Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla

1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26.0%, w/w); it is also relatively rich in proline (8.6%, w/w) but poor in cystine (0.35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the frictional ratio of the protein. 7. Optical-rotatory-dispersion measurements indicated that only a small part of the polypeptide chain was in an alpha-helical conformation. 8. These results are compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.     

Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562

We previously reported that insulin-like growth factor II (IGF-11) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-1), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with ¹²⁵I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immunoaffinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.     

High performance liquid chromatographic properties of peptides and proteins on a dihydroxyalkyl bonded silica stationary phase

The chromatographic behavior of biologically relevant peptides and proteins in the molecular weight range between 200 and 200,000 dalton units were studied on a size exclusion matrix column consisting of an aqueous compatible dihydroxyalkyl bonded silica support. The mechanism of separation appears to be dependent on hydrodynamic radius, hydrophobic and ionic interactions. Support for this contention is based on the chromatographic properties of these peptides and proteins at different mobile phase ionic strengths and pH, oxidation state of amino acid residues and total hydrophobicity of the peptide or protein. This column is also capable of separating native angiotensin-I from its iodinated congener. Recoveries of proteins and peptides from this column ranged between 70–100%. Unlike typical reverse phase separations, this modified silica chromatographic media allows for an alternative technique employing aqueous eluents for rapid separation/isolation and purification of peptides and proteins from natural or synthetic sources.     

Extended application of gel-permeation chromatography by spin column

Separation of small volumes of proteins from unbound ligands or reequilibration with buffer by passing through a 1-ml Sephadex G-50 column under mild centrifugal force is a popular technique. Here it has been demonstrated that other Sephadex matrix could similarly be used for complete or partial separation of protein molecules. Proteins to be eluted at void volume are recovered near quantitatively, while others are partly or almost completely retained depending on molecular size. Calibration curves using standard proteins of Mw 12.5 to 440 kDa with Sephadex G-50-G-200 representing recovery versus molecular weight show profiles as expected from the fractionation ranges of the column matrix. The procedure may be applied to follow protein association-dissociation reactions if the molecular weights of the species concerned are known and a proper matrix exists for separating them. Equilibrium unfolding transitions constructed with model proteins in presence of 0-8M urea using recovery as an index correspond to profiles obtained from other physical measurements. This may be a convenient approach to follow change of protein hydrodynamic volume quickly when a parallel methodology is not readily available.     

Purification of proteins of the Na/Cl cotransporter from membranes of Ehrlich ascites cells using a bumetanide-sepharose affinity column

Summary Bumetanide-binding proteins were isolated from membranes of Ehrlich ascites tumor cells by affinity chromatography. An affinity column was constructed with the active moiety of bumetanide as a ligand using 4-azidobumetanide, a photoactive analogue which inhibits Na/Cl cotransport in Ehrlich cells with high specificity. Covalent binding of the 4-azidobumetanide with Sepharose was promoted by photolysis. Membranes isolated from Ehrlich cells were solubilized withn-octylglucoside. Solubilized proteins retarded by the affinity column were readily eluted by bumetanide. In reducing gels the major proteins eluted by bumetanide were 76 kDa and 38–39 kDa. There were also two proteins of 32 to 35 kDa eluted in lesser amounts. No proteins retarded by the affinity column were eluted with extensive washing without bumetanide. Furthermore, bumetanide eluted no proteins from a control column lacking the specific ligand. Upon rechromatography with bumetanide in solution, bumetanide-eluted proteins were not retarded, but their purity was increased by the retardation of contaminating proteins. Bumetanide-binding protein purified in this manner were characterized further by electrophoresis in nonreducing, nondenaturing gels.     

Nonideal size-exclusion chromatography of proteins: Effects of pH at low ionic strength

Ideal size-exclusion chromatography separates molecules primarily on the basis of hydrodynamic volume. This is achieved only when the chromatographic support is neutral and the polarity nearly equal to that of the mobile phase. When this is not the case, the support surface may begin to play a role in the separation process. As the magnitude of surface contributions becomes larger, the deviation from the ideal increases. Because the separation mechanism is different than that of ideal size-exclusion chromatography, selectivity could be increased in nonideal size-exclusion chromatography. This paper explores the use of size-exclusion chromatography columns with mobile phases that cause proteins to exhibit slight deviations from the ideal size-exclusion mechanism. Although there are many ways to initiate nonideal size-exclusion behavior, the specific variable examined in this study is the influence of pH at low ionic strength. Individual proteins were chromatographed on SynChrom GPC-100, TSK-G2000SW, and TSK-G3000SW columns at low ionic strength. It was found that a protein could be selectively adsorbed, ion excluded, or chromatographed in an ideal size-exclusion mode by varying mobile-phase pH relative to the isoelectric point of the protein. In extreme cases, molecules could be induced either to elute in the void volume or beyond the volume of total permeation. It is postulated that these effects are the result of electrostatic interactions between proteins and surface silanols on the support surface. Optimization of size-exclusion separations relative to protein isoelectric points is discussed.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Chromatography of dodecyl sulphate-solubilized proteins of the human erythrocyte membrane on N-(3-carboxypropionyl)aminodecyl-sepharose.

Human erythrocyte 'ghosts' were solubilized in 0.5% (w/v) sodium dodecyl sulphate at pH 4.0(I = 0.012 mol/I). At a loading of 1-2 mg of protein/ml of column volume, all of membrane proteins were adsorbed to a column of CPAD [N-(3-carboxypropionyl)-aminodecyl]-Sepharose at pH 4.0 (I = 0-012 mol/1) and room temperature (22 degrees C). Many proteins were subsequently desorbed by raising the pH or by including sodium dodecyl sulphate continuously in the eluting buffer. Experiments with a series of adsorbents homologous with CPAD-Sepharose, in which the length of the hydrocarbon chain was varied, provided strong evidence of hydrophobic interactions, in addition to ionic interactions, in the binding of these proteins to CPAD-Sepharose. Elution with increasing-pH gradients at different concentrations of sodium dodecyl sulphate showed that glycophorin (the major sialoglycoprotein) was eluted in the void volume, at recoveries close to 100%, when the detergent concentration was greater than or equal to 0.3% (w/v). Protein E, the major protein, was desorbed late in the pH gradient even at a high (0.5%, w/v) concentration of the detergent, and was always incompletely desorbed, the maximum recovery recorded being 40%. Spectrin (the high-molecular-weight polypeptide pair) did not behave in a well-defined manner, and was found widely distributed among the effluent fractions under all the conditions that were tested.     

Chromatography of plasma proteins on immobilized Cibacron Blue F3-GA. Mechanism of the molecular interaction.

Fractionation of plasma proteins on immobilized Cibacron Blue F3-GA (Affi-gel Blue) under different conditions of pH, ionic strength and temperature was studied. At acidic pH the unbound proteins were eluted in order of increasing pI (the Affi-gel Blue behaving as ion-exchanger); at basic pH and at low ionic strength they were eluted in order of decreasing molecular weight (separation by diffusion-exclusion). For the proteins that were either retarded in comparison with substances of similar molecular characteristics, or that were bound to the resin, pseudo-ligand affinity or hydrophobic interactions were also implicated.     

Influence of native disulfide bonds of globular proteins on their retention behavior on reverse phase supports

The reduction of the disulfide bonds of globular proteins, for example, those of lysozyme or ribonuclease-A, results in an increase in the hydrodynamic volume of the polypeptide chain. This is reflected in an earlier elution of the reduced protein on gel filtration compared to that of the native disulfide-bonded form. The reduction of the four disulfide bonds of ribonuclease-A increased its retention time on reverse phase support, suggesting an increase in the apparent hydrophobicity of the protein molecule on reduction. Performic acid-oxidized ribonuclease-A eluted ahead of native disulfide-bonded ribonuclease on RP HPLC, suggesting a decrease in the hydrophobicity of the molecule. However, the hydrodynamic volume of performic acid-oxidized ribonuclease-A is similar to that of reduced protein as reflected in its gel filtration behavior. Thus, the increased retention of the reduced protein compared to that of native disulfide-bonded protein is not related to the increased hydrodynamic volume, and is a reflection of the stronger interaction of reduced protein with the reverse phase support. Reoxidation of the reduced ribonuclease-A regenerated the original chromatographic behavior of the protein on the reverse phase support. Similar results were also obtained with hen egg white lysozyme. The results of the present study are interpreted as indicating that the native disulfide bonds of a globular protein restrict the exposure of the hydrophobic amino acid residues of the polypeptide chain with a consequent lower retention on the reverse phase support compared to its reduced form.     

