Hydrophobic affinity chromatography of nucleic acids and proteins.

5' tritylated oligonucleotides binding hydrophobically to low trityl cellulose/sepharose (< 15 microMTr/ml) retain their hydrogen-bonding specificities for complementary sequences. This, constitutes a novel mode of attaching affinity ligands to solid supports, is more convenient than existing methods, and proceeds with 100% yield. The salt, dielectric constant and temperature dependence of these non-covalently anchored ligands permits the isolation of a variety of RNAs including fibroin mRNA. Medium trityl sepharose (15-40 microM Tr/ml) has a high binding specificity for poly A and poly A containing mRNA, equivalent to dT cellulose. Most proteins, including nucleic acid enzymes, bind to these columns and retain enzymatic activity, thus mimicking enzymes attached covalently to solid phases. A number of in vivo counterparts to this hydrophobically determined specificity are noted, as are homologies to nitro-cellulose filters.     

Interaction of tubulin with the macromolecular apolar probe, octyl sepharose

Binding of the microtubule protein, tubulin, to hydrophobic groups immobilized on octyl sepharose has been investigated. The results indicate that tubulin binds to octyl sepharose in a time-, temperature-, and concentration-dependent manner. Binding is multiphasic, with one fast phase and at least two slow phases, and is influenced by the presence of antimitotic drugs. Colchicine, vinblastine and podophyllotoxin enhance the fast binding of tubulin with very little effect on the slow binding. Pre-incubation of tubulin with the apolar probe, bis(1,8-anilinonaphthalenesulfonate) (BisANS) enhances both the rapid and slow phases of binding of tubulin to octyl sepharose. 1,8-Anilinonaphthalenesulfonate, the monomer of BisANS, has no effect. These results are consistent with a model for tubulin decay which involves the appearance of hydrophobic sites with time.     

Enzyme immobilization on tritylagarose

A method is described for the immobilization on tritylated agarose or Sepharose columns of a wide spectrum of enzymes, including types useful in contemporary biochemistry/molecular biology, many of which have never before been reported as immobilized. The method involves the formation of noncovalent hydrophobic bonds between the enzymes and trityl groups which are attached to the agarose by means of ether bonds. The immobilization of calf intestinal and E. coli alkaline phosphatases to tritylagarose is reported in detail. Their binding strength, binding capacity, and long-term stability (greater than six months) are described as a function of the salt concentration, pH, buffer type, and degree of agarose substitution. Homologies are noted between tritylagarose-bound and membrane-bound phosphatases. This method compares favorably with other methods, covalent or otherwise, reported to date, in terms of the enzyme immobilization yield (ca. 100%), the mildness of conditions, resulting, in most cases, in the retention of a high degree of activity, the ease and speed of the manipulations, and the long-term stability of the immobilized enzyme. Further, it is noted that highly tritylated and crosslinked Sephadex G10 selectively and mildly removes detergents from enzyme solutions.     

Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Reversible denaturation of carbonic anhydrase provides a method for its adsorptive immobilization

Palmityl-substituted sepharose 4B has been used for adsorptive immobilization of heat-denatured carbonic anhydrase. The native form of this enzyme does not show any affinity for binding to this hydrophobic support. However, through the process of denaturation-renaturation performed by heating and subsequent cooling of an enzyme solution in the presence of the matrix, it was possible to obtain a catalytically active immobilized preparation, which was used successfully in continuous catalytic transformations. It is suggested that this simple procedure may provide a convenient method of immobilization for proteins, which are not normally adsorbed on hydrophobic supports.     

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.     

Thermodynamic analysis of the effect of concentrated salts on protein interaction with hydrophobic and polysaccharide columns

An attempt was made to explain the effect of concentrated salts on protein interaction with hydrophobic columns. From the previously observed results of preferential interactions for salting-out salts with proteins, it was shown that the free energy of the protein is increased by addition of the salts and this unfavorable free energy is smaller for the proteins bound to the columns because of their smaller surface area exposed to solvent; i.e., the bound form of the proteins is thermodynamically more stable. This explains the protein binding to the hydrophobic columns at high salt concentrations and the elution by decreasing the salt concentration. The unfavorable interaction free energy was greater for Na2SO4 or (NH4)2SO4 than for NaCl, which explains the stronger effect of the former salts on the protein binding to the columns. The observed favorable interaction between KSCN or guanidine hydrochloride and the proteins explains the decreasing effect of these salts on the protein binding to the hydrophobic columns.     

