A study on heterogeneity in molecular species of shark cartilage chondroitin sulfate C. Fractionation of the polysaccharide on Sepharose CL-4B in the presence of high concentrations of ammonium sulfate

Shark cartilage chondroitin sulfate C was fractionated by chromatography on Sepharose CL-4B-2.5 to 1.5M ammonium sulfate in 10mM hydrochloric acid at 4 degrees. Both unit-disaccharide composition and molecular-size distribution clearly affected the fractionation. Comparison of this fractionation with the fractionation on Sepharose 6B gel in 0.2M sodium chloride revealed that the former is distinctly superior to the latter. The fractionation on Sepharose CL-4B in the presence of ammonium sulfate also showed that the chondroitin sulfate C molecules having a larger molecular size contain generally more chondroitin 6-sulfate units (as major constituent) and less chondroitin disulfate units (D type, as minor constituent) than those having a smaller molecular size).     

Hydrophobic interaction chromatography on octyl sepharose--an approach for one-step platform purification of cyclodextrin glucanotransferases

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55-0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.     

Purification and some properties of an extracellular halophilic 5'-nucleotidase from a moderate halophile, Micrococcus varians subsp. halophilus

Abstract An extracellular 5'-nucleotidase produced by a moderate halophile, Micrococcus varians subsp. halophilus was partially purified from the culture filtrate by ethanol precipitation and Sepharose 4B hydrophobic chromatography. The 5'-nucleotidase was a novel halophilic enzyme, requiring 2 M NaCl or 2.5 M KCl and 0.1 mM Co²⁺ or 0.1 mM Mn²⁺ for maximal activity.     

Hemoglobin & serum albumin: salt-mediated hydrophobic chromatography.

The binding of pure human serum albumin and pure human hemoglobin to L-phenylalinine Sepharose and aniline Sepharose columns, two chromatographic columns of differing hydrophobicity, has been investigated for various concentrations of ammonium sulfate salts.The binding of hemoglobin at a lower ammonium sulfate concentration than albumin in both hydrophobic support systems parallels the solubility and precipitation characteristics of these two proteins in solution and mirrors the phenomenon of salting out of proteins in solution. Both hemoglobin and albumin bind at lower concentrations on aniline Sepharose than on L-phenylalanine Sepharose, reflecting the greater efficiency of binding by the more hydrophobic support matrix.     

HYDROPHOBIC INTERACTION CHROMATOGRAPHY ON OCTYL SEPHAROSE—AN APPROACH FOR ONE-STEP PLATFORM PURIFICATION OF CYCLODEXTRIN GLUCANOTRANSFERASES

Cyclodextrin glucanotransferase (CGTase) from Bacillus circulans ATCC 21783 was concentrated by ultrafiltration and subsequently purified by hydrophobic interaction chromatography on Octyl Sepharose 4 fast flow. The matrix was able to bind selectively to the enzyme at a very low ammonium sulfate concentration of 0.67 M and enzyme desorption was performed by decreasing gradient of the salt. The overall recovery was 80% with 689-fold purity. CGTases derived from four soil isolates and Toruzyme, the commercial preparation of CGTase, also bound to Octyl Sepharose under similar conditions at 0.67 M and eluted at 0.55–0.5 M of ammonium sulfate. Octyl Sepharose chromatography can thus be used as a platform approach for purification of CGTases from various bacterial sources. Long stretches of sequence predominated by hydrophobic amino acids are reportedly present in the starch binding domains of CGTases. Starch binding experiments indicated the binding of the enzymes to the octyl matrix through these domains.     

Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts

Cultured human fetal lung fibroblasts produce some chondroitin sulfate proteoglycans that are extracted as an aggregate in chaotropic buffers containing 4 M guanidinium chloride. The aggregated proteoglycans are excluded from Sepharose CL4B and 2B, but become included, eluting with a Kav value of 0.53 from Sepharose CL4B, when Triton X-100 is included in the buffer. Conversely, some of the detergent-extractable chondroitin sulfate proteoglycans can be incorporated into liposomes, suggesting the existence of a hydrophobic membrane-intercalated chondroitin sulfate proteoglycan fraction. Purified preparations of hydrophobic chondroitin sulfate proteoglycans contain two major core protein forms of 90 and 52 kD. A monoclonal antibody (F58-7D8) obtained from the fusion of myeloma cells with spleen cells of BALB/c mice that were immunized with hydrophobic proteoglycans recognized the 90- but not the 52-kD core protein. The epitope that is recognized by the antibody is exposed at the surface of cultured human lung fibroblasts and at the surface of several stromal cells in vivo, but also at the surface of Kupffer cells and of epidermal cells. The core proteins of these small membrane-associated chondroitin sulfate proteoglycans are probably distinct from those previously identified in human fibroblasts by biochemical, immunological, and molecular biological approaches.     

Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 S thyroglobulin.

Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37 degrees C at pH 4.5 and 25 degrees C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol. wt. corresponding to the native 12 S were the major component, strongly suggesting a mol. wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.     

Fractionation and recovery of whey proteins by hydrophobic interaction chromatography

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.     

Variations in the composition of arterial wall isomeric chondroitin sulfate proteoglycans among different animal species

1. Isomeric chondroitin sulfate proteoglycans were extracted from human, bovine, swine and rabbit aortas by 4 M guanidine-HCl and were fractionated and purified by CsCl isopycnic centrifugation, Sepharose CL-4B gel filtration, DEAE-Sepharose ion-exchange chromatography and octyl-Sepharose hydrophobic interaction chromatography. 2. The molecular size and the composition of isomeric chondroitin sulfate proteoglycans varied among species. Variations were also noted in the composition and molecular weight of constituent glycosaminoglycan chains. 3. Observations made on chondroitinase ABC and chondroitinase AC digests of proteoglycans indicate that dermatan sulfate is linked to the core proteins through chondroitin sulfates.     

Hydrophobic-interaction chromatography of glycosaminoglycuronans: the contribution of N-acetyl groups in heparin and heparan sulfate to the affinity for hydrophobic gels, and variety of molecular species in beef-kidney heparan sulfate

Contribution of N-acetyl groups in heparin and heparan sulfate to their affinity for hydrophobic gels was examined by use of a series of semi-synthetic, N-acetylated, hog-intestinal heparins, a whale-intestinal heparin, and a beef-kidney heparan sulfate. Chromatography on Phenyl-Sepharose CL-4B in 3.8-1.0M ammonium sulfate-10mM hydrochloric acid indicated that an increasing N-acetyl content, which is correlated to a decreasing N-sulfate content, results in a marked increase in the affinity for the gels. The variety of molecular species in beef-kidney heparan sulfate, previously fractionated by conventional chromatographic procedures, was demonstrated by separating further, by hydrophobic-interaction chromatography, the polysaccharide into several fractions composed of molecular species distinctly different in N-acetyl and sulfate content, and in molecular size.     

Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes

A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.     

Detergent-solubilized proteoglycans in rat testicular Sertoli cells

Rat Sertoli cells were cultured for 48 h in the presence of [35S]sulfate and extracted with 4 M guanidine chloride. In this extract, a Sepharose CL-2B Kav 0.10 proteoheparan appeared lipid associated, since after addition of detergent it emerged at Kav = 0.65 on Sepharose CL-2B. Treatment of cells with 0.2% Triton X-100 released 35S-labeled material which was purified by ion-exchange chromatography and hydrophobic interaction chromatography on octyl-Sepharose. Proteoglycan with affinity for octyl-Sepharose (Kav = 0.30 and 0.12 on Sepharose CL-4B and CL-6B, respectively) mostly carried heparan sulfate chains with Kav = 0.38 and minor proportion of heparan chains with Kav = 0.77 on Sepharose CL-6B. An association with lipids was confirmed by intercalation into liposomes of this proteoheparan which might be anchored in the plasma membrane, via an hydrophobic segment and/or covalently linked to an inositol-containing phospholipid. Non-hydrophobic material consisted of: (i) proteoheparan slightly smaller in size than lipophilic proteoheparan and possibly deriving from this one and (ii) two heparan sulfate glycosaminoglycan populations (Kav = 0.38 and 0.86 on Sepharose CL-6B) corresponding to single glycosaminoglycan chains and their degradation products.     

Large-scale purification of halophilic enzymes by salting-out mediated chromatography

Large-scale purification of malate and glutamate dehydrogenases from extremely halophilic bacteria of the Dead Sea based on salting-out mediated chromatography techniques is described. Yields are in the range of hundreds of milligrams. Ammonium sulfate-mediated fractionation of halophilic enzymes on Sepharose is not affected by sodium chloride. The adsorption seems to weaken with increased temperatures. Higher sodium chloride concentrations, in the presence of ammonium sulfate, are necessary to elute halophilic enzymes from DEAE-cellulose as the sulfate concentration is increased. Possible applications of the saltingout mediated chromatography are discussed.     

Quaternary structure of higher plant glyceraldehyde-3-phosphate dehydrogenases.

