Application of poly-L-lysine to purification of leukocyte cathepsin G by affinity chromatography.

Poly-L-lysine with molecular masses of 3.3-290 kDa increased the amidolytic activities of leukocyte elastase and cathepsin G at low concentration, but had little effect on the activities of pancreatic elastase, alpha-chymotrypsin, plasmin and thrombin. Highly purified cathepsin G was obtained from column of EAH Sepharose 4B or Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose (affinity chromatography) by elution with poly-L-lysine solution (0.4 mg/ml, molecular weight (MW.) 290000 or 2.2 mg/ml, MW. 3300). Leukocyte elastase, adsorbed to Suc-L-Tyr-D-Leu-D-Val-pNA-Sepharose, was not eluted with poly-L-lysine solution. The amino acid composition of purified cathepsin G has been determined.     

Synthesis of affinity chromatography and electrophoresis matrices for the purification of bile acid transport proteins

Summary For the purification of the bile acid transport proteins from rat hepatocytes high affinity taurocholic acid derivatives were synthesized. One type was designed for coupling to a Sepharose matrix and the other for copolymerization with acrylamide. The resulting matrices were found to be suitable for affinity chromatography and affinity electrophoresis. In both syntheses, 3, 12-dihydroxy-7-amino (tauro) cholic acid derivatives were used as the starting material, since substitution in this position had previously been shown not to alter transport affinity constant.     

Novel affinity chromatography method for the efficient purification of recombinant Binder of SPerm homolog proteins

In mammalian species, a family of proteins named the Binder of SPerm proteins, which are expressed in the male reproductive tract, have been shown to play a role in epididymal sperm maturation and sperm capacitation. Recently, one homolog from human and two homologs from mouse were characterized. In order to further investigate the biochemical activity of these proteins, efficient purification procedures are required to isolate the proteins. Since these proteins are produced in very minute quantities, we exploited the high capacity of Escherichia coli to produce larger quantities of recombinant proteins that were subsequently purified using affinity chromatography on a diethylaminoethyl‐Sephadex A‐25 column. Binder of SPerm proteins have been shown to interact with pseudo‐choline groups such as diethylaminoethyl through affinity rather than ionic interactions. The aim of the current study was to develop a novel method for purifying these recombinant proteins, produced in Escherichia coli cells. Diethylaminoethyl is positively charged and is a weak anion exchanger, but binder of sperm proteins interacts with affinity to this resin. This study presents a new, rapid, and cost‐effective purification method that provides with an exceptional purity level, which can be used to study their roles in mammalian fertilization.     

Monosized adsorbents for high-performance affinity chromatography. Application to the purification of calf intestinal alkaline phosphatase and human urine urokinase

Affinity adsorbents comprising monodisperse spherical synthetic macroporous beads offer the prospect of high-capacity, high-resolution separation of proteins at low operating pressures. Purpose-designed biomimetic dyes were covalently attached to Dynospheres XP-3507 beads and exploited for the purification of calf intestine alkaline phosphatase and human urine urokinase from crude extracts. This study demonstrates that the combination of specifically designed affinity ligands with monosized support materials is a powerful approach to the resolution of proteins by high-performance affinity chromatography.     

Estradiol affinity chromatography: application to purification of murine alpha-fetoprotein

A one-step batch procedure is described for purification of murine alpha-fetoprotein (AFP) by estradiol affinity chromatography. Various ratios of carbodiimide (C), diaminononame (D) and estradiol hemisuccinate (E) were tested to determine optimal conditions for AFP purification. Although yields of AFP ranged from 15 to 44% depending on the reagent ratio employed, AFP isolates free of other protein contaminants were achieved at C:D:E ratios of 10:10:1 with a 29% yield. Both estrone and estradiol proved efficient as elution agents to free AFP bound to the estradiol-Sepharose beads, but higher yields were produced with estrone. After isolation the estrogen-eluted AFP preparations were analyzed by (1) estradiol-binding assays, (2) third-party radiocoprecipitation, (3) inhibition of radioimmunoassay for estrone and estradiol and (4) exchange of unlabeled for radiolabeled estradiol. These results indicated that the steroid remained attached to the eluted AFP molecule.     

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.     

Membrane affinity chromatography for analysis and purification of biopolymers

Summary Affinity columns suitable for HPLC were prepared by immobilization of various ligands of protein A, human IgG, human IgM and pectinase on GMA modified cellulose membrane. The adsorption capacity, affinity efficiency and activity recovery of various IgGs on these affinity columns were measured. It was observed that the length of the coupling arm plays a very important role in affinity efficiency, and the effect of eluent flow-rate on adsorption capacity was very small. The protein A column was exploited for the process monitoring of dog IgG in clinical experiments on immuno-adsorption therapy. A pectinase column was used for the determination of polygalacturonase inhibiting proteins first purified on a hydroxyapatite column. It took only about 2.5 min for analysis at a flow-rate of 1.0 mL min⁻¹. The high speed analysis of biopolymers could be performed at a flow rate of 6.0 mL min⁻¹ within 15 s. Membrane affinity chromatography gives good reproducibility, high efficiency, low column-pressure and is rapid. It can also be used for micro-scale purification of biopolymers.     

