Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

Glycoprotein-surfactant interactions: a calorimetric and spectroscopic investigation of the phytase-SDS system

The interactions of sodium dodecyl sulfate (SDS) and two glyco-variants of the enzyme phytase from Peniophora lycii were investigated. One variant (Phy) was heavily glycosylated while the other (dgPhy) was enzymatically deglycosylated. Effects at 24 degrees C of titrating SDS to Phy and dgPhy were studied by Isothermal Titration Calorimetry (ITC) and Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. Comparisons of results for the two variants were used to elucidate glycan-surfactant interrelationships. The CD spectra suggested that both the native and the SDS-denatured states of the two variants were mutually similar, and hence that the denaturation process was structurally equivalent for the two glyco-variants. The denatured state was far from fully unfolded and probably retained a substantial content of native-like structure. Furthermore, it was found that the glycans brought about only a small increase in the resistance towards SDS induced denaturation. The SDS concentration required to denature half of the protein molecules differed less than 1 mM for the two variants. The affinity for SDS of both variants was unusually low. The amount of bound SDS (w/w) at different stages of the binding isotherm was 3-10 times lower than that reported for the most previously investigated globular proteins. Analysis of the relative affinity of the glycan and peptide moieties suggested that the carbohydrates bind much less surfactant. At saturation, glycans adsorbed about half as much SDS (in g/g) as the peptide moiety of Phy and about five times less than average proteins.     

Influence of liposome charge and composition on their interaction with human blood serum proteins

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [³H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80–95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (K_d) and capacity (B_max). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [³H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [³H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [³H]CP-55,940 binding to the cannabinoid receptor.     

Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.     

Effect of salt concentration of buffer on the binding of sodium dodecyl sulfate and on the viscosity behavior of the protein polypeptide derived from bovine serum albumin in the presence of the surfactant

The complex between SDS and a protein polypeptide derived from bovine serum albumin was characterized with respect to binding of SDS and viscosity behavior. The amount of bound SDS increased from 1.0 to 2.2 g/g with increase of the buffer concentration from 10 to 220 mM. A logarithmic plot of the amount of bound SDS against the buffer concentration gave a linear relation like in the plot where the number of SDS molecules constituting a spherical micelle of SDS is plotted similarly. The increase in the buffer concentration up to 25 mM, from 25 to 100 mM and beyond 100 mM, was accompanied by a sharp rise, monotonic decrease and levelling-off of the intrinsic viscosity in the respective region. In the region 45-175 mM, a linear relation was found between the intrinsic viscosity and reciprocal square root of the buffer concentration. The observed changes can be interpreted as follows: (1), the electrostatic repulsion between charges introduced by the bound SDS caused the initial increase; (2), shielding of the charges as the result of ion condensation with further increase in ionic strength caused the viscosity drop and subsequent levelling-off. The characteristics of the plots are consistent with the necklace model proposed previously for such complexes in which SDS is bound to a protein polypeptide forming micelle-like clusters and which behave like a flexible polyelectrolyte (Shirahama, K., Tsujii, K. and Takagi, T. (1974) J. Biochem. 75, 309-319).     

pH and ionic strength dependence of protein (un)folding and ligand binding to bovine beta-lactoglobulins A and B

Formation of complexes between bovine beta-lactoglobulins (BLG) and long-chain fatty acids (FAs), effect of complex formation on protein stability, and effects of pH and ionic strength on both complex formation and protein stability were investigated as a function of pH and ionic strength by electrophoretic techniques and NMR spectroscopy. The stability of BLG against unfolding is sharply affected by the pH of the medium: both A and B BLG variants are maximally stabilized against urea denaturation at acidic pH and against SDS denaturation at alkaline pH. The complexes of BLGB with oleic (OA) and palmitic acid (PA) appear more stable than the apoprotein at neutral pH whereas no differential behavior is observed in acidic and alkaline media. PA forms with BLG more stable complexes than OA. The difference between the denaturant concentration able to bring about protein unfolding in the holo versus the apo forms is larger for urea than for SDS treatment. This evidence disfavors the hypothesis of strong hydrophobic interactions being involved in complex formation. Conversely, a significant contribution to FA binding by ionic interactions is demonstrated by the effect of pH and of chloride ion concentration on the stoichiometry of FA.BLG complexes. At neutral pH in a low ionic strength buffer, one molecule of FA is bound per BLG monomer; this ratio decreases to ca. 0.5 per monomer in the presence of 200 mM NaCl. The polar heads of bound FA appear to be solvent accessible, and carboxyl resonances exhibit an NMR titration curve with an apparent pK(a) of 4.7(1).     

