Dodecyl Sulfate Complexes Research Articles

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: A study by oxygen quenching and fluorescence depolarization

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 °C as compared to that at 5 °C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC Complexes.

Urokinase binding to bovine corneal endothelial cells

Previously, we showed that the clotting factor thrombin binds to bovine corneal endothelial cells forming a covalent complex with a protein released by these cells into the culture media. We report that the plasminogen activator, urokinase, an enzyme involved in dissolution of blood clots, binds specifically to bovine corneal endothelial cells. [125I]-urokinase is rapidly bound by corneal endothelial cells. The serine active site of urokinase is required for binding since [125I]-urokinase inactivated with diisoprophylfluorophosphate does not bind to the cells. Pre-incubation of corneal cells with unlabeled urokinase for 20 hr results in increased release of binding sites for [125I]-urokinase into the culture media and at least a 3.5-fold increase in binding by the cells. [125I]-urokinase forms a 73 300 dalton sodium dodecyl sulfate complex with a corneal endothelial cell protein. Although thrombin competes with urokinase for binding to corneal endothelial cells, urokinase has at least a 10-fold higher affinity for binding to the cells. Furthermore, these cells make a plasminogen activator identical in molecular weight to urokinase. Thus, under physiological conditions, urokinase rather than thrombin binds to corneal endothelial cells. Binding may serve to regulate endogenous urokinase activity in the anterior chamber of the eye by limiting its extracellular proteolytic activity.

Kinetic studies of the quenching reactions of photoexcited ruthenium(II) sodium dodecyl sulfate complexes by dialkylviologens in sodium dodecyl sulfate micellar solution

Dynamique des reactions de desexcitation des etats photoexcites de Ru(phen) 3 2+ et Ru(dppphen) 3 2+ par C n V 2+ (n=1, 2, 3, 4 et 6 pour CH 3 , C 2 H 5 , C 3 H 7 , C 4 H 9 et C 6 H 13 ) en solution micellaire de SDS. Etude par photolyse laser eclair. C n V 2+ est totalement solubilise en phase micellaire. La reaction de desexcitation est un processus de transfert d'electron du complexe de Ru (II) excite a C n V 2+ sur la surface du micelle SDS

Helix formation upon acidification of protein-dodecyl sulfate complexes

The pH dependence of circular dichroism spectra has been studied for dodecyl sulfate complexes formed by 25 proteins and for a random copolypeptide of glutamic acid and alanine. The pH range covered is that in which titration of side-chain carboxyl groups is to be expected. Circular dichroism spectra signify an increase in helical content upon acidification, although in many cases the increase is quite small. For all but three of the proteins studied, the spectral changes are in reasonable agreement with those expected because helix propagation by glutamyl and aspartyl residues is enhanced when the state of the side-chain carboxyl changes from COO − to COOH. This simple explanation seriously underestimates conformational changes reported for gastrin, Kunitz trypsin inhibitor and tropomyosin. Changes in charge density appear to play an important role in these proteins.

Electrophoretic elution of proteins from polyacrylamide gel slices

A method for electrophoretic elution of proteins from polyacrylamide gel slices is described. Eluted proteins were retained by a discontinuous conductivity gradient (M. Otto and M. Snejdárková, Anal. Biochem. 111, 111–114 (1981)). The method has been adapted to slices from slab gels and gels that have been stained and destained. Proteins were eluted as their sodium dodecyl sulfate complexes. Minute amounts of proteins (0.1 μg) were recovered in high yield (85–95%) in 2 h in less than 0.1 ml volume.

Conformational change of protein sodium dodecylsulfate complexes in solution: A study of dynamic light scattering

The binding of an ionic detergent [sodium dodecyl sulfate (SDS)] to two proteins [bovine serum albumin (BSA) and ovalbumin (OA)] has been studied by dynamic light scattering to determine the changes of the hydrodynamic size and shape of the proteins as a function of binding. This study has been correlated to a previous viscosity study (Ref. 1) in which the increase in the effective hydrodynamic radius of the detergent–protein complex as the gram ratio of detergent to protein increases has been interpreted as the unfolding of the protein as a direct result of the disruption of the intramolecular hydrophobic associations in the proteins by the SDS molecules. At the maximum binding, the BSA–SDS complex exhibits a hydrodynamic radius of 72 Å while the effective size of the native BSA is 27 Å. The OA–SDS complex has an effective hydrodynamic radius of 64 Å at the maximum binding while that of native OA is 22 Å. The dynamic light scattering experiments allow us to detect the presence of two different size species in the solution, the SDS micelles and the unfolded proteins combined with SDS. However, the shape of the protein–SDS complex at maximum binding cannot be definitely determined from the present measurements alone.

Direct renaturation of the dodecyl sulfate complexes of proteins with triton X-100

A simple procedure is described for renaturing dodecyl sulfate-unfolded enzymes. The method involves the direct addition of a large molar excess of the non-ionic detergent Triton X-100 to protein-dodecyl sulfate complexes either in solution or as a band on a polyacrylamide gel. The cytoplasmic enzymes lactate dehydrogenase and malate dehydrogenase have been renatured by this protocol. On the other hand, no recovery of activity was found with the mitochondrial isoenzyme of malate dehydrogenase or the mitochondrial enzymes glutamate dehydrogenase or fumarase. Possible implications of the differences in the ability of cytosolic and mitochondrial enzymes to renature under these conditions are discussed in terms of their biosynthesis.

Somatostatin-detergent interaction

The effect of cationic, anionic and nonionic detergents on the EPR spectrum of spin-labeled somatostatin has been studied. At detergent concentrations well above the critical micelle concentration, nonionic detergents do not alter the EPR spectrum. Sodium dodecyl sulfate markedly alters both the line height ratio and the hyperfine splitting constant, whilst dodecyltrimethylammonium bromide alters only slightly the hyperfine splitting constant and line height ratio. The somatostatin-sodium dodecyl sulfate complex appeared monodisperse by sedimentation equilibrium with about 17 g bound detergent per g peptide. Circular dichroic and difference spectra of the dodecyl sulfate-somatostatin complex show that the tryptophanyl residue is buried in a nonpolar environment and that the secondary and tertiary structure of the peptide is markedly altered. Sedimentation equilibrium studies suggest that two types of dodecyltrimethylammonium-somatostatin complex exist. One type resembles the dodecyl sulfate-peptide complex, whilst the other appears to include several peptide units with only about one gram bound detergent per gram peptide.

Characterization of Porins from the Outer Membrane of Salmonella typhimurium 2. Physical Properties of the Functional Oligomeric Aggregates

We have purified to homogeneity, from mutant strains of Salmonella typhimurium, the small oligomers of porin that confer permeability channels to artificial vesicle membranes reconstituted from phospholipids and lipopolysaccharide. The molecular weights of the porin oligomers from the strains SH5551 and SH6017 appeared to be 130000 and 125000, respectively, and those of the monomers were 41000 and 37500, respectively, when determined by sedimentation equilibrium in the presence of dodecylsulfate. It was thus concluded that the functional porin oligomers consisted of three identical subunits. The Stokes' radius of the trimer . dodecylsulfate complex was around 5 nm. The trimer bound less dodecylsulfate than the monomer. The trimer . dodecylsulfate complex retained at room temperature the native conformation of porin, which is rich in beta-structure. When the trimers were dissociated further by various treatments, only the porin monomers were recovered in significant amounts, and the permeability-conferring activity was lost simultaneously. We propose, therefore, that the trimer is the minimal functional unit of porin that is capable of forming permeability channels in the outer membrane of Salmonella typhimurium.

The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose.

Equilibrium and kinetic aspects of the binding of several proteins to N-(3-carboxypropionyl)aminodecyl-Sepharose, an amphiphilic ampholytic adsorbent, were studied at 22 degrees C, pH 7.0, I 0.10--0.12. In the absence of detergents Scatchard plots are linear for human haemoglobin and soya-bean trypsin inhibitor, but non-linear for bovine serum albumin, which is also adsorbed more tightly than the other two proteins. The introduction [corrected] of 3.5mM-sodium dodecyl sulphate causes dramatic increases in the amounts and affinities of serum albumin and haemoglobin adsorbed, but has relatively little effect on the trypsin inhibitor. At concentrations of sodium dodecyl sulphate greater than about 10mM there is a fall in the binding of all proteins, owing to competition from the detergent for binding sites on the adsorbent, and a tendency towards more uniform behaviour by different proteins. Kinetic experiments suggest that in the absence of the detergent haemoglobin and serum albumin are adsorbed initially by mainly ionic forces, but that subsequently hydrophobic forces become dominant. Addition of 3.5 mM-sodium dodecyl sulphate causes pronounced changes in the time course of adsorption of haemoglobin and serum albumin, the nature of the changes being different for each protein. The significance of these results is discussed.

Anomalous behavior of the major avian myeloblastosis virus glycoprotein in the presence of sodium dodecyl sulfate

The sodium dodecyl sulfate (SDS) complex of the major glycoprotein of avian myeloblastosis virus exhibited an anomalously low free electrophoretic mobility compared with those of non-glycosylated protein standards. The apparent molecular weight of the glycoprotein calculated from the relation between log molecular weight and electrophoretic mobility depended on the acrylamide concentration and reached a lower limit of 80,000. The molecular weight was also estimated from the retardation coefficients of protein standards and the viral glycoprotein. This method yielded a molecular weight of 64,000 for the avian myeloblastosis virus glycoprotein. When gel chromatography in SDS was used to determine the apparent molecular weight of the glycoprotein from its hydrodynamic properties alone, the estimated value was 50,000. The generally assigned value of 80,000 daltons for the avian myeloblastosis virus major glycoprotein, as determined by SDS electrophoresis, may be an overestimate due to its relatively low free electrophoretic mobility and peculiar conformation in SDS.

