Selective,reversible inhibition of the lactose phosphotransferase system of Staphylococcus aureus by dodecyl sulfate and deoxycholate

Low concentrations of sodium dodecyl sulfate (0.015%) and sodium deoxycholate (0.33%) completely inhibit phosphorylation of β-galactosides by the lactose phosphotransferase system of Staphylococcus aureus. Inhibition is reversible, even after prolonged detergent treatment. Phosphorylation of methyl-α-glucoside by the same preparations is only slightly inhibited by 0.015% dodecyl sulfate. The membrane-bound component, Enzyme IF^lac, is not solubilized by 0.015% dodecyl sulfate, nor is its ability to bind [¹⁴C]lactose affected. The results are consistent with hypotheses of selective binding of anionic detergent to Enzyme II^lac or to Factor III^lac, the detergent serving in the latter case as a membrane analog.     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

Charge separation of proteins complexed with sodium dodecyl sulfate by acid gel electrophoresis in the presence of cetyltrimethylammonium bromide.

Globular proteins, casein, and membrane proteins which were reacted with sodium dodecyl sulfate were studied by acid urea gel electrophoresis. The sodium dodecyl sulfate bound tightly to the proteins, producing a more acidic charge which prevented migration into the gel. When cetyltrimethylammonium bromide was added to the sodium dodecyl sulfate-protein complexes, the sodium dodecyl sulfate apparently reacted with cetyltrimethylammonium bromide and dissociated so that the proteins migrated in acid gel in a normal manner as compared to the proteins without any added detergent. The sodium dodecyl sulfate-cetyltrimethylammonium bromide complex could be removed from the proteins by centrifugation. Thus, cetyltrimethylammonium bromide used in conjunction with acid gel electrophoresis allows direct comparison by charge of proteins fractionated in the presence of sodium dodecyl sulfate with the starting mixture of proteins not exposed to detergent. The reaction of cetyltrimethylammonium bromide with sodium dodecyl sulfate in acidic urea also provides a simple convenient method of removal of sodium dodecyl sulfate from proteins.     

Effect of the composition of sodium dodecyl sulfate preparations on the renaturation of enzymes after polyacrylamide gel electrophoresis

The extent of renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate depended on the source of the detergent. Analysis of commercial preparations of sodium dodecyl sulfate revealed appreciable amounts of tetradecyl and hexadecyl sulfates in some preparations. Inhibition of renaturation was correlated with the amount of hexadecyl sulfate and, to a much lesser extent, of tetradecyl sulfate present. The higher alkyl sulfates appeared to bind more tenaciously to proteins in the gel. More extensive washing was required to remove them than to remove dodecyl sulfate, and they were inhibitory to enzyme activity at lower detergent concentrations. A system is described for gas chromatographic analysis of alkyl sulfates containing chains of 10 to 16 carbon atoms in length.     

Proteome of the human HaCaT keratinocytes: Identification of the oxidative stress proteins after sodium dodecyl sulpfate exposur

Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 μg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.     

Characterization of the apolipoprotein B polypeptide of human plasma low density lipoprotein in detergent and denaturation solutions.

Apolipoprotein B, the polypeptide moiety of human serum low density lipoprotein, is subject to degradation (as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) both in the intact particle and after delipidation. Protease inhibitors, sodium azide, and nitrogen saturation did not influence the rate or degree of degradation. Lipid-free apolipoprotein B prepared by gel exclusion chromatography in sodium dodecyl sulfate bound a limited number of detergent molecules (up to 300) in monomeric sodium dodecyl sulfate solutions; circular dichroic spectra of this complex were similar to spectra of the intact lipoprotein. Near the critical micelle concentrations, a large, cooperative increase in detergent binding occurred, accompanied by circular dichroic changes indicating increased alpha helicity. By sucrose density centrifugation, lysopalmitoyl phosphatidylcholine could be substituted for the anionic detergent; about 300 mol of lysolipid were bound to the polypeptide. Replacement of detergent with guanidine hydrochloride by dialysis produced a soluble polypeptide with no ordered structure at denaturant concentrations above 7 M. At lower guanidine hydrochloride concentrations, structural elements were regained in a broad, reversible transition. It appears that apolipoprotein B is an easily degraded polypeptide with regions resembling water-soluble proteins but other regions which interact with lipid (or synthetic amphiphiles) and produce an overall insolubility in aqueous solution in the absence of amphiphilic ligands.     

