Effect of ethyleneoxide groups of anionic surfactants on lipase activity

The use of enzymes in laundry and dish detergent products is growing. Such tendency implies dedicated studies to understand surfactant‐enzyme interactions. The interactions between surfactants and enzymes and their impact on the catalytic efficiency represent a central problem and were here evaluated using circular dichroism, dynamic light scattering, and enzyme activity determinations. This work focuses on this key issue by evaluating the role of the ethyleneoxide (EO) groups of anionic surfactants on the structure and activity of a commercial lipase, and by focusing on the protein/surfactant interactions at a molecular level. The conformational changes and enzymatic activity of the protein were evaluated in the presence of sodium dodecyl sulfate (SDS also denoted as SLE₀S) and of sodium lauryl ether sulfate with two EO units (SLE₂S). The results strongly suggest that the presence of EO units in the surfactant polar headgroup determines the stability and the activity of the enzyme. While SDS promotes enzyme denaturation and consequent loss of activity, SLE₂S preserves the enzyme structure and activity. The data further highlights that the electrostatic interactions among the protein groups are changed by the presence of the adsorbed anionic surfactants being such absorption mainly driven by hydrophobic interactions. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1276–1282, 2016     

Correlation between surfactant/micelle structure and the stability of bacteriorhodopsin in solution.

The rate of solubilization and isothermal bleaching of bacteriorhodopsin (bR) in a series of nine alkylammonium surfactants is studied by using time-resolved optical spectroscopy. The surfactant series RN(+)R'(3) covers a range in tail length (R = C(12)H(25), C(14)H(29), or C(16)H(33)) and headgroup size and hydrophobicity (R' = CH(3); C(2)H(5), or C(3)H(7)). The rate of bleaching increases initially with increasing surfactant concentration but decreases at higher concentrations. Possible explanations for this behavior are discussed. The kinetic data are consistent with the penetration of the surfactant into the protein interior. Interaction of the surfactants with the protein is a complicated, multistep process, and the rate curves are a function of at least four variables: 1) the micellar environment, 2) the length of the surfactant tail, 3) the size of the headgroup, and 4) the hydrophobicity of the headgroup. Our data provide new insights into the molecular characteristics that help define the performance of surfactants in the solubilization and denaturation of membrane-bound proteins.     

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.     

Influence of coprecipitation and interactions with anionic surfactants on the activity of plant phospholipase D.

The effects of two surfactants (sodium dodecyl sulfate and sodium dodecyl dioxyethylene sulfate) of similar structure but differing water solubility (of their calcium salts) on the enzymatic activity of cabbage phospholipase D have been studied. The solubility difference is insignificant because the two surfactants activate phospholipase D similarly. To elucidate the mechanism of their influence on the enzyme, the phase behavior in the reaction media and the interactions of the surfactants with the enzyme were investigated by potentiometry and by light scattering and UV spectroscopy. Calcium dodecyl dioxyethylene sulfate (which is more soluble in water than calcium dodecyl sulfate) precipitates in the presence of phosphatidylcholine, the substrate of the enzymatic reaction. In the reaction media phospholipase D was involved into a precipitate consisting of calcium salts of the surfactants and phosphatidylcholine that might be interpreted as its immobilization. In addition, the surfactants were adsorbed on the enzyme, unfolding the globular enzyme molecule due to electrostatic repulsion between adsorbed surfactant anions. The observed increase in the functional activity of phospholipase D is accounted for by transfer to an optimal tertiary structure for the enzyme molecule in the course of consecutive conformational transitions induced by the surfactants.     

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.     

Reactivity of trypsin in reverse micelles: neglected role of aggregate size compared to water-pool components

The catalytic efficiency of trypsin was estimated in cationic reverse micelles as a function of the concentration of water-pool components and aggregate size to determine their independent influence on enzyme activity. The variation in the aggregate size/water-pool size was achieved by changing both the W0 (mole ratio of water to surfactant) and the headgroup area of surfactant through introduction of hydroxyethyl groups at the polar head. The local molar concentrations of water present inside the water-pool ([H2O]wp) of different cationic reverse micelles across varying W0 was estimated using a modified phenyl cation-trapping protocol. The [H2O]wp in cationic reverse micelles (surfactant/isooctane/n-hexanol/water) increases with W0 and attains the molarity of normal water beyond W0=40 irrespective of the nature of headgroup. Concurrently, the catalytic activity of trypsin compartmentalized within the water-pool increases with the increase in [H2O]wp upto an optimal W0=40 in organized solutions of any surfactant. The aggregate size (determined by static light scattering) also increases expectedly with W0 and noticeably with the area of the surfactant headgroup at similar W0. Since the enzyme activity rises both with the increase in water-pool size and [H2O]wp, trypsin's efficiency was compared with these two parameters across reverse micelles of varying surfactant headgroup size at similar W0 to determine their probable independent influence in regulating the enzyme activity. Noticeably, the efficiency of trypsin rises two to ninefold in spite of the [H2O]wp being distinctly lower in case of hydroxyethyl group substituted surfactants compared to cetyltrimethylammonium bromide w/o microemulsions at similar W0. Thus, the influence of the aggregate size possibly plays an important role alongwith the [H2O]wp in modulating the enzyme activity.     

