elution method using DEAE Toyopearl 650^® was carried through. The concentration gradient was effected by changing the ionic strength of NaCl in the carrier buffer solution. Bovine serum albumin and hemoglobin were used as model proteins for separation. The experimental chromatogram was compared with theoretical results of Yamamoto et al. [1, 2]. Adsorption equilibria of the proteins onto the carrier were measured and expressed by a function of the ionic strength. The retention volume and peak width of the resulting chromatogram can be calculated from the equilibrium data using the Yamamoto theory. The calculated results agreed well with the experimental data.The method presented in this paper will be useful to predict the viability of ion-exchange chromatography in protein separation.List of Symbols c kg m^–3 concentration in the liquid phase - c _s kg m^–3 concentration in the solid phase - D _s m² s^–1 intraparticle diffusivity - d _p m particle diameter - E _z m² s^–1 longitudinal diffusivity of the protein - E _{z I} m² s^–1 longitudinal diffusivity of ionic strength - H /(1 – ) - I kmol m^–3 ionic strength - I _O kmol m^–3 initial ionic strength - I _p kmol m^–3 ionic strength at the peak - I _s kmol m^–3 ionic strength in the solid phase - I/V mol (dm³)^–2 slope of the ionic gradient elution - m distribution coefficient - m distribution coefficient at I - m _I distribution coefficient for ionic strength - Q cm³s^–1 flow rate - R m particle radius - R _s degree of separation - r m radial position inside particles - t s time - u m s^–1 linear velocity - V cm³ eluted volume of liquid - V _p cm³ eluted volume of liquid at the peak - V _T cm³ volume of the packed bed - W cm³ peak width - Z m bed height - z m vertical position in the bed - z _p m peak position from the inlet of the bed - (t) delta input at time - void fraction - ₁ s first moment - ₂ s² second central moment - s superficial space time     

Separation of cobalt binding proteins by immobilized metal affinity chromatography

BAY 43-9006 is a selective Raf-1 kinase inhibitor with antitumor activity against a variety of human cancers. A highly sensitive HPLC method for determination of BAY 43-9006 in small volumes of serum (30 microl) was developed. Sample preparation involved a liquid-liquid extraction procedure with tolnaftate as internal standard followed by linear gradient elution at a reversed phase C18 column and UV detection. The method was selective and the calibration curves were linear over the concentration range of 80-2000 ng/ml. The intra-day accuracy ranged from 99.9 to 107.6% and the inter-day accuracy from 94.6 to 115%. The lower limit of quantitation (LOQ) was 80 ng/ml with an accuracy of 105.8%. Thus, this method has been validated and can be applied for the drug monitoring or pharmacokinetic studies of BAY 43-9006 in small volumes of serum samples.     

Separation of insect hemolymph proteins by cascade-mode multi-affinity chromatography.

The hemolymph of the adult female Manduca sexta was fractionated by cascade-mode multi-affinity chromatography (CASMAC) on a main-line tandem column chain containing Zn(2+)-TED, T-gel, Ni(2+)-DPA, and phenylsepharose and a side-line column containing Zn(2+)-DPA. The technique separated some of the previously described major hemolymph proteins, and yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus, it appears that CASMAC would be a very useful fractionation technique for purification and characterization of the minor proteins of insect hemolymph.     

Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography

A rapid and simple method was developed to separate human vitamin K-dependent plasma proteins from each other, yielding virtually homogeneous pools. The purification technique is based on the single use of hydrophobic interaction chromatography, starting from prothrombin concentrate (PC or DEFIX, also termed factor IX concentrate) as initial material. Phenyl-sepharose HP demonstrated optimal separation by comparing several hydrophobic resins as well as resins used in standard procedures like immobilised heparin and Cibacron blue. Under ideal conditions, factor X could be separated in a single step as well as prothrombin. Factor IX co-eluted with other minor proteins. Focus was given only on these three proteins due to their relative abundance. Complete separation of all proteins present in the starting material was achieved by MonoQ anion-exchange chromatography following the phenyl-sepharose run. The resulting purified material could be demonstrated to be of equal or higher purity than using described methods. This strategy employing hydrophobic interaction chromatography for blood macromolecules could be of immense value for purifying the human vitamin K-dependent proteins and represents a considerable simplification over other purification schemes. It not only involves minimal sample handling but also can be readily up-scaled and is a cost-efficient alternative.     