A calorimetric comparison of the interaction of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins.

The interactions of sodium dodecyl sulfate with cytochrome c and erythrocyte glycoproteins have been studied by the method of titration calorimetry. It was found that the initial addition of sodium dodecyl sulfate to cytochrome c caused an endothermic unfolding of the protein, detectable by circular dichroism (CD). This was followed by the exothermic binding of sodium dodecyl sulfate to the protein, without further CD-detectable conformational changes. In contrast, sodium dodecyl sulfate bound directly to the erythrocyte glycoproteins in an exothermic reaction without any accompanying CD-detectable conformation changes. This indicates that the glycoproteins solubilized in aqueous media have exposed hydrophobic regions which can interact directly with this detergent. The enthalpy changes and stoichiometries of binding are reported.     

Amyloidogenic Propensity of a Natural Variant of Human Apolipoprotein A-I: Stability and Interaction with Ligands

A number of naturally occurring mutations of human apolipoprotein A-I (apoA-I) have been associated with hereditary amyloidoses. The molecular mechanisms involved in amyloid-associated pathology remain largely unknown. Here we examined the effects of the Arg173Pro point mutation in apoA-I on the structure, stability, and aggregation propensity, as well as on the ability to bind to putative ligands. Our results indicate that the mutation induces a drastic loss of stability, and a lower efficiency to bind to phospholipid vesicles at physiological pH, which could determine the observed higher tendency to aggregate as pro-amyloidogenic complexes. Incubation under acidic conditions does not seem to induce significant desestabilization or aggregation tendency, neither does it contribute to the binding of the mutant to sodium dodecyl sulfate. While the binding to this detergent is higher for the mutant as compared to wt apoA-I, the interaction of the Arg173Pro variant with heparin depends on pH, being lower at pH 5.0 and higher than wt under physiological pH conditions. We suggest that binding to ligands as heparin or other glycosaminoglycans could be key events tuning the fine details of the interaction of apoA-I variants with the micro-environment, and probably eliciting the toxicity of these variants in hereditary amyloidoses.     

Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22

The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.     

Comparative studies on superoxide anion production by polymorphonuclear leukocytes stimulated with various agents

Guinea-pig peritoneal polymorphonuclear leukocytes promoted superoxide anion (O2(-)) generation when stimulated with soluble antigen-antibody complex, concanavalin A or sodium dodecyl sulfate. The enhancement with antigen-antibody complex or concanavalin A was inhibited with diisopropyl fluorophosphate. On the other hand, the enhancement with sodium dodecyl sulfate was not affected by the inhibitor. L-1-pTosylamido-2-phenylethyl chloromethyl ketone (Tos-PheCH2Cl) and tetrahydrofuran also enhanced O2(-) generation even in the presence of diisopropyl fluorophosphate, while at low concentrations they inhibited O2(-) generation with antigen-antibody complex. These results indicate that a certain diisopropyl fluorophosphate-sensitive factor may be involved in the O2(-)-generating response of leukocytes to antigen-antibody complexes or concanavalin A, but not in that to sodium dodecyl sulfate, Tos-PheCH2Cl or tetrahydrofuran.     

Lectin binding and enzymatic deglycosylation of the cGMP-gated channel from bovine rod photoreceptors

In order to investigate the lectin-binding properties of the photoreceptor cGMP-gated channel, solubilized and purified channel protein was incubated with immobilized lectins followed by reconstitution of unbound proteins. Of the lectins tested, only concanavalin A (ConA) was able to specifically sediment channel activity. A 240-kDa protein, which copurifies with the 63-kDa channel protein but does not bind ConA, was also found to be sedimented by the ConA-affinity matrix, thereby implicating that it is associated with the channel complex. Treatment of the purified channel protein with the enzyme glycopeptidase F in the presence of the denaturing detergent sodium dodecyl sulfate resulted in a rapid reduction of the apparent molecular mass by 1.90 kDa, and the abolition of ConA-binding. No intermediate molecular weight species were observed, suggesting that the channel protein is N-glycosylated at one site only. Under nondenaturing conditions, the kinetics of deglycosylation were distinctly two-phased: 50-60% deglycosylation was achieved rapidly; however, prolonged incubation was required to arrive at complete deglycosylation. Reconstitution experiments showed that deglycosylation had no significant effect on the kinetics of channel protein activation by cGMP.     