A multitechnique approach in protein/surfactant interaction study: physicochemical aspects of sodium dodecyl sulfate in the presence of trypsin in aqueous medium

Interaction of sodium dodecyl sulfate (SDS) with a globular protein, trypsin, has been physicochemically studied in aqueous medium in detail using tensiometric, conductometric, calorimetric, fluorimetric, viscometric, and circular dichroism techniques. The results indicate that SDS-trypsin aggregates start to form at a surfactant concentration higher than the critical micelle concentration of pure SDS micelle. In contrast, the counterion binding decreases in the presence of trypsin. The free energies and enthalpies of micellization, interfacial adsorption, and entropy of micellization associated with the interaction have also been calculated. The values show that the interaction phenomenon is entropy controlled and endothermic in nature. The increase in viscosity is observed for the system of SDS-trypsin cluster above the critical micelle concentration of SDS micelle only. The aggregation number and interface polarity decrease compared to the values of micelles without protein. Circular dichroism spectra show the high alpha-helical content and unfolded structure of trypsin in the presence of SDS due to strong electrostatic repulsion leading to a probable "necklace and bead" model in the case of biopolymer-surfactant complexes.     

Diffusion NMR study of complex formation in membrane-associated peptides

Pulsed-field-gradient nuclear magnetic resonance (PFG-NMR) is used to obtain the true hydrodynamic size of complexes of peptides with sodium dodecyl sulfate SDS micelles. The peptide used in this study is a 19-residue antimicrobial peptide, GAD-2. Two smaller dipeptides, alanine–glycine (Ala–Gly) and tyrosine–leucine (Tyr–Leu), are used for comparison. We use PFG-NMR to simultaneously measure diffusion coefficients of both peptide and surfactant. These two inputs, as a function of SDS concentration, are then fit to a simple two species model that neglects hydrodynamic interactions between complexes. From this we obtain the fraction of free SDS, and the hydrodynamic size of complexes in a GAD-2–SDS system as a function of SDS concentration. These results are compared to those for smaller dipeptides and for peptide-free solutions. At low SDS concentrations ([SDS] ≤ 25 mM), the results self-consistently point to a GAD-2–SDS complex of fixed hydrodynamic size R = (5.5 ± 0.3) nm. At intermediate SDS concentrations (25 mM < [SDS] < 60 mM), the apparent size of a GAD-2–SDS complex shows almost a factor of two increase without a significant change in surfactant-to-peptide ratio within a complex, most likely implying an increase in the number of peptides in a complex. For peptide-free solutions, the self-diffusion coefficients of SDS with and without buffer are significantly different at low SDS concentrations but merge above [SDS] = 60 mM. We find that in order to obtain unambiguous information about the hydrodynamic size of a peptide-surfactant complex from diffusion measurements, experiments must be carried out at or below [SDS] = 25 mM.     

Unfolding of beta-sheet proteins in SDS

Beta-sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two beta-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds around the critical micelle concentration. Isothermal titration calorimetry confirms that unfolding of TII27 sets in at lower SDS concentrations, although the total number of bound SDS molecules is similar at the end of unfolding. In mixed micelles with the nonionic detergent dodecyl maltoside, where the concentration of monomeric SDS is insignificant, the behavior of the two proteins converges. TII27 unfolds more slowly than TNfn3 in SDS and follows a two-mode behavior. Additionally TNfn3 shows inhibition of SDS unfolding at intermediate SDS concentrations. Mutagenic analysis suggests that the overall unfolding mechanism is similar to that observed in denaturant for both proteins. Our data confirm the kinetic robustness of beta-sheet proteins toward SDS. We suggest this is related to the inability of SDS to induce significant amounts of alpha-helix structure in these proteins as part of the denaturation process, forcing the protein to denature by global rather than local unfolding.     

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.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins. Correspondence to: J. M. Sanchez-Ruiz     

Denaturation of α-lactalbumin and myoglobin by the anionic biosurfactant rhamnolipid

Glycolipid biosurfactants (GBS) are promising environmentally friendly alternatives to chemical surfactants. Surfactants interact with proteins in many applications, often leading to significant changes in protein properties. Given GBS' marked difference in structure compared to traditional chemical surfactants, it is of interest to investigate their impact on protein structure and stability. Here we combine spectroscopic and calorimetric studies to analyze the interactions between the anionic GBS rhamnolipid (RL) and two model proteins α-lactalbumin in the Ca^2 +-free apo-form (αLA) and myoglobin (Mb), whose interactions with traditional surfactants are well known. RL denatures αLA at sub-cmc concentrations (0.1–1 mM) while Mb is only denatured above the cmc, i.e. in the presence of RL micelles. Denaturation leads to increased α-helicity, similar to the effect of SDS. The proteins bind approximately the same amount of RL by weight as SDS. However, RL employs a denaturation mechanism which combines features from non-ionic surfactants (very slow unfolding kinetics and few unfolding steps) with those of SDS (unfolding below the cmc in the case of αLA and the ability to unfold stable proteins in the case of Mb). We ascribe these features to RL's weakly acidic carboxylic head group and complex hydrophobic tail, which lead to a low cmc and low protein affinity. These features restrict the concentration range where RL monomers can bind and denature proteins while still allowing micelles to bind and denature to a significant extent.     

