Sodium dodecyl sulfate in protein chemistry

This review summarizes in a brief manner the main aspects of the application of sodium dodecyl sulfate (SDS) to protein chemistry. The principal problems of SDS-polyacrylamide gel electrophoresis are described, as well as the anomalous behavior of protein-SDS complexes and the inactivation of enzymes due to variable binding of SDS to the polypeptides studied. The particular value of SDS in elucidating the protein composition of biological membranes and in membrane-reconstitution experiments is discussed.     

Sodium dodecyl sulfate enhancement of quantitative immunoenzyme dot-blot assays on nitrocellulose

Treating proteins with low concentrations of sodium dodecyl sulfate (SDS) and boiling for 2-3 min increased the linear range and total amount of protein that could be bound to nitrocellulose. Human serum albumin (HSA) and cathepsin G (Cat G) were both optimally bound at an SDS concentration of 10 micrograms/ml, while bronchial leukocyte proteinase inhibitor (BLPI) required 50 micrograms/ml SDS for optimum binding, corresponding to SDS-to-protein weight ratios of 0.5 and 2.5, respectively. Ionic strength and pH of the blotting buffers had a greater effect on the binding of SDS-treated proteins than on native proteins, with the linear binding range and total capacity for SDS-treated proteins being increased. Boiling SDS-treated human leukocyte extracts inactivated endogenous peroxidases, eliminating their interference with peroxidase-linked secondary antibodies in immunoassays. The nonionic detergents, Tween 20 and Nonidet P-40, were shown to rapidly wash both native and SDS-treated HSA off the filters, but these HSA samples were stable to washing with SDS. Although SDS-treated Cat G was more stable with nonionic detergents than was native Cat G, it was less resistant to washing with SDS. The substitution of SDS for nonionic detergents improved the response of immunoassays with native and SDS-treated proteins. Affinity-purified antibodies to human mast cell tryptase cross-reacted with native Cat G, but not with SDS-treated Cat G, indicating that SDS treatment can improve the specificity of immunoassays employing polyclonal antisera. These effects appear to be the result of partial denaturation and increases in the hydrophobicity of SDS-treated relative to native proteins.     

Sodium dodecyl sulfate hypersensitivity of clpP and clpB mutants of Escherichia coli

We studied the hypersensitivity of clpP and clpB mutants of Escherichia coli to sodium dodecyl sulfate (SDS). Both wild-type E. coli MC4100 and lon mutants grew in the presence of 10% SDS, whereas isogenic clpP and clpB single mutants could not grow above 0.5% SDS and clpA and clpX single mutants could not grow above 5.0% SDS. For wild-type E. coli, cellular ClpP levels as determined by Western immunoblot analysis increased ca. sixfold as the levels of added SDS increased from 0 to 2%. Capsular colanic acid, measured as uronic acid, increased ca. sixfold as the levels of added SDS increased from 2 to 10%. Based on these findings, 3 of the 19 previously identified SDS shock proteins (M. Adamowicz, P. M. Kelley, and K. W. Nickerson, J. Bacteriol. 173:229-233, 1991) are tentatively identified as ClpP, ClpX, and ClpB.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption

A simple method, sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with direct protein adsorption analysis (SDS–PAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentrations (in pg/μl). We used this method to characterize the effects of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures and its utility in developing preventive strategies of adsorption.     

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the alpha subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).     

十二烷基硫酸钠辅助提取布渣叶中的黄酮类物质

以布渣叶为研究体系,采用表面活性剂辅助热回流法提取黄酮类物质.考察了表面活性剂种类及浓度、提取时间、液固比和溶剂pH等因素,并通过实验数据进行了正交试验设计,得到最优的工艺条件:表面活性剂选择十二烷基硫酸钠(SDS),质量浓度0.8 g/L,提取时间105 min,液固比45 mL/g,溶剂pH 6.3.在此条件下,布渣叶黄酮得率为15.73％,与其他方法相比具有显著优势.     