Separation of nucleic acids and chromatin proteins by hydrophobic interaction chromatography

A procedure by which chromatin proteins (histones and non-histones) can be rapidly separated from nucleic acids by hydrophobic interaction chromatography is described. The procedure is carried out under non-rigorous conditions that must be assumed to induce little irreversible change in the biological properties of most proteins. More than 90% (w/w) of the chromatin proteins can be retained by hydrophobic interaction while nucleic acids pass quantitatively through the columns. By gradient elution of the columns the histones can be divided into fractions containing H1, H2A/H2B and H3/H4, and at the same time a subfractionation of the non-histone proteins is obtained. Protein recovery depends on the type of column used, but exceeds 80% (w/w) with even the most strongly binding hydrophobic matrix investigated.     

Separation of the proteins present in pancreatic juice using hydrophobic interaction chromatography

We have previously described a method for separating pancreatic juice proteins by reversed-phase HPLC. However, the solvents used in such a system denature the proteins, whose characterization therefore depends solely on molecular weight determinations. We have therefore tested the suitability of hydrophobic interaction chromatography as an alternative method of separation. Using a TSK phenyl 5PW column with a decreasing, four-stage ammonium sulfate concentration gradient, it was possible to separate the major proteins in rat pancreatic juice. The identity of each protein was confirmed by measuring its molecular weight and by assaying its enzyme activity. Hydrophobic interaction chromatography represents an improved system for separating pancreatic secretory enzymes in active form.     

Separation and purification of individual neurofilament proteins by reverse-phase high-performance liquid chromatography

A reverse-phase HPLC method was developed to separate individual neurofilament proteins (210,000, 160,000, and 70,000 Da) from the glial fibrillary acid protein. It is useful for analytical or preparative methods, with yields higher than 80%. The method represents improvement over previous methods in speed, efficiency, and purity. Combining this HPLC method with the conventional chromatographic method on DEAE-cellulose, highly purified individual neurofilament proteins can be obtained in large scale.     

Separation of phenylthiohydantoin-amino acids by high-pressure liquid chromatography for sequence determination of radiolabeled proteins

Published procedures for the separation of ethyl acetate-extractable phenylthiohydantoin-amino acids by high-pressure liquid ehromatography with organic solvent gradients have been modified to facilitate sequence determination of proteins labeled with several radioactive amino acids at a time.     

Separation of both fibrous and globular proteins on the basis of molecular weight using high-performance size exclusion chromatography

A high-performance size exclusion liquid chromatographic system has been used to separate proteins with different shapes solely on the basis of their molecular weights. After the effects of ionic and hydrophobic interactions with the stationary phase have been overcome, protein elution is normally governed by their effective size in solution. Conditions are described under which proteins, with isoelectric points within the normal operating pH range of the columns, are eluted independent of their Stokes' radii. Even fibrous proteins with axial ratios of 50 elute according to their known molecular weights over the range 2000–2,000,000.     

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 M guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of "white yolk" and "yellow yolk."     

Characterization of human retroplacental blood plasma proteins isolated by biospecific chromatography on immobilized cortisol]

Human retroplacental blood plasma proteins with affinity for cortisol were isolated by biospecific chromatography and identified by electrophoretic and immunochemical methods as alpha1- and beta1-globulins and IgG. IgM and IgA immunoglobulins. A high specific affinity for cortisol (Kas = 1,5 . 10(8) M-1 at 23 degrees C) and progesterone (Kas = 2,0 . 10(8) M-1 at 23 degrees C) was observed only for alpha-globulin; other proteins had a low affinity for cortisol. The molecular weight of alpha1-globulin (transcortin) was found to be 50,000-55,000. The amino acid and monosaccharide compositions of this glycoprotein were studied. Its N-terminal and C-terminal amino acid sequences are: Met-Asx-Pro-Asx-Ala- and (Val, Gln)-Leu, respectively. It was concluded that under normal physiological conditions and during pregnancy transcortin is the only specific corticosteroid-binding plasma protein. A complete removal of bound cortisol from the protein mixture and subsequent hydroxylapatite chromatography resulted in homogeneous transcortin retaining more than 90% of its binding capacity. The formation of the transcortin-steroid complex and its complete dissociation are accompanied by conformational changes of the protein globule. Significant changes of the spectral properties of the tryptophane residue of protein and the steroid delta4-3-keto group are indicative of the possibility of their direct interaction.