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.     

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.     

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.     

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.     

Use of nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.     

Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp

Significant differences in cysteine-containing proteins and detergent-related solubility properties were observed between outer membrane protein complexes of reproductive (reticulate body) and infective (elementary body) forms of Chlamydia psittaci (6BC). Elementary bodies harvested at 48 h postinfection possessed a 40-kilodalton major outer membrane protein and three extraordinarily cysteine-rich outer membrane proteins of 62, 59, and 12 kilodaltons, all of which were not solubilized by sodium dodecyl sulfate in the absence of thiol reagents. Intracellularly dividing reticulate bodies harvested at 21 h postinfection were severely deficient in the cysteine-rich proteins but possessed almost as much major outer membrane protein as did the elementary bodies. Most of the major outer membrane protein of reticulate bodies was solubilized by sodium dodecyl sulfate and was present in envelopes as monomers, although a proportion formed disulfide-cross-linked oligomers. By 21 to 24 h postinfection, reticulate bodies commenced synthesis of the cysteine-rich proteins which were found in outer membranes as disulfide-cross-linked complexes. The outer membranes of reticulate bodies of Chlamydia trachomatis (LGV434) also were found to be deficient in cysteine-rich proteins and to be more susceptible to dissociation in sodium dodecyl sulfate than were outer membranes of elementary bodies.     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

Isolation of coconut storage proteins by polyacrylamide-gel electrophoresis

By combining sodium dodecyl sulfate (SDS)-gel electrophoresis with a new chilling technique for visualization of protein-SDS complexes in polyacrylamide gels, a process has been developed which will permit the isolation of milligram quantities of pure polypeptides. Using this technique, we have isolated two molecular weight classes of polypeptides from coconut storage globulins and determined the amino acid composition of each. When the two amino acid compositions were summed on a molar basis, the result agreed reasonably well with the amino acid composition of the starting material with the exception of cystine. Apparently, some contaminant from the polyacrylamide caused its destruction to be accelerated during hydrolysis.     

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.     

Subunit analysis of bovine cytochrome c oxidase by reverse phase high performance liquid chromatography

Bovine cytochrome c oxidase subunits were separated by reverse phase high performance liquid chromatography using a C4 column eluted with water and an acetonitrile gradient, both containing 0.1% trifluoroacetic acid. Subunits I and III precipitated in this solvent and could not be analyzed; the remaining eleven subunits were dissociated, denatured, soluble and could be resolved by elution from the column. The protein subunit eluting in each chromatographic peak was identified by a combination of polyacrylamide gel electrophoresis in sodium dodecyl sulfate, NH2-terminal amino acid sequencing, and amino acid analysis. Each subunit produced a single elution peak with the exception of subunit VIc (nomenclature of Kadenbach et al., 1983, Anal. Biochem. 129, 517-521), which eluted from the column as two well-resolved peaks. Sequence analysis showed that the two subunit VIc elution peaks resulted from partial chemical blockage of the alpha-amino serine residue of subunit VIc. The C4 reverse phase HPLC was used to document specific subunit removal from bovine cytochrome c oxidase either by tryptic digestion or by dodecyl maltoside extraction. The described HPLC method for separating cytochrome c oxidase subunits should be applicable for the analysis of other multisubunit proteins, especially other multisubunit membrane protein complexes.     

Protein complexes in bacterial and yeast mitochondrial membranes differ in their sensitivity towards dissociation by SDS

Previously, a 2D gel electrophoresis approach was developed for the Escherichia coli inner membrane, which detects membrane protein complexes that are stable in sodium dodecyl sulfate (SDS) at room temperature, and dissociate under the influence of trifluoroethanol [R. E. Spelbrink et al., J. Biol. Chem. 280 (2005), 28742-8]. Here, the method was applied to the evolutionarily related mitochondrial inner membrane that was isolated from the yeast Saccharomyces cerevisiae. Surprisingly, only very few proteins were found to be dissociated by trifluoroethanol of which Lpd1p, a component of multiple protein complexes localized in the mitochondrial matrix, is the most prominent. Usage of either milder or more stringent conditions did not yield any additional proteins that were released by fluorinated alcohols. This strongly suggests that membrane protein complexes in yeast are less stable in SDS solution than their E. coli counterparts, which might be due to the overall reduced hydrophobicity of mitochondrial transmembrane proteins.     

