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1.
When sodium dodecyl sulfate (SDS) is added to a high-performance gel chromatographic column equilibrated with a buffer solution containing SDS at a level above the critical micelle concentration, the surplus SDS migrates as micelles giving a sharp peak. The presence of an unfolded protein in the sample solution gives a polypeptide peak in advance of the SDS micelle peak. As the result of SDS binding to the polypeptide, the SDS micelle peak is attenuated in comparison to that in the absence of protein. Thus the amount of SDS bound to the polypeptide can be determined accurately and simply from the decrease in the area of the SDS micelle peak. This approach is particularly useful for precise determination of bound SDS, which is pertinent to understanding the state of the protein polypeptide-SDS complex under the conditions of SDS-polyacrylamide gel electrophoresis.  相似文献   

2.
Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
The effect of different families of detergents on the solubilization and purification of the pore-forming protein (porin) of the mitochondrial outer membrane of bovine heart was investigated in detail. With Tritons, dimethylamine oxides and zwittergents, porin solubilization with respect to total mitochondrial membrane protein was more efficient with the more hydrophobic members of each series. With most detergents the protein eluted as protein-detergent micelles in the void volume of hydroxyapatite/celite columns. In contrast, the protein was bound to the column material and was eluted after the addition of salt to the elution buffer when the detergents octylglucoside, zwittergent Z-314 and lauryl(dimethyl)-amine oxide were used. The protein purified in the presence of the latter detergent had a higher pore-forming activity in lipid bilayer membranes compared to porin isolated in the presence of Triton X-100. The binding of porin to the hydroxyapatite/celite column was used to study the lipid content of the active pore-forming complex. The analysis revealed that the complex contained no phospholipid but rather five molecules of cholesterol/polypeptide chain.  相似文献   

4.
J Luka  T Lindahl    G Klein 《Journal of virology》1978,27(3):604-611
The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.  相似文献   

5.
Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.  相似文献   

6.
Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta- octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine- specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.  相似文献   

7.
The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.  相似文献   

8.
Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.  相似文献   

9.
Lecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37 degrees C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.  相似文献   

10.
The binding of an anionic surfactant to a protein polypeptide has been studied by the proton magnetic resonance (PMR) technique to form a part of our studies on the principles of SDS-polyacrylamide gel electrophoresis. Sodium 4-(p-butylphenyl) butane-1-sulfonate (CH3-(CH2)3-0-(CH2)4-SO3-Na+) was employed as an anionic surfactant, and reduced and carbosyamidomethylated (RCAM) bovine serum albumin as a typical protein polypeptide. The binding isotherm of the surfactant to RCAM bovine serum albumin was similar to that of sodium dodecyl sulfate (SDS). The surfactant could replace SDS in SDS-polyacrylamide gel electrophoresis without affecting the wellknown mode of spearation of protein bands. These results gave a sound basis for the assumption that the investigation of the complex between a surfactant with a benzene ring and RCAM bovine serum albumin would provide useful knowledge concerning the principles of SDS-polyacrylamide gel electrophoresis. Aggregation of the aromatic surfactant necessarily brings benzene rings together. A benzene ring is a strong source of the ring current effect on chemical shifts in nuclear magnetic resonance (NMR). Chemical shifts of the surfactant in NMR are, therefore, sensitive to whether the surfactant molecules are single-molecularly dissolved or aggregated. Full advantage was taken of the above fact in the present PMR study of the binding of the surfactant to RCAM bovine serum albumin. The chemical shifts of the phenyl and methyl protons both for the single-molecular and micellar aggregated states were estimated from measurements of the shifts as a function of the surfactant concentration. They shifted to a higher magnetic field on micelle formation, due to the increase of the ring current effect. Corresponding measurements for the complex between the surfactant and RCAM bovine serum albumin gave estimates of the chemical shifts of the phenyl and methyl groups of the surfactant bound to the protein polypeptide. They were found to shift to a magnetic field somewhat higher than that for the micellar state throughout the concentration range of the surfactant examined. These results strongly suggest that the surfactant molecules bind to the protein polypeptide in the form of micelle-like clusters, and that PMR of the groups are further influenced by the diagmagnetic effect of the protein polypeptide present as a core. No appreciable change in the mode of binding, corresponding to the steep increase in the amount of binding in the binding isotherm, was observed from the PMR studies. Taking the observed similarity between SDS and the aromatic surfactant in the binding and the gel electrophoresis into consideration, the present results strongly suggest that SDS also binds to protein polypeptides in the form of micelle-like clusters under the conditions of SDS-polyacrylamide gel electrophoreses, and support our "necklace model".  相似文献   

11.
The interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24 degrees C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships. The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants. The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants. The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.  相似文献   

12.
Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3--CaCl2, and K2HPO4-KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3-CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (greater than 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to action were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris--HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3--CacCl2 appeared to be the best buffer and Tris--HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.  相似文献   

13.

