Differential refractometric determination of binding of sodium dodecyl sulfate to protein using high-performance gel chromatography

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.     

Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assay

It is thought that sodium dodecyl sulfate (SDS), an anionic detergent, binds to hydrophobic moieties of peptide to destroy the conformational structure of protein. Because of this property, it is involved in many biochemical procedures such as separations of protein and proteolytic digestion. In the course of our study on a solid-phase protein assay, we found that SDS acts as an effective reagent for protein blotting onto a hydrophobic membrane of polyvinylidene difluoride with a manifold dot-blot apparatus. At least 0.1% SDS in an acid-ethanol blotting solution, while reducing the bias of pronounced interferers for protein assay to protein-membrane interaction, quantitatively retains protein on the membrane. Presumably, protein denatures by SDS to become an unfolded state and adsorbs into the membrane by hydrophobic interaction, even in the presence of excess SDS. Therefore, bolts stained with a pyrogallol red-molybdate complex (Pyromolex) reagent unreactive to the membrane allowed a precise protein determination without significant interference of materials, especially detergents in the sample solution. The filtration-blotting with SDS would be a crucial procedure for quantitative analyses such as immunoblotting in detergent-containing samples, together with the solid-phase protein assay with limited sample volumes, such as 20 microL or less.     

Precipitation of egg white proteins below their isoelectric points by sodium dodecyl sulphate and temperature.

The precipitating of effect of sodium dodecyl sulphate (SDS) on the egg white proteins ovalbumin, conalbumin and lysozyme was studied at 25 degrees C and at different pH values. The proteins precipitated below their respective isolectric points, provided the detergent to protein ratio was appropriate. The pH profile of precipitation was different for the three proteins reflecting net charge differences. The binding of SDS to the proteins was studied with [35S]-labelled SDS and for ovalbumin a ratio of 21--28 SDS molecules/protein molecule, dependent on the concentration of SDS initially used, seem to be required for precipitation at pH 4.5. This number, however, is dependent on pH and increases with an increased positive net charge of the protein. The precipitating effect of SDS was identical for ovalbumin, conalbumin and lysozyme when compared on a gram to gram basis (0.1--0.15 g SDS/g precipitated protein). The precipitated protein was denatured as measured by differential scanning calorimetry, but was also completely redissolved if pH was increased to above the isoelectric point. The precipitating effecto f SDS was also examined at elevated temperatures. The two-phase systems of the proteins induced by SDS at 25 degrees C were heated from 25 degrees C to 90 degrees C at a rate of 1.25 degrees C/min. The precipitation behaviour was similar for the three proteins upon heating. When the SDS concentration was increased the precipitation curves were transferred towards lower temperatures and the courses of precipitation became less sharp. The synergistic effect of SDS and heat on protein precipitation was differentiated by denaturation measurements and radioactive labelling. The ratio SDS to precipitated protein gradually diminished towards higher temperatures but no purely thermal precipitation was found.     

Partitioning of Proteins using a Hydrophobically Modified Ethylene Oxide/SDS Aqueous Two-phase System

Summary A novel aqueous two-phase system containing hydrophobically modified ethylene oxide (HM-EO) and sodium dodecyl sulphate (SDS) was developed to enhance the selectivity of protein partitioning in two phases. Phase diagrams of HM-EO/H₂O and HM-EO/SDS/H₂O were measured, and the mechanism of interaction between HM-EO polymer and the anionic surfactant sodium dodecyl sulphate (SDS) was also discussed. It was found that the improvement of selectivity of protein partitioning was related to the increase of electrostatic potential difference between the two phases because of the charged network formed by mixed micelles of HM-EO and SDS in the bottom phase. With bovine serum albumin (BSA) and lysozyme as model proteins, some factors, such as pH, SDS concentration, conductivity and temperature of the system, were investigated for the influences of protein partition in HM-EO/SDS/H₂O systems. The results showed that the addition of SDS not only changed the phase behaviour, but also played an important role in protein partitioning.     