Application of high performance liquid chromatography to the analysis of cytoplasmic and nuclear oestrogen receptors

Soluble and nuclear preparations of fresh human endometrium have been analysed by HPLC on a gel permeation column. With high ionic strength eluants recoveries of receptor binding were less than 40% while low ionic strength eluants gave recoveries of 70% or greater. Non-specific binding by endometrial and plasma proteins was largely lost at both high and low ionic strength. Soluble binding components saturable by diethylstilboestrol (DES) were resolved into two components differing in heat lability and response to variations in ionic strength. Molybdate appears to alter the receptor protein rather than the activity of inactivating enzymes. At low ionic strength nuclear binding components saturable by DES could be resolved into two components.     

The subunits of chemically treated proteoglycan isolated from bovine nasal cartilage

1. The light fraction of the proteoglycan of bovine nasal cartilage was split by treatment with 0.1m-hydrochloric acid in acetone. The products were separated by gel filtration on 4% agarose and two retarded fractions were detected and isolated. These two fractions were found to have a Stokes radius of 134 and 47 A respectively, as determined by calibration of the column against proteins of known hydrodynamic volumes. 2. The 47 A fraction had a protein content of 4% and a glucosamine/galactosamine ratio 1:23. The 134 A fraction had a protein content of 20% and a glucosamine/galactosamine ratio 1:4.8. 3. The results of the viscometric studies on both fractions suggested that the 134 A fraction alone exhibited the property of undergoing reversible pH-dependent aggregation with a transition point at pH4.9. 4. It was concluded that these fractions could represent subunits of the native cartilage proteoglycan.     

Multiple parameter kinetic studies of the oxidative folding of reduced lysozyme

We have compared the oxidative renaturation of reduced hen egg white lysozyme promoted by Cu(II) + O2 with that promoted by a glutathione redox buffer. The progress curves for protein fluorescence, circular dicroism, thiol oxidation, hydrodynamic volume, and enzymic activity were determined for both regeneration systems. All of these processes were more rapid in the glutathione regeneration than in the copper-catalyzed. Comparison of the two systems was carried out by normalizing the progress curves with a coordinate system where "time" is replaced by "extent of protein thiol oxidation." While similar progress curves were obtained for circular dichroism, the two systems produced distinctly different progress curves for enzymic activity, fluorescence, and gel permeation chromatographic reflection of protein hydrodynamic volume. We infer that all these differences result from differences in relative amounts and/or kind of reaction intermediates. Thus, there are substantial differences between the renaturation mechanisms of the glutathione- and the copper-promoted systems.     

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.     

Influence of protein dynamics on the metal-sites of ovotransferrin

Using the perturbed angular correlations (PAC) technique, the formation of hafnium-ovotransferrin complexes has been studied. Two binding configurations at each of the two specific binding-sites of the protein have been observed. They are characterized by well-defined electric quadrupole frequencies. Information about the dynamics of the protein was derived from temperature dependent measurements of the relaxation constant. The well-resolved spectra taken with fast BaF₂-detectors allow a precise determination of the relaxation behaviour of the protein. The results are compared with the predictions from a hydrodynamic model for the reorientation of macromolecules. Thus the hydrodynamic volume of ovotransferrin and its N-terminal half-molecule were determined. The ovotransferrin volume is in agreement with a value derived for human serum transferrin from small angle neutron scattering. From experiments with immobilized protein material there is evidence for internal protein dynamics which is probed by the Hf-ion bound to the specific metal-sites.Abbreviations PAC perturbed angular correlations technique - TF serum transferrin - LF lactoferrin - OTF ovotransferrin - OTF/2N N-terminal half-molecule of ovotransferrin - NQR nuclear quadrupole resonance - EFG electric field gradient - NQI nuclear quadrupole interaction - NTA nitrilotriacetate - MES 2-(N-morpholino)ethanesulphonic acid - HEPPS N-2-hydroxyethyl-piperazine-N-3-propanesulphonic acid - TRIS tris(hydroxymethyl)aminomethane Correspondence to: F. J. Schwab.     

Adsorption chromatography of proteins

A method for adsorption chromatography of proteins is proposed. A protein solution is passed through a cellulose column at a pH value corresponding to an isoelectric point of the protein. Depending on the charge of unwanted proteins, they either remain at the origin (if charges of protein and ion-exchanger are opposite) or are released from the column (if charges of protein and ion-exchanger coincide). Elution volume of the purified protein is higher than for the second group of unwanted proteins because movement of the uncharged protein of interest includes its adsorption on cellulose followed by subsequent desorption caused by the elution buffer. Problems of optimization of buffers and adsorbents are discussed. Applicability of the method of adsorption chromatography is illustrated using purification of horseradish peroxidase as an example.     

Interactions of lysozyme in guanidinium chloride solutions from static and dynamic light-scattering measurements

The interactions of partially unfolded proteins provide insight into protein folding and protein aggregation. In this work, we studied partially unfolded hen egg lysozyme interactions in solutions containing up to 7 M guanidinium chloride (GdnHCl). The osmotic second virial coefficient (B(22)) of lysozyme was measured using static light scattering in GdnHCl aqueous solutions at 20 degrees C and pH 4.5. B(22) is positive in all solutions, indicating repulsive protein-protein interactions. At low GdnHCl concentrations, B(22) decreases with rising ionic strength: in the absence of GdnHCl, B(22) is 1.1 x 10(-3) mLmol/g(2), decreasing to 3.0 x 10(-5) mLmol/g(2) in the presence of 1 M GdnHCl. Lysozyme unfolds in solutions at GdnHCl concentrations higher than 3 M. Under such conditions, B(22) increases with ionic strength, reaching 8.0 x 10(-4) mLmol/g(2) at 6.5 M GdnHCl. Protein-protein hydrodynamic interactions were evaluated from concentration-dependent diffusivity measurements, obtained from dynamic light scattering. At moderate GdnHCl concentrations, lysozyme interparticle interactions are least repulsive and hydrodynamic interactions are least attractive. The lysozyme hydrodynamic radius was calculated from infinite-dilution diffusivity and did not change significantly during protein unfolding. Our results contribute toward better understanding of protein interactions of partially unfolded states in the presence of a denaturant; they may be helpful for the design of protein refolding processes that avoid protein aggregation.     

Diffusion of charged ions in mucus gel: effect of net charge

The interposition of a neutral starch gel greatly retarded bulk ionic movement by free flow. A mucus (charged) gel preparation of identical concentration and thickness further retarded ionic diffusion. The findings suggest that the charges in the mucus matrix may exert an ionic exclusion effect (Donnan Exclusion), thus retarding other ionic diffusion. We speculate that a mucus layer may physiologically behave as an ion exchange gel column and modify the traffic of charged ions through it.     

The use of gel filtration to follow conformational changes in proteins. Conformational flexibility of bovine myelin basic protein.

The hydrodynamic behavior of bovine myelin basic protein was studied by gel filtration through Sephadex G-100 under conditions which included variations in pH from 2 to 12, variations in ionic strength from 0.01 to 1.5 M at pH 2 and from 0.1 to 2 M at pH 7, and variations in guanidinium chloride concentration from 0 to 6 M. A number of well characterized compact globular proteins were subjected to the same conditions for comparison. Compact globular proteins showed major conformational transitions due to acid, alkali, and guanidinium chloride denaturation and, possibly, minor transitions as well. Myelin basic protein behaved like a flexible linear polyelectrolyte, expanding continuously between pH 11 and pH 2 to 3 at ionic strength 0.1 M and contracting continuously with increase in ionic strength at pH 2 and at pH 7 to the point of salting-out. Relatively low concentrations of guanidinium chloride (approximately 0.5 M) were sufficient to cause the basic protein to expand. With increasing concentration of the denaturant the molecule continued to expand, but in a noncooperative manner. These results demonstrated the lack of significant intramolecular stabilization in the protein.     

Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain

1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem. 121, 579–585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.