Nuclear proteins. V. Studies of histone-binding proteins from mouse liver by affinity chromatography

We examined four extracts of mouse liver for histone-binding proteins using histone affinity chromatography and positively charged resins. The extracts used were cytoplasm and washes from isolated nuclei with buffers containing 0.05 M Tris, 0.15 M NaCl or 0.35 M NaCl. Proteins from the nuclear washes showed greater binding to the columns than proteins from the cytoplasm. The binding fractions were heterogeneous in gel electrophoresis systems. Proteins bound to affinity columns of individual histones were similar to those bound to columns of whole histone, polylysine and DEAE. A 25,000 dalton polypeptide (J2), found only in nuclear washes was a prominent histone-binding protein. It could be competitively eluted from DEAE with histones, suggesting polypeptide J2 may show a specific affinity for histones. Polypeptide J2 has an acidic to basic amino acid ratio of 1.58, and its amino acid composition is not similar to that of the high mobility group 1 protein. Polypeptide J2 binds to hydrophobic columns and may play a role in modifying histone-histone and histone-DNA interactions.     

Localization of fragments binding nuclear proteins in the promotor region of the human alpha-1-antitrypsin gene]

Human and mice nuclear extracts from livers and mice spleen extract were analysed in an attempt to find any proteins capable of binding to the human alpha 1-antitrypsin gene promoter. The nuclei of all studied tissues contain such proteins. The proteins were partially purified on DEAE-trisacryl, heparin sepharose and phosphocellulose columns. The multiple sites for liver nuclear proteins binding to the human alpha 1-antitrypsin gene promoter were found by the DNAse I footprinting technique.     

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.     

High-performance hydrophobic interaction chromatography of proteins

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.     

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: modulation of affinity binding to fibronectin

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hydrophobic interaction chromatography of myosin fragments: Potential use in purification

Myosin fragments were fractionated on columns of the hydrophobic gel phenyl-Sepharose CL-4B. In the presence of high NaCl concentrations the fragments bound tightly to the columns; they could be eluted by decreasing the ionic strength, by increasing the pH, or by applying various concentrations of ethylene glycol. In myosin subfragment-1 (S-1), the light chains underwent partial dissociation from the heavy chain and bound separately to the column matrix. The order of strength of binding of the various species to the column was heavy chain > A1 light chain > A2 light chain > native S-1 > denatured heavy chain or S-1. Thus the hydrophobic gel appears to be able to differentiate between enzymatically active and inactive S-1. Under appropriate elution conditions it was possible to obtain S-1 preparations depleted from nicked heavy chains and with specific ATPase activities 34–130% higher than those of untreated S-1. When S-1(A2) was fractionated on phenyl-Sepharose a fivefold enrichment of the heavy chain with respect to the light chains was obtained, while the ATPase activity was equal or larger than that of the original S-1, implying that the light chains are not essential for ATPase activity. Thus, it seems that chromatography of S-1 on phenyl-Sepharose is a potentially useful method for obtaining a purified myosin heavy-chain fragment with a high ATPase specific activity.     

Affinity purification of ubiquitin-protein ligase on immobilized protein substrates. Evidence for the existence of separate NH2-terminal binding sites on a single enzyme