Sodium dodecyl sulfate activation of a plant polyphenoloxidase. Effect of sodium dodecyl sulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase

Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS). Further characterization of the enzyme was carried out in the presence and absence of SDS. Activation of the enzyme increased in a sigmoidal manner with increasing SDS concentrations up to a maximum of 1.75 mM. The presence of SDS eliminated a low pH optimum induced by acid shocking. Increased thermolability of the enzyme was observed in the presence of SDS as well as an increased binding of [14C]dihydroxy-phenylalanine. Size exclusion chromatography on high performance liquid chromatography showed that the size and apparent molecular mass of the enzyme were slightly altered in the presence (48 kDa) versus absence (47 kDa) of SDS. Although the estimations were larger than those obtained by size exclusion techniques, no large differences in molecular weight were observed after sedimentation equilibrium of the enzyme in the presence (53.9 kDa) and absence (52.3 kDa) of SDS. Relative electrophoretic mobility and intrinsic fluorescence of tyrosine and tryptophan residues increased in a complex fashion as the SDS concentration was increased. Plateau regions in these latter experiments corresponded to concentrations of SDS needed for activation. The ability of SDS to activate the enzyme alters both its enzymatic and physical characteristics and suggests that a limited conformational change, due to binding of small amounts of SDS, may induce or initiate the activation of latent enzyme.     

The effect of sodium dodecyl sulfate on the structure and function of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor (SSI) has been shown to exist as a dimer of molecular weight of 23,000 in 25 mm phosphate buffer, at pH 7.0 (the ionic strength 0.1 m with NaCl), 25.0 °C in the concentration range of 0.01–10 mg/ml. In the present paper, the effects of an anionic detergent, sodium dodecyl sulfate (SDS), on the structure and function of SSI has been examined, [a]The molecular weight of SSI was measured in the SDS solution with the sedimentation equilibrium method of the multicomponent-polydisperse system under the conditions described above, and thereby it has been shown that SSI dissociates into monomers with SDS of 0.03–0.12% ($\text{w}\text{v}$) when the concentration of SSI is 1.00 mg/ml (87.0 μm as monomer), [b]As SSI dissociates into monomers, there were observed blue-shift troughs at 293 nm and 300 nm due to a tryptophyl residue and a red-shift of phenylalanyl residues in the absorption difference spectrum induced by the binding of SSI and SDS. [c] The inhibitory activity of SSI against subtilisin BPN′-catalyzed hydrolysis of p-nitrophenyl acetate was measured under the conditions that SSI is in monomer in the SDS solution. Unexpectedly half of the inhibitory activity of SSI against subtilisin BPN′ is lost in the SDS solution.     

Sodium dodecyl sulfate potentiates collagen degradation by proteases from Porphyromonas (Bacteroides) gingivalis

Abstract Collagen degradation by protease(s) from Bacteroides gingivalis was estimated by spectrophotometry with insoluble, type I, collagen a substrate. When 1% sodium dodecyl sulfate (SDS) was included in the assay, there was a 7-fold increase in reaction velocity. The protease(s) were extracted from the bacteria with one percent Triton X-100 and partially purified by gel chromatography on Superose HR 12. The SDS-potentiated enzyme (tentatively named proteinase D(odecyl)) eluted immediately after the void volume and migrated at a position corresponding to 100 kDa by localization of enzyme activity after SDS-polyacrylamide gel electroporesis. The temporal degradation of collagen fibrils by proteinase D was illustrated by phase contrast microphotography of fibrils dispersed in 1% SDS and addition of concentrated enzyme from the edge of the coverslip. During 30 min, the diameter of the fibrils gradually diminished, and some fibrils exhibited a zig-zag profile. After 45 min, most of the fibrils had disappeared. Incubation of proteinase D with 1% SDS for 6 h at 37°C did not diminish the activity of the enzyme. The collagen was completely degraded to small peptides when SDS was present.     

Multiple-layer substrate zymography for detection of several enzymes in a single sodium dodecyl sulfate gel

We have developed a system to detect three hydrolytic enzymes (cellulase, lipase, and protease) using a single sodium dodecyl sulfate (SDS) gel and an electrotransfer system. After electrophoresis, proteins in the gel were transferred to three sandwiched substrate gels containing glycerol tributyrate, azo-carboxymethyl cellulose (Azo-CMC), and fibrin for detection of cellulase, lipase, and protease, respectively. We show that three cellulases (from a Paenibacillus sp. and two Bacillus sp. strains), one lipase (from a Staphylococcus sp.), and two proteases (from two Bacillus sp. strains) can be detected simultaneously with our zymogram system.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis as a method for studying the stability of subtilisin