Cell aggregation of Pseudomonas aeruginosa strain PAO1 as an energy-dependent stress response during growth with sodium dodecyl sulfate

Pseudomonas aeruginosa strain PAO1 grew with the detergent sodium dodecyl sulfate (SDS). The growth started with the formation of macroscopic cell aggregates which consisted of respiring cells embedded in an extracellular matrix composed of acidic polysaccharides and DNA. Damaged and uncultivable cells accumulated in these aggregates compared to those cells that remained suspended. We investigated the response of suspended cells to SDS under different conditions. At high energy supply, the cells responded with a decrease in optical density and in viable counts, release of protein and DNA, and formation of macroscopic aggregates. This response was not observed if the energy supply was reduced by inhibiting respiration with KCN, or if cells not induced for SDS degradation were exposed to SDS. Exposure to SDS caused cell lysis without aggregation if cells were completely deprived of energy, either by applying anoxic conditions, by addition of CCCP, or by addition of KCN to a mutant defective in cyanide-insensitive respiration. Aggregated cells showed a more than 100-fold higher survival rate after exposure to SDS plus CCCP than suspended cells. Our results demonstrate that cell aggregation is an energy-dependent response of P. aeruginosa to detergent stress which might serve as a survival strategy during growth with SDS.     

The synthesis of a photolabile detergent and its use in the isolation and characterization of protein

A new ionic detergent sodium 4-(3,3-dimethyl-1-oxotridecyl)benzenesulfonate similar to sodium dodecyl sulfate has been synthesized which is photodegradable to sodium p-acetyl-benzenesulfonate and a simple olefin mixture upon irradiation in aqueous solution with light 300 nm and above in wavelength. This photodegradable detergent can be used to solubilize many proteins and provide information as to the molecular weight and subunit composition of proteins by electrophoresis. The removal of this detergent by photolysis results in no apparent damage to protein.     

NADH: (acceptor) oxidoreductase from bovine adrenal medulla chromaffin granules

The NADH:(acceptor) oxidoreductase from membranes of bovine adrenal medulla chromaffin granules has been purified by column chromatography. After solubilization of the membranes with emulphogen, a nonionic detergent, the enzyme was purified by dye-ligand chromatography and gel filtration. The oxidoreductase appeared essentially homogeneous on two gel electrophoretic systems. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme revealed a dimeric structure with a combined molecular weight of about 55,000. The enzyme eluted as a detergent-lipid-protein aggregate with a Stoke's radius of 43 Å on gel filtration columns in the presence of emulphogen. The amino acid composition of the oxidoreductase was found to be distinct from that of similar enzymes from other organelles. Topographical experiments indicated that the enzyme is a transmembrane protein.     

Over two hundred polypeptides resolved from the human erythrocyte membrane

A modification of O'Farrell's method of two-dimensional polyacrylamide gel electrophoresis has allowed for the resolution of erythrocyte membranes showing up to 200 individual components. Data is presented which indicates that this protein heterogeneity is not produced by artifactual protein-protein aggregation, ednogenous protease activity of secondary charge modification. Similar patterns are obtained when the samples are added to the unpolymerized isoelectric focusing gel, and isolated and stored in protease inhibitor. Individual spots could be eluted off of stained gels, resolubilized under extreme detergent solubilization conditions and run on one-dimensional gels; these run as sodium dodecyl sulfate in the solubilization procedure. The method chosen for solubilization prior to isoelectric focusing appears to cause selective aggregation of all or most of the spectrin and band 3 proteins. This further allows for excellent resolution of more components.     

Lysolecithin--a potent activator of prophenoloxidase from the hemolymph of the lobster, Homarus americanas

The phenoloxidase system, which is involved in encapsulation and melanization of foreign objects in crustacean, is found to be present in an inactive proenzyme form in the hemocytes of the lobster, Homarus americanas. Activation of the enzyme could be achieved either by treatment with an anionic detergent such as sodium dodecyl sulfate, or by a cationic detergent such as cetylpyridinium chloride, but not by either nonionic detergent or zwitterionic detergent. In addition, a number of fatty acids also activated the proenzyme. However, phospholipids, especially lysolecithin proved to be the most potent activator of prophenoloxidase. Therefore, it is proposed that apart from the well established proteolytic mode of activation, prophenoloxidase can also be activated by this alternative mode involving lipids.     