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na+, whereas Ca2+ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.  相似文献   

14.
Different folding states of the small, globular milk protein bovine alpha-lactalbumin (BLA) induced by the anionic surfactant sodium dodecylsulphate (SDS) have been examined by fluorescence spectroscopy, CD and NMR. The solution structure of the protein in the absence of SDS was also determined, indicating fluidity even under native conditions. BLA is partly denatured to a molten globule (MG)-like state by micromolar concentrations of SDS, and the transitions from native to MG-like state are dependent on pH, the protein being more sensitive to the surfactant at pH 6.5. As indicated by measurements of the intrinsic emission fluorescence, the tertiary structure disappears at lower concentrations of SDS than most of the secondary structure, as estimated from CD data. The MG-like state induced by low concentrations of SDS is not observable by NMR, and is probably fluctuating and/or aggregating. At higher concentrations of SDS above the critic concentration of micelles, an NMR-observable state reappears. This micelle-associated conformer was partially assigned, and found to bear strong resemblance to the acid-tri-fluoroethanol state, retaining weakened versions of the A and C helix of native BLA. We discuss the results in terms of the inherent flexibility of the protein, and its ability to form multiple folding states and to bind to membranes. Also, we propose that proteins with stable MG-like conformers can have these states stabilized by low levels of compounds with surfactant properties in vivo.  相似文献   

15.
The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).  相似文献   

16.
Phycocyanin (PC) prepared from a cyanobacterium Spirulina platensis by the DEAE-DE52 cellulose column chromatography that was developed by gradient elution of 50-250 mM phosphate buffer (pH 7.0) was stabilized by its subunits cross-linked covalently with formaldehyde. The single blue band that the chemically stabilized PC showed in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the stabilized PC still maintained its trimeric aggregate form even after its incubation at 60 degrees C for 3h and at 100 degrees C for 10 min in the denatured buffer containing 5% (w/v) SDS. Moreover, the stabilized PC exhibited similar spectroscopic properties of absorption and fluorescence to those of the native PC, and showed adequate energy coupling with R-phycoerythrin (R-PE) after it was conjugated with R-PE via glutaraldehyde.  相似文献   

17.
High-speed gel filtration of polypeptides in sodium dodecyl sulfate   总被引:2,自引:0,他引:2  
The separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 M sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3,000 to 50,000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).  相似文献   

18.
DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.  相似文献   

19.
The aim of the investigation was to obtain large amounts of the bacteriolytic enzymes of Myxococcus virescens and to separate these enzymes from the non-bactcriolytic protemases produced by this organism. The bacteria were grown in Casitone broth. When the bacteriolytic activity had reached its maximal value, the cells were removed from the culture medium by centrifugation. Polyethylene glycol 4000 (20 g/1) and potassium phosphate (about 210 g/1) were added to the cell-free solution. The additions resulted in the formation of two liquid phases. The bottom layer was removed, and polyethylene glycol was added to it at a final concentration of 10 g/1. Again two liquid phases formed. The two top phases thus obtained were pooled and 1.6 volumes of cold acetone were added to the mixture. The precipitate formed was dissolved in water and desalted on a Sephadex G-25 column. The desalted protein solution was applied to a carboxymethyl-cellulose column equilibrated with 0.025 M sodium phosphate buffer of pH 6.0. Most of the proteins and the proteinases but none of the bacteriolytic enzymes passed the column unadsorbed. The column was washed with 0.05 M glycine-NaOH buffer of pH 8.8, whereupon the adsorbed bacteriolytic enzymes together with small amounts of proteinases and other proteins were eluted with 0.2 M ammonium carbonate. The material not adsorbed on the CM-cellulose column contained 22 % of the proteolytic activity of the initial cell-free solution and had a 26-fold higher specific activity. The enzyme solution eluted with carbonate contained 24 and 0.3 %, respectively, of the initial bacteriolytic and proteolytic activities. The specific activity of the bacteriolytic enzyme system was about 5000-fold higher than that of the original solution.  相似文献   

20.
Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4 degrees C resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca(2+) caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity inthe protein.  相似文献   

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