Facile purification of rare cucurbiturils by affinity chromatography

A practical method for the separation and purification of cucurbituril (CB) hexamers was developed on the basis of affinity chromatography using aminopentylaminomethylated polystyrene beads. This recyclable resin, which can be used repeatedly, facilitates the general preparation of cucurbituril derivatives and compensates for the usually moderate yields and mixed products that characterize the acid-catalyzed synthesis of CB derivatives. This technique allows convenient, rapid isolation of rare substituted cucurbiturils, including hexacyclohexanocucurbit[6]uril and dodecamethylcucurbit[6]uril. [reaction: see text]     

Affinity purification of plasminogen by radial-flow affinity chromatography

A method for the purification of plasminogen using immobilized L-lysine on a membrane, the whole system being constructed in a radial flow cartridge, is described. Human plasma was applied to the cartridge at 20 ml/min. The results showed that under the chromatographic conditions chosen, in a single pass, greater than 85% recovery of plasminogen was attained with a 110-fold increase in specific activity.     

Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney.

Ca(2+)-dependent carbohydrate-binding proteins were purified from bovine kidney by two-step affinity chromatography on fetuin and heparin columns and subsequent anion-exchange high-performance liquid chromatography. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the purified fraction gave two protein bands corresponding to proteins of relative molecular mass 33,000 (p33) and 41,000 (p41), respectively. Although the proteins had no haemogglutinating activities towards human and rabbit erythrocytes, their carbohydrate-binding activity was examined by a newly developed method using horseradish peroxidase (HRP) and/or biotin-labelled glycoconjugates as affinity probes. They could bind in a Ca(2+)-dependent manner to labelled fetuin and heparin in a specific and dose-dependent manner by solid-phase assay after immobilization on plastic plate surface. Inhibition assay of the binding revealed that N-acetylneuraminic acid is the most potent inhibitor of the proteins among the monosaccharides tested. Fucoidin and heparan sulphate most strongly inhibited the binding of the proteins to labelled heparin. Direct binding assay to acidic glycolipids prepared from bovine kidney showed that the proteins react with the ganglioside fraction but not with sulphatide [Gal(3-SO4) beta 1-1Cer]. These results indicated that the purified proteins have a significant affinity to charged oligosaccharides linking to glycoproteins, glycolipids and charged polysaccharides in a Ca(2+)-dependent manner.     

Isolation and purification of biopolymers by affinity chromatography. VI. Preparation and properties of an affinity adsorbent with a polysaccharide spacer for the purification of proteolytic enzymes

A new affinity sorbent has been synthesized — soybean trypsin inhibitor (STI)-amylopectin-hydrazidosuccinyl-Sepharose — and its properties have been studied in comparison with those of an analogous adsorbent without the spacer STI-Sepharose. The STI-amylopectin-hydrazidosuccinyl-Sepharose adsorbent has been used for the purification of trypsin from porcine pancreas and of callicrein from human blood plasma.     

Isolation and purification of biopolymers by affinity chromatography. VI. Preparation and properties of an affinity adsorbent with a polysaccharide spacer for the purification of proteolytic enzymes

A new affinity sorbent has been synthesized — soybean trypsin inhibitor (STI)-amylopectin-hydrazidosuccinyl-Sepharose — and its properties have been studied in comparison with those of an analogous adsorbent without the spacer STI-Sepharose. The STI-amylopectin-hydrazidosuccinyl-Sepharose adsorbent has been used for the purification of trypsin from porcine pancreas and of callicrein from human blood plasma.For Communication V, see [1].Institute of Biological and Medicinal Chemistry, Academy of Medical Sciences of the USSR, Moscow. I. M. Sechenov First Moscow Medical Institute. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 556–560, July–August, 1980.     

An easy purification of glycoluril clips by affinity chromatography

A convenient method for separation and purification of glycoluril clips was developed on the basis of affinity chromatography using a resorcinol-functionalized Merrifield resin. The modified resin was readily prepared via immobilization of phlroglucinol on Merrifield resin. It was used for successful isolation of the desired clip products from their corresponding reaction mixtures. Also, the resin could be repeatedly recycled and reused without any noticeable decrease in its effectiveness.     

Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification.

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.     

Application of countercurrent chromatography to the purification of chemical reagents

Countercurrent chromatography has been employed for the purification of solid chemical reagents, such as (NH₄)₂SO₄, NH₄HSO₄, NH₄F and NH₄Cl from a number of most common metal impurities (Fe, Al, Zn, Cu, Co, Cd, Ni, Cr, Ca, Mg, K) in order to gain high-purity reagents. After evaporation these can be used for fusion decomposition purposes in trace analysis of various refractory materials (e.g. high tech ceramics). N,N-hexamethylenedithiocarbamic acid, 8-hydroxyquinoline, dibenzo-18-crown-6 and dicyclohexano-18-crown-6 were used as extracting reagents.