Prevention of aggregation of synthetic membrane-spanning peptides by addition of detergent

In our initial attempts to solubilize and purify a chemically synthesized 22-amino acid, membrane-spanning peptide, we encountered numerous difficulties. The peptide was not soluble in dilute acids, organic solvents, or chaotropic agents (+/- detergent) following standard HF cleavage protocols. The insolubility was a direct result of the formation of peptide-(peptide)n aggregates that occurred during the initial phase of isolation, i.e., during the HF cleavage. Eliminating the ether precipitation and subsequent washes did not decrease the degree of aggregation of the product. Inclusion of nonionic detergents in the HF-cleavage reactions displayed little ability in preventing aggregation. Cleavage in the presence of sodium dodecyl sulfate, however, dramatically reduced the degree of aggregation, even after washing with organic solvents. The cleaved peptide was purified to homogeneity using a detergent-based HPLC protocol. This column procedure also permits the quantitative exchange of the sodium dodecyl sulfate for n-octyl-beta-D-glucopyranoside. Combined use of the two protocols results in high-yield isolations for a class of peptides that is generally difficult to handle.     

Anomalies in the electrophoretic resolution of Na+/K(+)-ATPase catalytic subunit isoforms reveal unusual protein--detergent interactions

Three different isozymes of the Na+/K(+)-ATPase have slightly different different electrophoretic mobilities in sodium dodecyl sulfate (SDS). Certain procedures (reduction and alkylation, heating, and the use of sodium tetradecyl sulfate) have been reported either to improve the electrophoretic separation of isoforms or to reveal the presence of new isoforms. The variables affecting gel electrophoretic mobility were investigated here. Reduction and alkylation decreased the mobility of all three isozymes, and slightly improved the separation of alpha 1 from alpha 2 and alpha 3 without causing a qualitative change in the alpha isoforms detected. Heating the enzyme in SDS caused splitting into two bands. Both bands were intact polypeptides but migrated differently in 5% and 15% polyacrylamide, disclosing an anomalous conformation in detergent. The use of sodium tetradecyl or decyl sulfate instead of dodecyl sulfate altered the relative mobilities of the isozymes, revealing differences in detergent affinity, but no new isoforms were found. In conclusion, Na+/K(+)-ATPase alpha-subunit mobility reflects complex detergent-protein interaction that can be affected by experimental conditions. The existence of more than one band on gels may reflect different conformations in detergent, but should not be accepted alone as evidence for subunit structural heterogeneity.     

Structure of the Staphylococcus aureus cell wall determined by the freeze-substitution method.

The fine structure of the Staphylococcus aureus cell wall was determined by electron microscopy with the new technique of rapid freezing and substitution fixation. The surface of the cell wall was covered with a fuzzy coat which consisted of fine fibers or an electron-dense mass. Morphological examination of the cell wall, which was treated sequentially with sodium dodecyl sulfate, trypsin, and trichloroacetic acid, revealed that this coat was partially removed by trypsin digestion and was completely removed by trichloroacetic acid extraction but was not affected by sodium dodecyl sulfate treatment, suggesting that the fuzzy coat consists mostly of a complex of teichoic acids and proteins. This was confirmed by the application of the concanavalin A-ferritin technique for teichoic acid and antiferritin immunoglobulin G technique for protein A.     

Use of a zwitterionic detergent for the restoration of the antibody binding capacity of immunoblotted Francisella tularensis lipopolysaccharide.

A method for the partial restoration of the antibody binding capacity of Francisella tularensis lipopolysaccharide (LPS) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS) is described. The method relies on the presence of a zwitterionic detergent in the matrix of an SDS-polyacrylamide gel and in the transfer buffer during an immunoblot. F. tularensis LPS, which had lost its earlier capacity to bind to a particular monoclonal antibody in the normal blot procedure, did bind following the addition of the zwitterionic detergent to the polyacrylamide gel and transfer buffer. A number of detergents were tested but most success in restoring antibody binding was achieved with Zwittergent 3-08. This simple modification to the immunoblot procedure proved helpful in identifying a monoclonal antibody specific to hot phenol-extracted F. tularensis LPS.