Refolding SDS-denatured proteins by the addition of amphipathic cosolvents

Sodium dodecyl sulfate (SDS) is a highly effective and widely used protein denaturant. We show that certain amphipathic cosolvents such as 2-methyl-2,4-pentanediol (MPD) can protect proteins from SDS denaturation, and in several cases can refold proteins from the SDS-denatured state. This cosolvent effect is observed with integral membrane proteins and soluble proteins from either the α-helical or the β-sheet structural classes. The SDS/MPD system can be used to study processes involving native protein states, and we demonstrate the reversible thermal denaturation of the outer membrane protein PagP in an SDS/MPD buffer. MPD and related cosolvents can modulate the denaturing properties of SDS, and we describe a simple and effective method to recover refolded, active protein from the SDS-denatured state.     

Electrophoretic properties of sodium dodecyl sulfate and related changes in its concentration in SDS-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate(SDS) in a protein sample solution migrates in SDS-polyacrylamide gel electrophoresis as a band with a mobility higher than those of protein bands. Behind this band, which is mostly composed of SDS micelles, SDS concentration is raised uniformly in a gel column as a result of the retardation effect of the gel matrix on SDS micelles. Electrophoretic patterns of SDS were obtained when SDS was omitted from various portions of the gel electrophoretic system.     

Isolation of a membrane protein from R rubrum chromatophores and its abnormal behavior in SDS-polyacrylamide gel electrophoresis due to a high binding capacity for SDS.

A membrane protein insoluble in water was isolated by gel chromatography in the presence of 0.1% sodium dodecyl sulfate (SDS) from chromatophores of a photosynthetic bacterium, Rhodospirillum rubrum. This is one of the major membrane proteins of the chromatophore. The protein was found to bind about four grams of SDS per gram, a value which is more than twice the amount generally observed with protein polypeptides derived from water-soluble globular proteins. The electrophoretic behavior of the complex between the membrane protein and SDS is abnormal due to this high capacity for binding SDS. Estimation of the molecular weight of this protein by SDS-polyacrylamide gel electrophoresis was thus impossible. Such an anomaly in SDS binding is unlikely to be restricted to the particular membrane protein described in this paper. The possibility of such a deviation from standard behavior in the interaction with SDS should be taken into consideration in studies of other membrane proteins, since SDS is often used both in analytical and preparative procedures.     

A low-angle laser light scattering study of the association behavior of a major membrane protein of Rhodospirillum rubrum chromatophore at various concentrations of sodium dodecyl sulfate where polypeptides derived from water-soluble globular proteins are solubilized monomerically

The major membrane protein of Rhodospirillum rubrum chromatophore could be solubilized in the presence of free sodium dodecyl sulfate (SDS) in concentration above 0.8 mM. At this concentration, the protein was highly associated to give a weight-averaged molecular weight as high as one million as determined by the low-angle laser light scattering technique. With the increase of free SDS concentration, the aggregates were progressively dissociated to give a molecular weight of 8300 at the critical micelle concentration of SDS. Three protein polypeptides derived from typical water-soluble globular proteins, bovine serum albumin, ovalbumin and beta-lactoglobulin, were found to be solubilized monomerically even at 0.8 mM free SDS. The results obtained suggest that there is substantial difference in the mode of solubilization between polypeptides derived from intrinsic membrane proteins and those from water-soluble globular proteins.     

Acid-base and optical properties of heparin.

Sodium dodecylsulfate (SDS) can be removed from protein by gel electrophoresis. This principle is useful for separating protein bands which are close to each other in SDS gel electrophoresis. We accomplished this by “two-stage” gel electrophoresis. In this system, SDS gel electrophoresis was carried out as the first step. Gel electrophoresis was then continued (after replacing the buffer) without SDS. SDS was then eluted from the gel into the lower buffer during the second stage. Separation of the subunits was significantly improved relative to simple SDS gel electrophoresis.     

Solution structure of human saposin C in a detergent environment

Saposin C is a lysosomal, membrane-binding protein that acts as an activator for the hydrolysis of glucosylceramide by the enzyme glucocerebrosidase. We used high-resolution NMR to determine the three-dimensional solution structure of saposin C in the presence of the detergent sodium dodecyl sulfate (SDS). This structure provides the first representation of membrane bound saposin C at the atomic level. In the presence of SDS, the protein adopts an open conformation with an exposed hydrophobic pocket. In contrast, the previously reported NMR structure of saposin C in the absence of SDS is compact and contains a hydrophobic core that is not exposed to the solvent. NMR data indicate that the SDS molecules interact with the hydrophobic pocket. The structure of saposin C in the presence of SDS is very similar to a monomer in the saposin B homodimer structure. Their comparison reveals possible similarity in the type of protein/lipid interaction as well as structural components differentiating their quaternary structures and functional specificity.     

A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances

We describe a simple assay for small amounts of protein that is insensitive to sodium dodecyl sulfate (SDS) or many common interfering substances including Tris and reducing sugars. For this reason, it is particularly useful in the analysis of protein content of samples prior to SDS electrophoresis. The assay consists of the following steps: (i) absorption of protein to nitrocellulose; (ii) fixation of the bound protein with methanol; (iii) staining of the bound protein with amido black; and (iv) elution and spectrophotometric measurement of the bound dye. The assay is sensitive to as little as 0.5 microgram of protein in 1 microliter of solution. Although SDS does not interfere appreciably with measurement, Nonidet-P40 does.     

SDS removal from protein by polystyrene beads

The applications of sodium dodecyl sulfate (SDS) to molecular weight determination (1,2) and for the separation of protein subunits (3) have been of immense value in biochemical studies [see Waehneldt (4) for a general review]. The tight stoichiometric binding of SDS to polypeptide chains has proven to be a nuisance if one desires to recover the activity of the isolated polypeptides. Removal of the SDS has been affected by the use of anion exchangers in the presence (5,6) and absence of urea (6). However, the residual levels of SDS or urea are often quite unsatisfactory for further protein studies.We have attempted to adapt the procedure of Holloway for the removal of Triton X-100 by Bio-Beads to the removal of SDS. We chose bovine serum albumin as a test protein since it has well-established strong binding properties for linear-chain fatty acids (7).     

Inhibition of amyloid aggregation of bovine serum albumin by sodium dodecyl sulfate at submicellar concentrations

Sodium dodecyl sulfate (SDS), as an anionic surfactant, can induce protein conformational changes. Recent investigations demonstrated different effects of SDS on protein amyloid aggregation. In the present study, the effect of SDS on amyloid aggregation of bovine serum albumin (BSA) was evaluated. BSA transformed to β-sheet-rich amyloid aggregates upon incubation at pH 7.4 and 65°C, as demonstrated by thioflavin T fluorescence, circular dichroism, and transmission electron microscopy. SDS at submicellar concentrations inhibited BSA amyloid aggregation with IC₅₀ of 47.5 μM. The inhibitory effects of structural analogs of SDS on amyloid aggregation of BSA were determined to explore the structure–activity relationship, with results suggesting that both anionic and alkyl moieties of SDS were critical, and that an alkyl moiety with chain length ≥10 carbon atoms was essential to amyloid inhibition. We attributed the inhibitory effect of SDS on BSA amyloid aggregation to interactions between the detergent molecule and the fatty acid binding sites on BSA. The bound SDS stabilized BSA, thereby inhibiting protein transformation to amyloid aggregates. This study reports for the first time that the inhibitory effect of SDS on albumin fibrillation is closely related to its alkyl structure. Moreover, the specific binding of SDS to albumin is the main driving force in amyloid inhibition. This study not only provides fresh insight into the role of SDS in amyloid aggregation of serum albumin, but also suggests rational design of novel antiamyloidogenic reagents based on specific-binding ligands.     

Quantification of protein-bound sodium dodecyl sulfate by Rhodamine B: a method for identification of kinetically stable proteins

Sodium dodecyl sulfate (SDS) bound to proteins in solution could be estimated by passing through Extracti-Gel that removes free SDS followed by specific interaction of the fluorophore Rhodamine B with protein-bound SDS. The resulting fluorescence intensity is compared with a calibration curve. Whereas globular proteins respond to binding of 1.4 mg SDS/mg protein under native conditions, “kinetically stable” proteins that are otherwise resistant to denaturation due to structural integrity show a low level of SDS binding. Analysis of the circular dichroism spectrum shows that in spite of the low level of SDS binding to kinetically stable proteins under nondenaturing conditions, the detergent generates considerable secondary structure in these proteins. Because the low level of SDS binding is a general feature of kinetically stable proteins, the protocol may fulfill one of the criteria to classify a protein as kinetically stable.     