1. NAD(P)+-induced changes in the aggregational state of prepurified NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were used to isolate the enzyme from Spinacia oleracea, Pisum sativaum and Hordeum vulgare. Each of the three plant species contains two separate isoenzymes. Isoenzyme 1 (fast moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 55--70% saturation. It shows two separate subunits in dodecylsulfate gels, which are probably arranged as A2B2 in the native enzyme molecule. Isoenzyme 2 (slow moving during conventional electrophoresis) precipitates with the ammonium sulfate fraction 70--95%. It contains a sigle subunit of the same Mr as subunit A in isoenzyme 1 and is apparently a tetramer (A4). The molecular weights of subunits A/B for spinach, peas and barley were determined as 38,000/40,000, 38,000/42,000 and 36,000/39,000 respectively. 2. The NAD-specific glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) was purified from Spinacia oleracea and Pisum sativum by affinity chromatography on blue Sepharose CL-6B. The enzyme from both plant species is shown to be a tetramer of subunits with Mr 39,000. 3. The present findings contrast with heterogeneous results obtained previously by other authors. These results suggested that there are considerable interspecific differences in the quaternary structure of glyceraldehyde-3-phosphate dehydrogenases from higher plants.     

一种改进的纯化和鉴定牛脑皮层Gs蛋白的方法

用１％胆酸钠和１５％孢和硫酸铵相结合的方法,从牛脑皮层细胞膜中抽提得到主要含激活型Ｇ－蛋白和腺苷酸环化酶两种蛋白组分的制剂,然后通过Ｓｅｐｈａｒｏｓｅ６Ｂ柱将两者分开,将含Ｇｓ高活力的级分用庚胺－Ｓｅｐｈａｒｏｓｅ４Ｂ柱进一步分离,即可获得高活力的Ｇｓ,ＳＤＳ－ＰＡＧＥ显示为分子量４５０００和３６０００的两条蛋白带,该法具简便,快速,重复性好、产率高等优点,且可同时获得无Ｇｓ污染的ＡＣ。用无Ｇｓ污     

General aspects of hydrophobic chromatography. Adsorption and elution characteristics of some skeletal muscle enzymes.

If the degree of substitution of Sepharose 4 B with alpha-alkylamines is varied gels of different hydrophobicity are produced. Proteins can be adsorbed when a critical hydrophobicity (ca. 10-12 alkyl residues/Sepharose sphere) is reached. The enzymes phosphorylase kinase, phosphorylase phosphatase, 3',5'-cAMP dependent protein kinase, glycogen synthetase, and phosphorylase b are successively adsorbed as the hydrophobicity of the Sepharose is increased. The capacity of the gels for these enzymes and protein in general increases exponentially reaches plateau values as a function of the degree of substitution. There is no indication of a restriction of the hydrophobic centers for a given protein. The critical hydrophobicity needed to adsorb proteins can either be otained in the above manner or by elongation of the employed alkylamine at a constant degree of substitution. Additonally, as the hydrophobicity of a gel is increased higher binding forces result and desorption of proteins requires an augmentation of the salt concentration in the elution buffer. Elution of proteins from a hydrophobic matrix can be described in terms of salting-in phenomena since desorption is dependent on the type of salt employed and not on the ionic strength alone. This also rules out ionic interactions as a major factor in adsorption per se. By rationally controlling the hydrophobicity of a Sepharose gel the adsorption and elution of a protein may be thus establised that its purification or elimination can be optimally performed.     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

Purification of prostatic acid phosphatase from human seminal plasma

Prostatic acid phosphatase has been isolated from human seminal plasma. The purification method utilizes gel filtration on Sephadex G100, ammonium sulfate precipitation and a series of chromatographical steps including concanavalin A Sepharose 4B, anion exchange and gel filtration chromatography. The final product appears homogenous when analyzed by gel filtration on Sephadex G100. It gives one major band on SDS polyacrylamide gels. The specific activity is similar to that obtained by other purification schemes. The yield of the method described above has allowed to set up a sensitive radioimmunoassay of prostatic acid phosphatase.     

Refolding of lysozyme in hydrophobic interaction chromatography: Effects of hydrophobicity of adsorbent and salt concentration in mobile phase

The effects of salt concentration in mobile phase, elution strategy, and hydrophobicity of stationary phase on lysozyme refolding in hydrophobic interaction chromatography (HIC) were investigated. Butyl Sepharose 4 Fast Flow, the least hydrophobic HIC resin among the tested adsorbent, showed the best refolding yield. The binding efficiency of unfolded lysozyme on the adsorbent was maximized when 1 and 0.4 M of initial and final concentration of ammonium sulfate was used in mobile phase, respectively. The optimum gradient strategy for refolding and elution of lysozyme was determined as linear increase of urea concentration to 4M. The optimized condition suggests the less hydrophobic environment than conventionally used salt solutions and HIC resins. Consequently, total refolding yield was improved 1.6 times comparing with optimized dilution-based batch refolding method.     

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.