Unsaturation at the surfactant head: influence on the activity of lipase and horseradish peroxidase in reverse micelles

Influence of unsaturation present at the surfactant head on the activity of interfacially located enzyme, lipase, and horseradish peroxidase (HRP) is investigated in cationic reverse micelles of a series of surfactants having unsaturated (allyl and pyridinium moieties) as well as analogous saturated (n-propyl and piperidinium moieties) polar head. Lipase activity increases with n-propyl (saturated) substitution as the increase in the headgroup area (A(min)) presumably provides greater space for the enzyme to attain flexible conformation and increases the local concentrations of enzyme and substrate at the interface. In contrast, activity of lipase decreases with increasing number of allyl (unsaturated) substitution though A(min) gradually increased. Similar trend in deactivation was observed when unsaturation is present in cyclic ring (pyridine) at the surfactant head in comparison to the saturated analogue, piperidine. Circular dichroism (CD) spectra of lipase in reverse micelles indicate that ellipticity in the far-UV region increases with increasing unsaturation. Thus, lipase probably loses its alpha-helix content and thereby its activity. Inhibition of biocatalyst with increasing unsaturation at the polar head of surfactant is also observed in case of HRP, an oxidoreductase enzyme.     

The phospholipid headgroup specificity of an atp-dependent calcium pump

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37°C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids.We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.     

Competition between Anion Binding and Dimerization Modulates Staphylococcus aureus Phosphatidylinositol-specific Phospholipase C Enzymatic Activity

Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) is a secreted virulence factor for this pathogenic bacterium. A novel crystal structure shows that this PI-PLC can form a dimer via helix B, a structural feature present in all secreted, bacterial PI-PLCs that is important for membrane binding. Despite the small size of this interface, it is critical for optimal enzyme activity. Kinetic evidence, increased enzyme specific activity with increasing enzyme concentration, supports a mechanism where the PI-PLC dimerization is enhanced in membranes containing phosphatidylcholine (PC). Mutagenesis of key residues confirm that the zwitterionic phospholipid acts not by specific binding to the protein, but rather by reducing anionic lipid interactions with a cationic pocket on the surface of the S. aureus enzyme that stabilizes monomeric protein. Despite its structural and sequence similarity to PI-PLCs from other Gram-positive pathogenic bacteria, S. aureus PI-PLC appears to have a unique mechanism where enzyme activity is modulated by competition between binding of soluble anions or anionic lipids to the cationic sensor and transient dimerization on the membrane.     

The phospholipid headgroup specificity of an ATP-dependent calcium pump.

We have replaced the lipid associated with a purified calcium transport protein with a series of defined synthetic dioleoyl phospholipids in order to determine the effect of phospholipid headgroup structure on the ATPase activity of the protein. At 37 degrees C the zwitterionic phospholipids (dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) support the highest activity, while a phospholipid with two negative charges (dioleoyl phosphatidic acid) supports an activity which is at least twenty times lower. Dioleoyl phospholipids with a single net negative charge support at intermediate ATPase activity which is not affected by the precise chemical structure of the phospholipid headgroup. The protocol used to determine the phospholipid headgroup specificity of calcium transport protein is novel because it establishes the composition of the lipid in contact with the protein without the need to isolate defined lipid-protein complexes. This allows the lipid specificity to be determined using only very small quantities of test lipids. We also determined the ability of the same phospholipids to support calcium accumulation in reconstituted membranes. Two requirements had to be met. The phospholipid had to support the ATPase activity of the pump protein and it had to form sealed vesicles as determined by electron microscopy. Since a number of phospholipids met those requirements it is clear that in vitro the lipid specificity of the calcium-accumulating system is rather broad.     

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.     

Monovalent lectin as a novel tool for the resolution of microheterogeneity in glycoproteins

We have devised a method for the determination of the long-term (days) uptake of radiolabeled surfactants by intact mammalian cells in monolayer culture. We have utilized the method to investigate several characteristics of the uptakes of three highly purified synthetic zwitterionic surfactants by confluent hamster embryo fibroblasts in culture medium containing serum. The uptakes reach a steady state after several hours and appear to be nonsaturable at sublytic concentrations. Surfactant structure was found to greatly influence the rate and extent of cellular uptake as well as the extent of the in vitro surfactant biotransformation.     

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.     

A new and simple procedure for the evaluation of the association of surfactants to proteins

A new and simple method useful for the evaluation of the association of surfactants to proteins is proposed. The method is based on an analysis of the effect promoted by surfactant addition upon the fluorescence intensity of the intrinsic tryptophan chromophore and its dependence with protein concentration. The proposed methodology is applied to quantify the binding of an anionic (sodium dodecylsulfate), a zwitterionic (N-hexadecyl-N,N′-dimethyl-3-ammonio-1-propane-sulfonate) and a neutral (Triton X-100, reduced) surfactant to bovine serum albumin (BSA).     

