High-performance affinity chromatography of human serum concanavalin A binding proteins

A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.     

High-performance liquid chromatography of proteins: purification of alpha-fetoprotein from fetal calf serum

High-performance liquid chromatography was utilized for the purification of bovine alpha-fetoprotein (BAFP) from fetal calf serum (FCS). An initial step in the purification involved absorption of charcoal delipidated FCS on Cibacron Blue F3GA gel. The Cibacron Blue pre-purified FCS was then chromatographed on a Polyanion SI weak anion-exchange column. The BAFP isolated had a purity of greater than 93% with an overall yield of 48% from FCS. The procedure was applicable for semi-preparative scale purification of BAFP.     

High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G

High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.     

High-performance liquid chromatography: purification and chromatographic behaviour of molecular variants of pepsinogen A from human urine.

By combining conventional DEAE chromatography with high-performance liquid chromatography on Sephacryl S-200 HR and Mono-Q columns, we have been able to isolate and fractionate human pepsinogen A (PGA) isozymogens from large amounts of urine. This method of fractionation is simple and allows one to obtain pepsinogen in a native non-denatured conformation. The isozymogens are homogeneous by electrophoretic and chromatographic criteria; this was confirmed by N-terminal amino acid sequencing. Purified PGA-3 and PGA-5 can be converted into an additional, more anionic, isoform on incubation at 37 degrees C. This isoform exists not only in vitro but also in vivo. The net negative charge of the PGA isozymogens is in the order PGA-5 less than deamidated PGA-5 less than PGA-3 less than deamidated PGA-3. Surprisingly, the elution order on the Mono-Q column was PGA-5/PGA-3/deamidated PGA-5/deamidated PGA-3. We have performed molecular modelling on PGA to investigate this phenomenon in terms of surface charge (not net charge) of the proteins. The model provides evidence that (1) only a fraction of the protein surface interacts with the support and (2) regions of localized charge at the protein surface may allow portions of the external surface to dominate chromatographic behaviour, resulting in a steering of the proteins with respect to the oppositely charged matrix. Pepsinogens may serve as model proteins for elucidating some of the variables that determine the chromatographic behaviour of proteins on ion-exchange columns.     

High-performance affinity chromatography of DNA

The octadecamer of thymidylic acid, (dT)18, was synthesized with a primary amino group on the 5'-terminal phosphate and this was covalently coupled to 300 A pore macroporous silica. Coupling was performed inside a prepacked column to an activated N-hydroxysuccinimidyl ester silica. The (dT)18-silica column successfully separates mixtures of adenine oligomers differing in length by one nucleotide. The dependence upon salt concentration, temperature and length for elution of oligonucleotides was determined. Methods were also developed to selectively elute such columns using either salt or temperature gradients.     

Preparative purification of adenosine deaminase from human erythrocytes by affinity chromatography

The purification of adenosine deaminase from human erythrocytes is reported. By means of classical procedures and by using affinity chromatography as the last step, the enzyme is purified 760,000-fold with a yield of 32%. The affinity resin is composed of purine riboside (nebularine) linked to Sepharose CL6B. Since the compound has no leaving group at the C-6 position the affinity gel is stable and the chromatography can be repeated several times (up to fifteen times in eight months). Purine riboside was chosen because its potency as a reversible inhibitor of adenosine deaminase is greater than that of inosine (a low-affinity inhibitor), but lower than that of erythro-9-(2-hydroxy-3-nonyl)adenine (a high-affinity inhibitor).     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixture of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10(4) and 10(5). HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.     

High-performance liquid chromatography of aflatoxins in human urine

A method was developed for the extraction and determination of unconjugated aflatoxins in human urine by high-performance liquid chromatography. The analysis is based on the elimination of lipid-soluble constituents other than unconjugated aflatoxins in urine by light petroleum extraction. The unconjugated aflatoxins were subsequently extracted from the aqueous phase with chloroform-acetone. Chromatography was performed isocratically with a silica column at 40 degrees C. The resolved aflatoxins were detected and identified by ultraviolet and fluorometric detectors. The recoveries of aflatoxins B1 and G1 added prior to the extraction were 72% and 83%, respectively. This procedure is simple, sensitive and practically useful for epidemiological survey of unconjugated aflatoxins in human urine from areas with a high risk of aflatoxin consumption.     

High-performance liquid chromatographic assay of fleroxacin in human serum using fluorescence detection

A HPLC method has been developed for the direct assay of fleroxacin in serum, without previous extraction. Serum samples, after the addition of sodium dodecylsulfate (0.5%), were injected directly into an LC Hisep column. The mobile phase consisted of acetonitrile, water and triethylamine in a per cent volume ratio 18:80:2. The pH of the mobile phase was adjusted to 6.50 with the addition of phosphoric acid. The drug was detected fluorometrically at lambda (ex )=280 nm and lambda (em )=450 nm . The linear concentration range of fleroxacin was between 0.01 and 2.0mg/l with a detection limit of 1ng/ml.     

Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography

In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu‐iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14‐atom spacer arm. The absorption analysis of this medium revealed a desorption constant K_d of 21.5 μg/mL and a theoretical maximum absorption Q_max of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high‐performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases.     

Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds

We have developed a large-scale method for recovering the corn inhibitor of trypsin and activated Hageman factor from a trypsin-agarose column predominantly in the single-chain form. To do so, inhibitor retained by the column was eluted with 1.0 M glycine buffer, pH 2.1. We have used reversed-phase high-performance liquid chromatography to further purify the inhibitor eluted from the trypsin-agarose column by separating the single-chain inhibitor from two-chain inhibitor (a small amount of which is present in the preparation after trypsin-agarose chromatography) and from still smaller amounts of another protein (apparently trypsin) that appears as a contaminant during trypsin-agarose chromatography.     

High-performance liquid chromatography of proteins using chemically-modified silica supports

Summary Highly efficient and fast exclusion-chromatographic separations of proteins are possible on chemically-modified, silica stationary phases. By optimizing the pH and the ionic strength of the aqueous eluent secondary interactions of the samples with surface groups can be excluded. Bonded propylamide groups proved to possess optimum properties for exclusion chromatography. With other functional groups adsorption effects cannot be excluded totally. The optimum pore size distribution for protein separation up to relative molecular masses of 500,000 daltnons is between 10nm and 50nm. With these silica-based phases the pore size distribution, the pore volume and the packing characteristics are independent of the eluent, therefore the same column can be used with aqueous as well with organic eluents. It is possible to correlate the elution volume (molecular size) of proteins with those of polystyrene standars. The recovery of the proteins and their biological activity has always been better than 90%. The potentialities of adsorption chromatography of proteins on chemically-bonded stationary plases with different functional groups are demonstrated.     

Fluorescence immunological determination of immunoglobulin G in human serum by high-performance liquid gel-permeation chromatography

Summary A high-performance liquid gel-permeation chromatographic method is described for the determination of human serum immunoglobulin G (IgG) by separating the fluorescent immuno complex from the free fluorescence-labeled antibody. Fluorescence-labeled antibody used in this study was fluorescein isothiocyanate (FITC)-labeled Fab fragment goat anti-human IgG (anti-IgG Fab). Immuno complexes and antibody of different molecular sizes can be separated. FITC-labeled anti-IgG Fab was added to the serum and the mixture is passed through the column. An immuno complex separates as well-delineated peak in the column void volume, and was measured by the fluorescence of the column eluate (Ex=490nm, Em=520nm). The total analysis time for a serum sample was approximately 15min. The minimum detection limit was 25 mg/dl. The relative standard deviation was below 2% (peak area). The results of the HPL-GPC analysis correlate well with those obtained by laser nephelometric assay (r=0.992).     

High-performance liquid chromatography of alkaloids

Literature information on the HPLC of alkaloids, including adsorption, reversed-phase, ion-exchange, ion-pair, and other variants is generalized. Results are given of the chromatographic analysis of various classes of alkaloids with an indication of the conditions of separation, and the columns used, and also of the methods of detection. The advantages and disadvantages of the HPLC variants are discussed. The review includes the literature over the last 14 years. Scientific Center for Chromatography, Institute of Chemistry and Physics of Polymers, Uzbek Academy of Sciences, Tashkent. Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 595–613, September–October, 1991.     

High-performance liquid chromatography of alkaloids

Literature information on the HPLC of alkaloids, including adsorption, reversed-phase, ion-exchange, ion-pair, and other variants is generalized. Results are given of the chromatographic analysis of various classes of alkaloids with an indication of the conditions of separation, and the columns used, and also of the methods of detection. The advantages and disadvantages of the HPLC variants are discussed. The review includes the literature over the last 14 years.Scientific Center for Chromatography, Institute of Chemistry and Physics of Polymers, Uzbek Academy of Sciences, Tashkent. Institute of the Chemistry of Plant Substances, Uzbek Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 595–613, September–October, 1991.     

High-performance affinity chromatography of messenger RNA.

A 50-mer of thymidylic acid, (dT)50, was coupled to silica inside prepacked columns using the N-hydroxysuccinimide chemistry. The resulting (dT)50-silica columns were used to resolve oligomers of adenylic acid, (dA)19-24, and to separate poly(A) mRNA (messenger RNA) from Saccharomyces. Oligomers which differed in length by a single nucleotide base were readily resolved. Using either (dT)50- or (dT)18-silica, poly(A) mRNA could be purified in as little as 8 min. The poly(A) mRNA isolated appeared to be full length and could be used directly for T4 RNA ligase and RNAse A and T1 enzymatic reactions. The (dT)50-silica column was used to fractionate total poly(A) mRNA by tail length. While the separation was primarily due to poly(A) tail length, most fractions appeared to contain multiple tail lengths. Whether this represents an intrinsic feature of the RNA or a limitation of the method is discussed. These studies show that polynucleotides in the kilobase size range can be separated rapidly and with good resolution on DNA-silica.