High performance liquid chromatography,chemical laboratory practice: By Heinz Engelhardt. Springer-Verlag,New York/Berlin, 1979. 248 pp. $29.80

A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, $\text{v}\text{v}$). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more.     

Structure of heat-induced beta-lactoglobulin aggregates and their complexes with sodium-dodecyl sulfate

We report on the conformation of heat-induced bovine beta-lactoglobulin (betalg) aggregates prepared at different pH conditions, and their complexes with model anionic surfactants such as sodium dodecyl sulfate (SDS). The investigation was carried out by combining a wide range of techniques such as ultra small angle light scattering, static and dynamic light scattering, small angle neutron scattering, small-angle X-ray scattering, electrophoretic mobility, isothermal titration calorimetry (ITC) and transmission electron microscopy. Three types of aggregates were generated upon heating betalg aqueous dispersions at increasing pH from 2.0 to 5.8 to 7.0: rod-like aggregates, spherical aggregates, and worm-like primary aggregates, respectively. These aggregates were shown not only to differ for their sizes and morphologies, but also for their internal structures and fractal dimensions. The main differences between aggregates are discussed in terms of the ionic charge and conformational changes arising for betalg at different pHs. The formation of complexes between SDS and the various protein aggregates at pH 3.0 was shown to occur by two main mechanisms: at low concentration of SDS, the complex formation occurs essentially by ionic binding between the positive residues of the protein and the negative sulfate heads of the surfactant. At complete neutralization of charges, precipitation of the complexes is observed. Upon further increase in SDS concentration, complex formation of SDS and the protein aggregates occurs primarily by hydrophobic interactions, leading to (i) the formation of an SDS double layer around the protein aggregates, (ii) the inversion of the total ionic charge of each individual protein aggregate, and (iii) the complete redispersion of the protein aggregate-SDS complexes in water. Remarkably, the SDS double layer around the protein aggregates provides an efficient protective shield, preventing precipitation of the aggregates at any possible pH values, including those values corresponding to the isoelectric pH of the aggregates.     

Assessment study on the high-performance liquid chromatography-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate.

The HPLC-type hydroxyapatite chromatography in the presence of sodium dodecyl sulfate (SDS) was assessed with special attention to the behavior of the surfactant. A significant amount of SDS was found to be adsorbed to the hydroxyapatite packed in the column from the starting buffer, 50 mM sodium phosphate buffer, pH 7.0, only when the buffer contained SDS in a concentration at or above its critical micelle concentration. When the phosphate buffer concentration was increased while the SDS concentration was kept at 1 mg/ml, the adsorbed surfactant was desorbed in advance of the release of proteins. Polypeptides derived from proteins could be successfully separated only when the column had been thoroughly equilibrated with the above-mentioned starting buffer solution. When a protein polypeptide complexed with SDS, which had been similarly equilibrated, was applied to the column, an amount of SDS corresponding to 75-90% (w/w) of the surfactant originally bound to the polypeptide was released upon its binding to the hydroxyapatite. On the other hand, porin, an Escherichia coli outer membrane protein, retaining its trimeric native structure in the presence of SDS, released a significantly smaller amount of SDS. When the membrane protein was denatured to give a single polypeptide, it behaved in a manner similar to that of the other protein polypeptides. The mechanism of binding of the protein polypeptides was discussed on the basis of these results. The native and denatured entities of porin could be efficiently separated as the result of the difference in their mode of interaction with the hydroxyapatite.     

Analysis of protein-surfactant interactions--a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant

We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS]tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS]tot in which different modes of interactions dominated were identified. Going from low to high [SDS]tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS]tot>3-6 mM (depending on the protein concentration), the adsorption saturated, and no further protein-detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS]tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS]tot=[SDS]aq+[HiC] Nb where [SDS]aq and Nb are, respectively, the aqueous ("free") concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 degrees C and pH 7.0, HiC denatures with approximately 14 bound surfactant molecules at [SDS]aq=1.0 mM. Saturation is characterized by Nb approximately 39 and [SDS]aq=2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.     

Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.