Regulation of catalytic properties of enzymes in "inverse micelles"]

A triple system (inverse micellae) that simulates the membrane environment of the enzyme was studied. Inverse micellae were obtained using anionic (aerosol OT), synthetic (Brij 56), and natural (lecithin) surfactants. It was found that upon inclusion of an enzyme into inverse micellae, its activity can be regulated by changing the structure and nature of the surfactant matrix. It was shown that enzyme activity in micellar environment is much higher than in water solution. Moreover, the enzyme solubilized in inverse micellae (acid phosphatase) shows a superactivity. It was found that surfactants specifically interact with solubilized enzyme, and the activity of the enzyme is inversely proportional to surfactant concentration. The mechanisms of viscotropic regulation of enzyme activity are discussed.     

Importance of direct interactions with lipids for the function of the mechanosensitive channel MscL

We have studied the effects of lipid structure on the function of the mechanosensitive channel of large conductance (MscL) from Escherichia coli to determine whether effects follow from direct interaction between the lipids and protein or whether they follow indirectly from changes in the curvature stress in the membrane. The G22C mutant of MscL was reconstituted into sealed vesicles containing the fluorescent molecule calcein, and the release of calcein from the vesicles was measured following opening of the channel by reaction with [2-(triethylammonium)ethyl] methanethiosulfonate (MTSET), which introduces five positive charges into the region of the pore constriction. The presence of anionic lipids in the vesicle membrane changed the rates and amplitudes of calcein release, the effects not correlating with calculated changes in lipid spontaneous curvature. Mutation of charged residues in the Arg-104, Lys-105, Lys-106 cluster removed high-affinity binding of anionic lipids to MscL, and the presence of anionic lipid no longer affected calcein flux through MscL. Changing the zwitterionic lipid from phosphatidylcholine to phosphatidylethanolamine resulted in a large decrease in the rate of calcein release, the change in rate varying linearly with lipid composition, as expected if spontaneous curvature affected the rate of release. However, rates of release of calcein measured in the presence of phosphatidylethanolamine- N-methyl and phosphatidylethanolamine- N, N-dimethyl did not fit the correlation between rate and curvature established for the phosphatidylcholine/phosphatidylethanolamine mixtures. Rather, the effects of zwitterionic lipid headgroup on calcein flux suggested that what was important was the presence of a proton in the headgroup, able to take part in hydrogen bonding to MscL. We conclude that the function of MscL is likely to be modulated by direct interaction with the surrounding, annular phospholipids that contact the protein in the membrane.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Activation and stabilization of alpha-chymotrypsin by cationic additives.

alpha-Chymotrypsin activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide (GPNA) in aqueous media in the presence of synthetic surfactants, which differ in the flexibility of their bulky head groups. Superactivity can be ascribed to the presence of the tributylammonium residue on the surfactant head group, as in p-octyloxybenzyltributylammonium bromide (pOOTBABr), while in the presence of a more rigid moiety, i.e. a cyclic one, no activation was found. A nonmicellizable quaternary ammonium salt, the tetrabutylammonium bromide (TBABr), which has a head group structure very similar to pOOTBABr, not only induces a remarkable superactivation, at a concentration 80-fold higher than pOOTBABr, but also allows the enzyme to retain a high residual activity for long periods of time. The presence of a lipophilic chain, which by interacting with apolar residues on the enzyme surface, probably penetrates into hydrophobic pockets of the protein and causes a rapid inactivation. In 0.4 m TBABr, a 20-fold increase both in kcat and Km values, with respect to buffer alone, was found. The increase of Km could be attributed either to a true decrease in affinity between enzyme and substrate or alternatively to the presence of TBA+ ions near the catalytic region. They could interact with protein residues around the active site and bind to negatively charged GPNA molecules, lowering the local substrate concentration. Spectroscopic experiments (CD and fluorescence) show minor changes of protein conformation in 0.4 m TBABr, while at 1 m a strong modification of both spectra was observed.     

Folding and aggregation of TEM beta-lactamase: analogies with the formation of inclusion bodies in Escherichia coli.

The enzyme TEM beta-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM beta-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1.0-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-8-naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM beta-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM beta-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM beta-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7:131-136). Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo.     

The conformation of fusogenic B18 peptide in surfactant solutions.

The interaction of B18 peptide with surfactants has been studied by circular dichroism spectroscopy and fluorescence measurements. B18 is the fusogenic motif of the fertilization sea urchin protein. The peptide forms an alpha-helix structure when interacting with positively or negatively charged surfactants below and above the critical micellar concentration (CMC). The alpha-helix formation is due to binding of surfactant monomers rather than the formation of surfactant micelles on the peptide. Fluorescence measurements show that the CMC of the negatively charged surfactant increases in the presence of B18, supporting the fact that there is a strong interaction between the peptide and monomers. Nonionic surfactant monomers have no effect on the peptide structure, whereas the micelles induce an alpha-helical conformation. In this case the helix stabilization results from the formation of surfactant micelles on the peptide.