Previous studies have indicated the existence of separate binding sites of ubiquitin-protein ligase, E3, specific for basic (Type I) or bulky hydrophobic (Type II) NH2-terminal amino acid residues of proteins. Another class (Type III) of protein substrates appeared to interact with E3 at regions other than the NH2 terminus (Reiss, Y., Kaim, D., and Hershko, A. (1988) J. Biol. Chem. 263, 2693-2698). In the present study we have used affinity chromatography on immobilized protein substrates to examine the question of whether the different binding sites belong to one E3 enzyme, or to different E3 species. Another objective was to develop a procedure for the extensive purification of E3. When a crude extract of reticulocytes is applied to Type I or Type II protein substrates linked to Sepharose, E3 becomes strongly bound to the affinity columns and is not eluted with salt at high concentration. However, the enzyme can be specifically eluted by a dipeptide that has an NH2-terminal residue similar to that of matrix-bound protein substrate. A 350-fold purification is obtained in this single step. Preparations of E3 purified on either Type I or Type II protein substrate affinity columns act on both types of protein substrates, indicating that the separate binding sites for basic and hydrophobic NH2-terminal residues belong to one enzyme. Another species of E3 that acts strongly on some Type III protein substrates does not bind to Type I or Type II protein substrate affinity columns.     

On the mode of adsorption of proteins to “hydrophobic columns”

α-Lactalbumin and ovalbumin were found to bind tightly to alkyl amino agaroses with hydrocarbon chains of 4–6 carbon atoms. These proteins were not adsorbed when the columns were acetylated. The binding of protein to the hydrophobic column appears to occur in two phases. The first of these is suggested to involve binding of the protein anions to the cationic site on the columns. The second phase is suggested to involve binding of the hydrophobic portions of the column with the hydrophobic amino acid residues of the protein.     

Depressed cytotoxic T-cell responses in previously treated Hodgkin's and non-Hodgkin's lymphoma patients

Summary Peripheral blood lymphocytes from 62 previously treated Hodgkin's and non-Hodgkin's lymphoma patients were tested for their ability to generate cytotoxic T lymphocytes in response to stimulation with allogeneic cells in mixed leukocyte culture. In most patients, including some in long-term unmaintained remission, extremely low cytotoxic responses were generated. To test whether these patients have circulating cells that suppress autologous lymphocytes from responding to alloantigens, patients' responding cells were passaged over columns of sepharose beads conjugated with histamine-rabbit serum albumin (Hist-RSA). This procedure has been shown to remove mouse suppressor cells and Concanavalin A(ConA)-induced human suppressor cells. Passage of patients' cells, prior to allogeneic stimulation, over columns of sepharose beads conjugated with Hist-RSA but not over control RSA columns, resulted in the isolation of lymphocytes that generated increased cytotoxic responses to alloantigens in 18 of 22 patients with initially low cytotoxic responses. These results suggest that the impaired ability of treated Hodgkin's and non-Hodgkin's lymphoma patients' lymphocytes to differentiate into cytotoxic T lymphocytes is at least in part due to the presence of circulating suppressor cells that bear histamine receptors.Scholar of the Leukemia Society of America     

Studies on the interaction between actin and cofilin purified by a new method.

Cofilin is a 21,000-Mr actin-binding protein that widely exists in mammalian tissues. (1) A new purification procedure for porcine brain cofilin has been developed that involves (NH4)2SO4 fractionation and sequential chromatographies on Toyo Pearl and butyl-Toyo Pearl hydrophobic columns, hydroxyapatite, phosphocellulose and Sephadex G-75 gel-filtration columns. The purified cofilin bound to F-actin and increased the amount of G-actin to a limited extent, as previously reported [Nishida, Maekawa & Sakai (1984) Biochemistry 23, 5307-5313]. (2) The binding of cofilin to F-actin was scarcely affected by Mg2+, Ca2+ or by calmodulin. However, the binding was diminished by increasing concentrations of KCl, but was only slightly affected by temperature. (3) Cofilin and either alpha-actinin or filamin could bind to F-actin simultaneously with some competition, but the binding of caldesmon to F-actin was markedly inhibited by cofilin. Phalloidin inhibited the binding of cofilin to F-actin, and protected F-actin from depolymerization by cofilin.     

A new method of isolating lysyl-tRNA-synthetase from the rat liver

The methods of gel filtration on sepharose 6B, hydrophobic chromatography, chromatofocusing were used to isolate the preparation of lysyl-tRNA-synthetase. The enzyme activity at the terminal stage of purification is 1800 times as high. The isolated preparation is homogeneous with electrophoresis in 4-30% PAAG.     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium