Multiphasic kinetics of myoglobin/sodium dodecyl sulfate complex formation

We have carried out a kinetic analysis of the conformational changes that myoglobin (Mb) undergoes in the presence of the anionic surfactant sodium dodecyl sulfate (SDS). The time-resolved results have been combined with steady-state circular dichroism (CD) and resonance Raman (RR) spectroscopy. Time-resolved absorption spectra indicate that SDS induces changes in the heme coordination with the formation of three different Mb species, depending on SDS concentration. The formation of the Mb/SDS complex involves three or four phases, depending on surfactant concentration. The kinetic data are analyzed assuming two modes of interaction according to whether SDS is monomeric or micellar. The two pathways are separated but interconnected through free Mb. At the lowest concentrations a six-coordinated, low-spin form dominates. Two distinct five-coordinated species are formed at higher SDS concentrations: one is a protein-free heme and the other reequilibrates slowly with the six-coordinated, low-spin form. The resulting complexes have been characterized by CD and RR. In addition, CD spectra show that the local changes in the heme environment are coupled to changes in the protein structure.     

Inactivation of prions by acidic sodium dodecyl sulfate

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

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.     

Changes in tyrosinase specificity by ionic liquids and sodium dodecyl sulfate

Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. The ability of tyrosinases to convert monophenols into diphenols has stimulated studies regarding the production of substituted catechols, important intermediates for the synthesis of pharmaceuticals, agrochemicals, polymerization inhibitors, and antioxidants. Despite its enormous potential, the use of tyrosinases for catechol synthesis has been limited due to the low monophenolase/diphenolase activity ratio. In the presence of two water miscible ionic liquids, [BMIM][BF₄] and ethylammonium nitrate, the selectivity of a tyrosinase from Bacillus megaterium (TyrBm) was altered, and the ratio of monophenolase/diphenolase activity increased by up to 5-fold. Furthermore, the addition of sodium dodecyl sulphate (SDS) at levels of 2–50 mM increased the activity of TyrBm by 2-fold towards the natural substrates l-tyrosine and l-Dopa and 15- to 20-fold towards the non-native phenol and catechol. The R209H tyrosinase variant we previously identified as having a preferential ratio of monophenolase/diphenolase activity was shown to have a 45-fold increase in activity towards phenol in the presence of SDS. We propose that the effect of SDS on the ability of tyrosinase to convert non-natural substrates is due to the interaction of surfactant molecules with residues located at the entrance to the active site, as visualized by the newly determined crystal structure of TyrBm in the presence of SDS. The effect of SDS on R209 may enable less polar substrates such as phenol and catechol, to penetrate more efficiently into the enzyme catalytic pocket.     

Effect of sodium dodecyl sulfate on polar lobe formation and function in Ilyanassa obsoleta embryos

Polar lobes, anucleate vegetal pole protrusions formed by Ilyanassa obsoleta embryos, serve as a mechanism for shunting morphogenetic determinants to one cell during the first two cleavages. Polar lobe material becomes segregated in the CD cell during first cleavage and in the D cell during second cleavage, resulting in a very unequal four-cell stage. Larval structures including external shell, foot, operculum, statocysts, and eyes develop only when polar lobe material is present. Treatment with the anionic detergent sodium dodecyl sulfate (SDS) before and during the first cleavage inhibited polar lobe formation and equalized cleavage, as the lobe material was distributed to two cells. No polar lobes formed during second clevage in SDS-equalized embryos, and the four-cell stage consisted of four equal cells with reduced cell contacts. SDS inrreversibly inhibited polar lobe formation without affecting cytokinesis. Although 27% of the larvae from SDS-equalized embryos had one or more lobe-dependent structures duplicated, morphogenesis was impaired: more than 40% of such larvae failed to form shell and/or statocysts. When cells were separated after equalized first cleavage and raised as pairs, the pairs of resulting larvae duplicated lobe-dependent structures with the same frequency as whole equalized embryos. Possible explanations for impaired morphogenesis in SDS-treated embryos are discussed.     

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.     

Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Activation of tobacco leaf polyphenol oxidase by sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10 degrees C, suggesting that stabilization of the structure had been improved by the formation of complex.     

Circular dichroism studies on helical structure preferences of amino acid residues of proteins caused by sodium dodecyl sulfate

The extent of helical structure of 19 intact proteins and of 15 proteins with no disulfide bridges in the absence and presence of 10 mM sodium dodecyl sulfate (SDS) was determined using the curve-fitting method of circular dichroic spectra. The change in helicity caused by the addition of SDS was examined as a function of each amino acid fraction. An increase in the helicity upon the addition of SDS occurred in most of the proteins with no disulfide bridges (C proteins) and containing more than 0.06 Lys fraction. In most of the intact proteins (B proteins), most of which contained disulfide bridges, helicity in SDS decreased with an increase in Lys fraction. The helicity of the C proteins in SDS also tended to increase with an increase in the Leu and Phe fractions, while it decreased with an increase in the Gly fraction. For the helicity of the B proteins in SDS, there was a tendency to increase with increased Asn fraction and decrease with increased His fraction. On the other hand, amino acids were divided into eight groups according to their side-chain properties and the conformational preference for each of the amino acid groups of C proteins was calculated using a simple assumption.     

High-speed gel filtration of polypeptides in sodium dodecyl sulfate

The separation of polypeptides treated with SDS was studied using G3000SW packing prepared from silica for high-speed gel filtration. The peaks of ovalbumin, chymotrypsinogen A, cytochrome c, aprotinin, and insulin B chain were completely separated in the presence of 0.1% SDS and 0.05 M sodium phosphate buffer (pH 7.0). A plot of the logarithm of molecular weight of polypeptides versus Kd was linear over a molecular weight range of 3,000 to 50,000 at the above concentrations of SDS and sodium phosphate. The slopes of the plots of log molecular weight versus Kd depend to a significant extent on the concentration of the sodium phosphate buffer (pH 7.0).