The effects of diverse proteins on the solubilization of various hydrophobic probes by protein · detergent complexes

The solubilization behavior of various protein · detergent complexes with respect to a particular water-insoluble organic substance (“hydrophobic probe”) dimethylaminoazobenzene, was reported in earlier studies. The present report describes further the solubilization of other hydrophobic probes (e.g. Sudan II, naphthalene, anthracene and azobenzene) in various protein · sodium dodecyl sulfate complexes, in order to enlarge the scope of our understanding of these phenomena, which undoubtedly play a part in the transport of different water-insoluble organic substances in the living organisms. Solubilization by the various protein · SDS complexes is found to be specific for each probe. The amount of a particular probe solubilized is nearly always equal to the amounts which are solubilized by pure SDS micelles equivalent in amount to the SDS bound. Serious exceptions are found with two heme proteins (e.g. myoglobin and hemoglobin) and a few others. The hemeprotein · SDS complexes also exhibit regions of flat plateaus in the solubilization curves, whereas the binding equilibria show progressively larger amounts of SDS bound. The solubilization of probes by cationic detergent (cetylpyridinium chloride and cetyltrimethylammonium bromide) · protein complexes indicate that the solubilization phenomena are related to the environment of the binding sites (the cationic detergents are known to bind at different sites on the protein than the anionic detergents, i.e. SDS in the present case). With anionic detergents the effective chain length of the pseudo-micellar protein · detergent clusters is sufficient to cause an increase in solubilizing effectiveness of about 1.5 between the complexes and pure micelles. When small probes such as naphthalene are used such ratios are found. With larger probes the effectiveness ratio is reduced to 1.0 or even less as a result of steric interference with the formation of the protein · detergent · probe clusters. The solubilization energy exhibited by each protein · detergent complex is largely determined by the individual protein, and by the charge on the detergent.

The interaction of triton X-100 with soluble proteins: Possible implications for the transport of proteins across membranes

Dodecyl sulfate complexes of two soluble proteins, serum albumin and fumarase, have been “renatured” with large excesses of the nonionic detergent Triton X-100. The resulting complexes, essentially free of dodecyl sulfate, differ in their sedimentation properties relative to the native protein and in their interaction with Triton X-100. Albumin molecules refold to a form binding only very small amounts of Triton and have a sedimentation coefficient similar to that of the non-denatured protein. On the other hand, refolded fumarase molecules have a lower sedimentation coefficient than that of the native enzyme and bind up to 1.06 mg of Triton/mg protein. It is postulated that the fumarase molecule has been turned “inside-out” by the dodecyl sulfate/Triton treatment, and the implications of such large conformational changes for protein transport across membranes are discussed.

Differences in the solubilizing effectiveness of the sodium dodecyl sulfate complexes of various proteins.

The detailed reversible binding isotherms of sodium dodecyl sulfate (NaDodSO4) with 13 different initially native proteins are reported; the data were obtained at 20 degrees C and pH 7.1, ionic strength 0.033, with amounts bound with some proteins up to 1.1 g per g of protein. Although the isotherms of some of the proteins do not vary widely, extreme variations between certain classes are found. Thus, for example, hemoglobin and myoglobin both have high affinities and high binding capacities, while gamma-globulin, apoferritin, and transferrin have low initial affinities, and change drastically at higher concentrations. The protein-NaDodSO4 complexes solubilize the water-insoluble dye dimethylaminoazobenzene (DMAB) as effectively as micelles of pure NaDodSO4 when only small amounts (0.2 to 0.5 g/g of NaDodSO4) are bound. In most cases this effectiveness falls progressively as larger amounts are bound, and may even cease altogether at limits characteristic of the individual protein. With some of the latter, a second region of renewed solubilization occurs when substantially higher amounts of NaDodSO4 are present. In all cases, solubilization by ordinary micelles in normal amount occurs when the free NaDodSO4 concentration exceeds the critical micelle concentration, but the binding of NPADodSO4 to the protein also increases, in competition with formation of micelles. With some, but not all proteins the NaDodSO4 bound at concentrations above the cmc also solubilizes DMAB. In such cases the solubilizations by the protein-NaDodSO4 complexes and by the simple micelles are additive. The significance of the differences in binding and solubilizing encountered among these proteins is discussed in terms of surface structure, cooperativity of binding, and protein composition. No certain correlations with content of most amino acids, subunit structure, solubility, and hydrophobicity have been found, but there is a weak inverse dependence of solubilizing effectiveness on molecular size and indications of a strong dependence on content of cationic groups.