A simple method has been developed to study the stability of subtilisin. Protein incubated at various temperatures in the presence of proteinase inhibitor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and showed a transition from the intact state to the unfolded state between 55 degrees C and 65 degrees C. Additionally, autolysis was also observed above 65 degrees C. In the absence of inhibitor, similar results were obtained below 55 degrees C; however, above 65 degrees C no protein of any size was observed due to extensive autolysis. These results demonstrate that SDS-PAGE can trap subtilisin in the state in which the protein existed prior to the analysis. It can be used to identify the different forms, including autolysis products, of the protein generated by heat denaturation. This method was used to study SDS-induced unfolding of aprA-subtilisin. When the protein was incubated with 0.25% SDS at different NaCl concentrations, a gradual increase in unfolding was observed with increasing NaCl concentration. This change paralleled a decrease in the critical micelle concentration of SDS, indicating that the rate of unfolding of aprA-subtilisin increases with increasing SDS micelle concentration. No detectable unfolding was observed below the critical micelle concentration.     

Sodium dodecyl sulfate (SDS)-based whole-mount in situ hybridization of Xenopus laevis embryos

We describe a streamlined whole-mount in situ hybridization protocol that utilizes high concentrations of the detergent sodium dodecyl sulfate (SDS). Our results suggest that SDS is an effective blocking agent in Xenopus laevis embryos which permeabilizes membranes without disrupting morphology. Consequently, riboprobes appeared to disperse uniformly within the embryo and several arduous and/or laborious steps of conventional procedures could be eliminated without compromising the technique.     

Sodium dodecyl sulfate stability of HLA-DR1 complexes correlates with burial of hydrophobic residues in pocket 1

Certain class II MHC-peptide complexes are resistant to SDS-induced dissociation. This property, which has been used as an in vivo as well as an in vitro peptide binding assay, is not understood at the molecular level. Here we have investigated the mechanistic basis of SDS stability of HLA-DR1 complexes by using a biosensor-based assay and SDS-PAGE with a combination of wild-type and mutant HLA-DR1 and variants of hemagglutinin peptide HA306-318. Experiments with wild-type DR1 along with previously published results establish that the SDS-stable complexes are formed only when the hydrophobic pocket 1 (P1) is occupied by a bulky aromatic (Trp, Phe, Tyr) or an aliphatic residue (Met, Ile, Val, Leu). To further explore whether the SDS sensitivity is primarily due to the exposed hydrophobic regions, we mutated residue beta Gly86 at the bottom of P1 to tyrosine, presumably reducing the depth of the pocket and the exposure of hydrophobic residues and increasing the contacts between subunits. In direct contrast to wild-type DR1, the peptide-free mutant DR1 exists as an alpha/beta heterodimer in SDS. Moreover, the presence of a smaller hydrophobic residue, such as alanine, as P1 anchor with no contribution from any other anchor is sufficient to enhance the SDS stability of the mutant complexes, demonstrating that the basis of SDS resistance may be localized to P1 interactions. The good correlation between SDS sensitivity and the exposure of hydrophobic residues provides a biochemical rationale for the use of this assay to investigate the maturation of class II molecules and the longevity of the complexes.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.     

Sodium dodecyl sulfate solution is an effective between-run rinse for capillary electrophoresis of samples in biological matrices

It is common practice in capillary electrophoresis to perform some sort of capillary washing step(s) between separations. In many analyses little consideration is given to optimization of the wash, and typically a rather standard washing procedure is used involving a few minutes wash with 0.1M NaOH followed by a few minutes reconditioning with the run buffer. As an alternative to this procedure, we have investigated the use of wash solutions containing sodium dodecyl sulfate (SDS). This type of wash has been used in the analyses of both small molecules and proteins, with encouraging results. After the SDS wash, the electroosmotic flow has been shown to be restored to values close to normal in a capillary which had previously been coated with plasma proteins. Separation efficiency for a test compound (dextromethorphan) is improved if an SDS rather than a HCl---NaOH wash is used after injection of plasma. In a direct-injection analysis of plasma proteins using a pH 10 borate buffer, an SDS-based washing procedure (total time, 1 min) gave better migration-time reproducibility than an NaOH-based wash, which took 5 min in total.     

Gas chromatographic determination of sodium dodecyl sulfate

A gas chromatographic method has been developed for the determination of sodium dodecyl sulfate. The method is sensitive, reasonably accurate, and uninfluenced by the presence of protein. The method depends upon the formation of 1-dodecanol and inorganic sulfate by acidic hydrolysis of sodium dodecyl sulfate (4 n HCl, 2 hr, 100°C). The ether extracted 1-dodecanol is analyzed by standard gas chromatographic techniques.     

Stability of aprA-subtilisin in sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.