Cation Modulation of Hemoglobin Interaction with Sodium <Emphasis Type="Italic">n</Emphasis>-Dodecyl Sulfate (SDS). I: Calcium Modulation at pH 7.20

A comparative denaturation of HbA and HbS in the R states using sodium n-dodecyl sulfate (SDS) was carried out at pH 7.20 in the presence and absence of Calcium (0–40 μM) and monitored by UV–Vis spectrophotometry in the range of 250–650 nm. In the HbS spectra, the calcium alone caused little or no perturbation of the aromatic region but caused a decrease in oxygen affinity when compared to the HbA. The combinations of [SDS] and [Ca] perturbed the HbS the most, relative to the individual spectra of the [SDS] and [Ca]. However, the presence of Ca appeared to diminish the adverse effects of the SDS on HbA. The denaturation pathway of the HbA involved mainly the formation of heme dimers and some ferryl heme species. For the HbS, heme monomers and a large amount of ferryl species were formed. It is suggested that the greater monomer species formed by the HbS denaturation pathway would result in both Fenton and enhanced enzymatic reactions, compared to the dimer. This could lead ultimately to the formation of ferryl radicals. Thus, at physiological pH for the HbS, the Ca–SDS interaction increases the tendency for protein denaturation in comparison to the HbA.     

Quantification of protein-bound sodium dodecyl sulfate by Rhodamine B: a method for identification of kinetically stable proteins

Sodium dodecyl sulfate (SDS) bound to proteins in solution could be estimated by passing through Extracti-Gel that removes free SDS followed by specific interaction of the fluorophore Rhodamine B with protein-bound SDS. The resulting fluorescence intensity is compared with a calibration curve. Whereas globular proteins respond to binding of 1.4 mg SDS/mg protein under native conditions, “kinetically stable” proteins that are otherwise resistant to denaturation due to structural integrity show a low level of SDS binding. Analysis of the circular dichroism spectrum shows that in spite of the low level of SDS binding to kinetically stable proteins under nondenaturing conditions, the detergent generates considerable secondary structure in these proteins. Because the low level of SDS binding is a general feature of kinetically stable proteins, the protocol may fulfill one of the criteria to classify a protein as kinetically stable.     

Ionic strength-dependent denaturation of Thermomyces lanuginosus lipase induced by SDS

Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10 mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS–lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.     

Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties.

We have examined the binding of the fluorescent Ca indicator indo-1 to cellular protein in permeabilized ventricular myocytes and also to soluble and particulate myocyte protein. Using either a filtration technique or equilibrium dialysis, and conditions similar to those in a cardiac myocyte patch clamped with 100 microM indo-1 in the patch pipette, we found that 72% of the total indo-1 was bound to myocyte protein at a protein concentration of 100 mg/ml. This corresponds to a binding of 3.8 +/- 0.5 nmol indo-1/mg protein. Separation of the myocyte protein into a soluble and a particulate fraction showed that 63% of the bound indo-1 was bound to soluble protein, corresponding to a binding of 3.22 +/- 0.99 nmol/mg, whereas 37% of the bound indo-1 was bound to particulate protein (0.85 +/- 0.14 nmol/mg) at a low [Ca] (pCa approximately 9). Binding of indo-1 in permeabilized myocytes was approximately 60% higher at a saturating Ca concentration (pCa = 3), than under Ca free conditions (1 mM EGTA). Simultaneous measurements of free [Ca] with a Ca selective electrode and indo-1 fluorescence showed that, the dissociation constant (Kd) for Ca was increased 4-5 fold in the presence of permeabilized myocytes as compared to the value obtained in vitro. In agreement with the binding experiments we estimate that the true Kd and the apparent Kd (using ratiometric measurements) for Ca binding to indo-1 are increased approximately four fold, at a myocyte protein concentration of 100 mg/ml.     

A low-angle laser light scattering study of the association behavior of a major membrane protein of Rhodospirillum rubrum chromatophore at various concentrations of sodium dodecyl sulfate where polypeptides derived from water-soluble globular proteins are solubilized monomerically

The major membrane protein of Rhodospirillum rubrum chromatophore could be solubilized in the presence of free sodium dodecyl sulfate (SDS) in concentration above 0.8 mM. At this concentration, the protein was highly associated to give a weight-averaged molecular weight as high as one million as determined by the low-angle laser light scattering technique. With the increase of free SDS concentration, the aggregates were progressively dissociated to give a molecular weight of 8300 at the critical micelle concentration of SDS. Three protein polypeptides derived from typical water-soluble globular proteins, bovine serum albumin, ovalbumin and beta-lactoglobulin, were found to be solubilized monomerically even at 0.8 mM free SDS. The results obtained suggest that there is substantial difference in the mode of solubilization between polypeptides derived from intrinsic membrane proteins and those from water-soluble globular proteins.