Conformational properties of the complexes formed by proteins and sodium dodecyl sulfate

Circular dichroism spectra have been obtained for albumin, alpha-chymotrypsinogen, collagen, concanavalin A, elastase, hemoglobin, histone f2b, alpha-lactalbumin, lactate dehydrogenase, beta-lactoglobulin, lysozyme, myoglobin, papain, ribonuclease A, and thermolysin in the presence of sodium dodecyl sulfate and dithiothreitol. While all spectra have the shape anticipated for a mixture of random coil and alpha helix, the intensities differ markedly ([theta]222 ranges from --1400 to --15 000 deg cm2/dmol). The variation in the circular dichroism can be quantitatively explained by a model which assumes that the arginyl, histidyl, and lysyl residues have an enhanced probability of propagating a helical segment in the presence of the detergent. The model also permits the computation of dimensional properties (unperturbed end-to-end distance and radius of gyration) for polypeptides of known amino acid sequence. Such computations have been performed for 67 proteins. The computed dimensions are compatible with experimental values and with the molecular weight dependence of the transport properties of the complexes. Furthermore, the model can account for the abnormal transport properties of the sodium dodecyl sulfate complexes formed by ribonuclease A, collagen fragments, and histones f2a1, f2a2, f2b, and f3. Even though some of the protein--sodium dodecyl sulfate complexes have helical contents as high as 50%, their overall conformation more closely approximates that of a random coil than a rod.

Fluorescent labeling of proteins in sodium dodecyl sulfate complexes with fluorescamine

The effects of sodium dodecyl sulfate on the fluorescent labeling of proteins were studied. Of 57 primary amine groups in bovine serum albumin, no more than 7 are titrable by fluorescamine. Fluorescamine labeling does not cause appreciable conformational changes of proteins. The extent of labeling of proteins decreases as the concentrations of sodium dodecyl sulfate increases. The fluorescence properties of labeled primary amine are only slightly affected by the polarities of the solvents. The inhibitory effects of sodium dodecyl sulfate upon labelings are interpreted as the low permeability of fluorescamine toward the highly charged envelopes of sodium dodecyl sulfate-protein micelles.

Properties of the low-temperature Photosystem I primary reaction in the P-700-chlorophyll a-protein

The Photosystem I primary reaction, as measured by electron paramagnetic resonance changes of P-700 and a bound iron-sulfur center, has been studied at 15°K in P-700-chlorophyll a-protein complexes isolated from a blue-green alga. One complex, prepared with sodium dodecyl sulfate shows P-700 photooxidation only at 300°K, whereas a second complex, prepared with Triton X-100, is photochemically active at 15°K as well as at 300°K. Analysis of these two preparations shows that the absence of low-temperature photoactivity in the sodium dodecyl sulfate complex reflects a lack of bound iron-sulfur centers in this preparation and supports the assignment of an iron-sulfur center as the primary electron acceptor of Photosystem I.

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.

Controlled pore glass chromatography of protein-sodium dodecyl sulphate complexes

The inclusion of urea has been found to eliminate adsorption of proteinsodium dodecyl sulphate (SDS) complexes to controlled pore glass. Using buffer containing 6 M urea, 0.5% SDS and glass with pore diameter 12.3 nm, it is possible to determine protein molecular weights in the range 3500–12,000. Results with glass of larger pore diameter (25.5 nm) are similar to those reported in the absence of urea in the molecular-weight range 12,000–140,000. Controlled pore glass chromatography also permits the study of the relative importance of conformation free of charge effects for those proteins which deviate from the normal calibration curve for SDS-polyacrylamide gels.

Effect of temperature and pH on the β–helix transition of poly(L‐lysine) in sodium dodecyl sulfate solution

AbstractThe conformational phase diagram of poly(L‐lysine) (4.6 × 10−4 M, residue) in sodium dodecyl sulfate (1.6 × 10−2 M) solution was constructed from circular dichroism results at various temperatures and pH's. Poly(L‐lysine)–sodium dodecyl sulfate complexes undergo a β–helix transition upon raising the pH of the solution. The transition pH tends to shift downward at elevated temperatures. No helix–β transition can be detected for poly(L‐lysine) in sodium dodecyl sulfate solution (pH > 11) even after 1‐hr heating at 70°C. This is in marked contrast with uncharged poly(L‐lysine) solution without sodium dodecyl sulfate, which is converted into the β‐form upon mild heating of the solution above 50°C.