Spectroscopy and computational studies on the interaction of octyl,dodecyl, and hexadecyl derivatives of anionic and cationic surfactants with adenosine deaminase

Effects of sodium (octyl, dodecyl, hexadecyl) sulfate and their cationic analogous on the structure of adenosine deaminase (ADA) were investigated by fluorescence and circular dichroism spectroscopy as well as molecular dynamics simulation and docking calculation. Root-mean-square derivations, radius of gyration, solvent accessible surface area, and radial distribution function were obtained. The results showed that anionic and cationic surfactants reduce protein stability. Cationic surfactants have more effect on the ADA structure in comparison with anionic surfactants. More concentration and longer surfactants are parallel to higher denaturation. Furthermore, aggregation in the presence of anionic surfactants is more than cationic surfactants. Docking data showed that longer surfactants have more interaction energy and smaller ones bound to the active site.     

Effect of homologous series of n-alkyl sulfates and n-alkyl trimethylammonium bromides on low molecular mass protein tyrosine phosphatase activity

The effect of anionic and cationic surfactants on acid phosphatase denaturation has been extensively studied. Low molecular mass (LMr) protein tyrosine phosphatase (PTP), a key regulatory enzyme involved in many different processes in the cell, was distinctly affected by anionic (homologous series of n-alkyl sulfates (C8-C14)) and cationic (n-alkyl trimethylammonium bromides (C12-C16)) surfactants. At concentrations 10-fold lower critical micellar concentration (cmc) values, the enzyme was completely inactivated in the presence of anionic surfactants, in a process independent of the pH, and dependent on the chain length of the surfactants. Under the same conditions, the effect of cationic surfactants on the enzyme activity was pH-dependent and only at pH 7.0 full inactivation was observed at concentrations 10-fold higher cmc values. In contrast to cationic surfactants the effect of anionic surfactants on the enzyme activity was irreversible and was not affected by the presence of NaCl. Inorganic phosphate, a known competitive inhibitor of PTP, protected the enzyme against inactivation by the surfactants. Our results suggest that the inactivation of the LMr PTP by anionic and cationic surfactants involved both electrostatic and hydrophobic interactions, and that the interactions enzyme-surfactants probably occurred at or near the active site.     

Restoring the activity of serum-inhibited bovine lung extract surfactant (BLES) using cationic additives

In this work four cationic additives were used to improve the surface activity of lung surfactants, particularly in the presence of bovine serum that was used as a model surfactant inhibitor. Two of those additives were chitosan in its soluble hydrochloride form with average molecular weights of 113 kDa and 213 kDa. The other two additives were cationic peptides, polylysine 50 kDa and polymyxin B. These additives were added to bovine lipid extract surfactant (BLES) and the optimal additive-surfactant ratio was determined based on the minimum surface tension upon dynamic compression, carried out in a constrained sessile drop (CSD) device in the presence of 50 μl/ml serum. At the optimal ratio all the BLES-additive mixtures were able to achieve desirable minimum surface tensions. The optimal additive-surfactant ratios for the chitosan chlorides are consistent with a previously proposed patch model for the binding of the anionic lipids in BLES to the positive charges in chitosan. For the peptides, the optimal binding ratios were consistent with ratios established previously for the binding of these peptides to monolayers of anionic lipids. The optimal formulation containing these peptides were able to reach low minimum surface tension in systems containing 500 μl/ml of serum, matching the effectiveness of a lung surfactant extract that had not undergone post-separation processes and therefore contained all its proteins and lipids (complete lung surfactant).     

Solid phase extraction of BSA

Silica with immobilized polyoxyethyle-neisooctylphenol (SiO2-TX) was investigated as an adsorbent for solid phase extraction of bovine serum albumin (BSA). It was shown that efficient BSA extraction (up to 96%) takes place on SiO2-TX from water solution in the form of its ionic associates with cationic (at pH = 8) and anionic (at pH = 1.5) surfactants.     

Effects of surfactants on the activity of phospholipase D.

We studied the dependence of the activity of cabbage phospholipase A on the substrate (phosphatidylcholine) the aggregated state of which is regulated by addition of either anionic (sodium dodecyl sulfate, cholate or oleate) or cationic (cetyl-trimethylammonium bromide) surfactants. Activation of the enzyme induced by anionic surfactants was shown to correlate with the size of their polar groups. The phospholipase hydrolase activity correlated with the transformation of multilayer liposomes into micelles. The relationship between the processes was of a complex character. The dependence of the amount of enzymically released choline on the calcium concentration passed through a sharp maximum in the presence of the anionic detergents and monotonically increased in the presence of the cationic detergent. In the former case, the sharp increase in the enzyme activity was suggested to be caused by precipitation of phospholipase D with the anionic detergent calcium salt, which can be considered as a specific type of immobilization.     

Surfactant-induced refolding of lysozyme

The surfactant-lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C(n)SO3, C(n)SO4, C(n)NE and C(n)NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic-cationic surfactants and that of surfactant-lysozyme. It was thought that the formation of mixed micelles of anionic-cationic surfactants is a more favorable process than that of lysozyme-surfactant complexes, which induces the dissociation of lysozyme-surfactant complexes when cationic surfactants were added.     

Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Cytotoxic ribonucleases: the dichotomy of Coulombic forces

Cells tightly regulate their contents. Still, nonspecific Coulombic interactions between cationic molecules and anionic membrane components can lead to adventitious endocytosis. Here, we characterize this process in a natural system. To do so, we create variants of human pancreatic ribonuclease (RNase 1) that differ in net molecular charge. By conjugating a small-molecule latent fluorophore to these variants and using flow cytometry, we are able to determine the kinetic mechanism for RNase 1 internalization into live human cells. We find that internalization increases with solution concentration and is not saturable. Internalization also increases with time to a steady-state level, which varies linearly with molecular charge. In contrast, the rate constant for internalization (t1/2 = 2 h) is independent of charge. We conclude that internalization involves an extracellular equilibrium complex between the cationic proteins and abundant anionic cell-surface molecules, followed by rate-limiting internalization. The enhanced internalization of more cationic variants of RNase 1 is, however, countered by their increased affinity for the cytosolic ribonuclease inhibitor protein, which is anionic. Thus, Coulombic forces mediate extracellular and intracellular equilibria in a dichotomous manner that both endangers cells and defends them from the potentially lethal enzymatic activity of ribonucleases.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

A fluorescence study of the interactions between sodium alginate and surfactants

The interactions between the polysaccharide alginate with charged ionic surfactants (anionic and cationic) in aqueous solution have been investigated using pyrene as a photophysical probe. Static fluorescence determinations have been used to obtain information about the new microenvironments arising by these interactions. Micropolarity studies using the I(1)/I(3) ratio of the vibronic bands and I(E)/I(M) ratio between the excimer and monomer emissions of pyrene shows the formation of hydrophobic domains. The interactions between the natural polyelectrolytes and the oppositely charged surfactants lead to the formation of pre-micelles at surfactant concentrations lower than the CMC of the surfactants. The aggregation process is assumed to be due to electrostatic attraction. On the other side, systems containing an anionic surfactant do not show the same behaviour at low concentrations.     

Kinetic Studies on the Interaction of Ferricytochrome c with Anionic Surfactants

The kinetics of absorbance and fluorescence changes of cytochrome c as induced by an aqueous solution of the anionic surfactant sodium dodecyl sulfate (SDS) or sodium bis(2-ethylhexyl)sulfosuccinate (AOT) are studied. The results are compared with far-UV circular dichroism (CD) spectra. Both surfactants cause similar alterations in the secondary structure of cytochrome c, while their influence on the heme environment of cytochrome c is different. In the presence of AOT below and above critical micellar concentration a conversion of the low-spin native cytochrome c to a denatured low-spin protein not having methionine ligand takes place. In the presence of SDS micelles conversion of the native protein to a denatured mixed-spin form occurs. The changes in the heme group induced by both surfactants occur independently of the alterations in tertiary structure.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

An ethoxylated alkyl phosphate (anionic surfactant) for the promotion of activities of proteases and its potential use in the enzymatic processing of wool

Pretreatments of wool fabrics with cationic, anionic or non-ionic surfactants were investigated to reduce surface tension and improve the wettability of the fibres in order to promote protease activity on the fibres in subsequent processes. Results showed that an ethoxylated alkyl phosphate (specific anionic surfactant) as well as the widely used non-ionic surfactant was compatible with proteases in the enzymatic treatment of wool. There is therefore a potential for using specific anionic surfactants to achieve efficient enzymatic scouring processes.     

Inhibition of Mussel Suspension Feeding by Surfactants of Three Classes

Effects of three surfactants on the filtration rates by marine mussels were studied. The xenobiotics tested represented anionic, cationic and non-ionic surfactants (tetradecyltrimethylammonium bromide, a representative of a class of cationic surfactants; sodium dodecyl sulphate, a representative of anionic alkyl sulfates; and Triton X-100, a representative of non-ionic hydroxyethylated alkyl phenols). All three surfactants inhibited the clearance rates. The significance of the results for the ecology of marine ecosystems is discussed.     

Fluorescence quenching of beta-carboline alkaloids in micellar media. A study to select the adequate surfactant to use in analytical techniques.

The study of fluorescence quenching of the fluorophores allows the localization of the alkaloids (harmane and harmine) in the micelles (SDS, CTAB, Brij-35) to be established. In aqueous micellar solutions (SDS and Brij-35) at pH 13.0, emission corresponding to the neutral or zwitterionic forms can be observed. In the presence of CTAB (pH = 13.0) it was possible to observe the emission of anionic form. These species are not present in buffered aqueous solutions at these pH values. Bromide ion was added to the different surfactant solutions and the quenching effect was studied according to the Stern-Volmer equation. In the presence of SDS the quenching effect is considerably reduced compared to the aqueous solutions without surfactants, while for Brij-35 micelles were similar to those observed in homogeneous aqueous solution. For CTAB micelles a notable fluorescence quenching was observed for the different pH values studied. The fluorescence quenching studies show that the neutral species are associated inside the micelles, instead of the ionic species (cationic, zwitterionic or anionic) remaining on the surface of the micelles. The anionic surface of SDS micelles prevents the quenching effect by anionic quenchers for both neutral and charged species.     

The essential cationic charge of phospholipid polar head in the reactivation of -β-hydroxybutyrate apodehydrogenase revealed by cationic surfactants

Attempts to reactivate purified -β-hydroxybutyrate apodehydrogenase, a lecithin-requiring enzyme, have been carried out using neutral, anionic, cationic and zwitterionic surfactants. Cationic and zwitterionic compounds exclusively are able to partially replace phosphatidylcholine, the reactivating phospholipid. The extent of reactivation depends on the steric hindrance of the polar head and on the hydrophobic tail length. A molecule bearing a positive charge and an aliphatic chain is the sole structure absolutely required for activity. However the presence of a negative charge is important for enzyme binding to amphiphilic structures and for the efficiency of reactivation.     

The effects of pH and surfactants on the absorption and fluorescence properties of ochratoxin A and zearalenone

The pH and surfactant dependencies of the absorption and fluorescence properties of ochratoxin A (OTA) and zearalenone (ZEN), the main mycotoxins found as contaminants in foods and feeds, were evaluated. Three surfactants with different ionic properties were investigated, namely sodium dodecyl sulfate (SDS, anionic), Tween 20 (nonionic) and hexadecyltrimethylammonium bromide (CTAB, cationic). The results show that the effects of pH on the absorption wavelength maxima and fluorescence efficiencies of the mycotoxins, which are a consequence of the presence of acidic phenol and/or carboxyl containing fluorophores, are dependent on the ionic nature of the added surfactants. Specifically, the fluorescence responses to pH changes of OTA and ZEN are similar in the presence or absence of Tween 20 and SDS. By contrast, the pH‐dependent fluorescence properties of these mycotoxins are altered when CTAB is present in the solutions. Moreover, unlike OTA, ZEN in aqueous solution displays almost no fluorescence. However, fluorescence enhancement takes place when surfactants are present in aqueous solutions of this mycotoxin. The results of this study demonstrate that the different microenvironments, present in the organized micellar systems created by the individual surfactants, can be potentially employed to modulate the sensitivities and selectivities of the fluorescence detection of OTA or ZEN. Copyright © 2015 John Wiley & Sons, Ltd.     

Complexation of polymethine dyes with human serum albumin: a spectroscopic study

Non-covalent interactions between polymethine dyes of various types (cationic and anionic thiacarbocyanines as well as anionic oxonols and tetracyanopolymethines) and human serum albumin (HSA) were studied by means of absorption, fluorescence and circular dichroism (CD) spectroscopies. Complexation with the protein leads to a red shift of the dye absorption spectra and, in most cases, to a growth of the fluorescence quantum yield (Phif; for oxonols this growth is very small). The binding constants (K) obtained from changing the absorption spectra and Phif vary from 10(4) to (5-6) x 10(7) M(-1). K for the anionic dyes is much higher than for the cationic dyes (the highest K was found for oxonols). Interaction of meso-substituted anionic thiacarbocyanines with HSA results in cis-->trans isomerization and, as a consequence, an appearance and a steep rise of dye fluorescence. Binding to HSA gives rise to dye CD signals and in many cases is accompanied by aggregation of the dyes. These aggregates often exhibit biphasic CD spectra. The aggregates formed by the dyes alone are decomposed in the presence of HSA.     

Standard free energies of binding of solute to proteins in aqueous medium. Part 1: Thermodynamic analysis for multicomponent system

Using an equilibrium dialysis technique, moles (Gamma(2)(1)) of cationic and anionic surfactants bound per kilogram of proteins of various types in aqueous media have been measured previously in this laboratory under different physicochemical conditions. From a thermodynamic analysis in the present paper, Gamma(2)(1) has been shown to be equal to the Gibbs relative excess of surfactant per kilogram of protein at a measured value of solute activity, a(2). The values of relative solvent excesses, Gamma(2)(1) (which are negative for surfactants) can be estimated from values of Gamma(2)(1) and a(2). Using the Gibbs-Duhem relationship for protein solution inside the dialysis bag and dialysate solutions respectively at equilibrium, an integrated expression for the standard free energy change, DeltaG(o) (in kilojoules per kilogram of protein for binding with ligand as a result of the change of a(2) from zero to unity) can be calculated from experimental data. The isopiestic vapour pressure technique was used extensively for evaluation of negative binding (-Gamma(2)(1)) of inorganic salts to proteins of different types for various values of a(2) of salts present in the bulk media. With some modifications of our derived equations for free energy of binding in such a system, DeltaG(o) has been evaluated for the change of mean activity of electrolyte from zero to unity in the rational scale. DeltaG(o) is positive since Gamma(2)(1) is negative and Gamma(2)(1) is positive for such ionic systems. DeltaG(o) in all cases, however, are expressed in terms of the standard state of reference of unit activity so that their magnitudes and sign can be related to the relative affinities of a solute for binding with proteins in aqueous media.     

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin

Disintegration by surfactants of egg yolk phosphatidylcholine vesicles stabilized with carboxymethylchitin was investigated by measuring the amount released of a marker dye from the vesicles. In solutions of pH around 7, anionic and nonionic surfactants caused vesicle disintegration at very low concentrations, while cationic surfactants produced a breakdown of the vesicles at rather high concentrations. Increase in the alkyl chain-length of surfactant molecules brought about decrease in the surfactant concentration at which vesicle disintegration starts. As the length of the polyoxyethylene chain in nonionic surfactant molecules increased, the tendency of vesicle disintegration to occur decreased. Both anionic and cationic surfactants gave clear solutions above their critical micelle concentrations when they acted on the phospholipid vesicles, whereas nonionic surfactants left ghost cell-like debris consisting of carboxymethylchitin molecules in their micellar solutions. The effect of pH on vesicle disintegration was notable for ionic surfactants but not for nonionic surfactants. Thus, anionic surfactants increased the degree of disintegration as pH increased, while cationic surfactants produced an identical vesicle disintegration curve below pH 8 above which the curve started to shift toward the lower concentration region of the agents. These findings were explained in terms of surfactant penetration into phospholipid bilayers and solubilization of phospholipid molecules by surfactant micelles.