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.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.     

A comparison of the dodecyl sulfate-induced precipitation of the myelin basic protein with other water-soluble proteins

The interactions of sodium dodecyl sulfate with a number of proteins were examined at a variety of pH values ranging from 4.8 to 11.6 The dodecyl sulfate-induced precipitation of some of these proteins was observed within a relatively limited range of total dodecyl sulfate concentration. Most of the basic proteins precipitated at low pH but as the isoelectric point of the protein was approached the amount of protein that precipitated decreased. Bovine myelin basic protein was unique in that it precipitated at all pH values examined both above and below its isoelectric point. Thus, the dodecyl sulfate-induced precipitation of myelin basic protein appears to be different from the dodecyl sulfate-induced precipitation of most proteins. A comparison of protein precipitation at equivalent dodecyl sulfate: protein molar or weight ratios revealed very little difference in the precipitation behavior of the proteins studied. When the bovine myelin basic protein was cleaved at its single tryptophan residue, the N-terminal fragment (1–115) formed insoluble dodecyl sulfate complexes at pH values ranging from 4.8 to 9.2. The C-terminal fragment (116–169) precipitated almost completely at pH 4.8 but to a lesser extent at pH 7.4 and 9.2 Equimolar mixtures of the N- and C-terminal fragments precipitated in the presence of dodecyl sulfate at pH 7.4 and 9.2 to an extent greater than the C-terminal fragment alone but comparable to the N-terminal fragment alone or the whole basic protein. These results suggest: (a) that the mechanism by which dodecyl sulfate induces the precipitation of myelin basic protein may be unique compared to other proteins and (b) that the intact myelin basic protein is not necessary for its precipitation by dodecyl sulfate.     

Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.

A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

The action of proteolytic enzymes on chloroplast thylakoid membranes

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analyzed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.     

Arrangement of proteins O-8 and O-9 in outer membrane of Escherichia coli K-12. Existence of homotrimers and heterotrimers.

1. The molecular arrangement of major outer membrane proteins O-8 and O-9 that exist as trimers has been studied by means of cross-linking with dimethylsuberimidate. 2. The cross-linked samples were examined on a urea/sodium dodecyl sulfate/polyacrylamide gel which was developed to separate cross-linked trimer and dimer of O-8 from those of O-9. 3. Cells simultaneously synthesizing both O-8 and O-9 formed heterotrimers (trimers containing both proteins) as well as homotrimers. 4. Quantitative analyses revealed that there was no discrimination between O-8 and O-9 in the assembly process to form trimers. 5. When cells were grown sequentially under two different sets of conditions so that the cells synthesized either one of the two proteins in the first stage and the other in the second stage of growth, no heterotrimers were formed. This result indicates that subunit exchange did not take place between trimers which had been incorporated into the outer membrane.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by the Ionic Detergent Sodium-Lauryl Sarcosinate

The sensitivity of the outer and cytoplasmic membranes of Escherichia coli to detergent was examined by isopycnic sucrose density gradient centrifugation. Sodium lauryl sarcosinate (Sarkosyl) was found to disrupt the cytoplasmic membrane selectively under conditions in which Triton X-100 and dodecyl sodium sulfate solubilized all membrane protein. These results were verified by gel electrophoresis; membrane proteins solubilized by Sarkosyl were identical to those of the cytoplasmic membrane. The presence of Mg(2+) during treatment with Sarkosyl was found to afford partial protection of the cytoplasmic membrane from dissolution.     

Isolation of outer membrane proteins of Escherichia coli and their characterization on polyacrylamide gel

Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.