Detergent effects on enzyme activity and solubilization of lipid bilayer membranes

Over 50 detergents were tested to establish which would be most effective in releasing proteins from membrane-bounded compartments without denaturating them. Various concentrations of each detergent were tested for two activities: (1) solubilization of egg phospholipid liposomes as measured by reduction of turbidity and (2) effect of detergent concentration on the activities of soluble, hydrolytic enzymes. Those detergents must effective in solubilizing 0.2% lipid and least detrimental to enzymes were five pure, synthetic compounds recently introduced: CHAPS, CHAPSO, Zwittergents 310 and 312, and octylglucoside. Industrial detergents were generally much inferior, insofar as they solubilized membranes inefficiently and/or inactivated certain hydrolytic enzymes readily. The five detergents were characterized by (a) an unusually high critical micelle concentration and (b) a preference for forming mixed micelles with lipids instead of forming pure micelles, as indicated by an ability to solubilize lipid at concentrations of detergent significantly below the critical micelle concentration. This characteristic permits solubilization of high concentrations of membrane below the critical micelle concentration of the detergent so that protein denaturation is minimized. A generally applicable guideline that emerged from this study is that detergents should be used at approximately their critical micelle concentration which should not be exceeded by the concentration of membrane. Similar considerations should apply to the use of detergents in purifying and reconstituting intrinsic membrane proteins.     

Bile salt damage of egg phosphatidylcholine liposomes

Physiochemical damage of egg phosphatidylcholine liposomes, caused by the salts of three bile acids, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid, has been investigated. Of the three bile salts, that of chenodeoxycholic acid was the most destructive, and the effect of the damage was examined by monitoring the induced 6-carboxyfluorescein release from the liposomes. For all three of the bile salts and under the experimental conditions, the minimum (effective) concentrations causing the 6-carboxyfluorescein release were below their critical micelle concentrations. In the case of the salt of chenodeoxycholic acid, the presence of cholesterol in the liposomal bilayers did not show any significant effect on the induced 6-carboxyfluorescein release, while, for the salts of ursodeoxycholic acid and cholic acid, the presence of cholesterol tended to depress the release. Permeation of bile salts into the membranes of liposomal bilayers made these membranes more fluid, and this fluidity was monitored by measuring the change in fluorescence polarization using 1,6-diphenylhexatriene entrapped in the liposomes. Coating the liposomes with polysaccharides, to make them more hydrophobic, led to their easier lysis by the bile salts.     

Non-reducing trisaccharide fatty acid monoesters: novel detergents in membrane biochemistry

Three families of non-reducing trisaccharide fatty acid monoesters bearing C?? to C?? acyl chains have been prepared by enzymatic synthesis in organic media. Their critical micelle concentrations, determined by dye-inclusion measurements, cover a broad range from mM to μM. The new compounds are capable of dissolving phospholipid vesicles and have been characterized as detergents in membrane biochemistry. In a comparative screening test for solubilizing/extraction capacity under native conditions of an ABC transporter as model integral membrane protein, the novel detergents have shown an excellent behavior similar to other commercial carbohydrate-based detergents and in some cases even better than the commonly employed β-dodecylmaltoside. The new detergents are also efficient at extracting membrane proteins from different lipidic environments and are likewise compatible with common protein affinity chromatography purification. These compounds may also be used for the preparation of (proteo)liposomes by detergent removal, not only using the classical method of detergent adsorption on hydrophobic resins but also by enzyme-catalyzed hydrolysis of the ester bond. These results show the new detergents as promising tools to expand the arsenal for membrane protein studies.     

Interaction of sea cucumber saponins with multilamellar liposomes

The effect of three sea cucumber saponins, echinoside A, bivittoside D and holothurin A, on multilamellar liposomes was investigated. An ideal osmotic behavior of liposomes was described as a linear relationship between the reciprocal $\text{3}\text{2}\text{s}$ power of absorbance at 450 nm and the osmotic gradient across the membrane. Sea cucumber saponins at concentrations below critical micelle concentration (CMC) disturbed this linear relationship in liposomes composed of egg phosphatidylcholine, phosphatidic acid and cholesterol. Cholesterol-free liposomes were not susceptible to these saponins. Results of optical measurements were consistent with those of transmission electron microscopy, which showed saponin-induced changes in liposomal structure. The lytic activity of sea cucumber saponins on liposomes depended on their chemical structure.These results suggest that sea cucumber saponins as monomers can interact with liposomes and that cholesterol serve as a principal binding site for the sea cucumber saponins.     

Effect of covalent attachment of immunoglobulin fragments on liposomal integrity

Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.     

Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel.

The stability of the sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) of the electric eel, Electrophorus electricus, was studied in five detergents in an effort to establish conditions for reconstitution of this membrane protein into defined phospholipids. The Na,K-ATPase activity of purified electric organ membranes as well as the ATPase is stable for at least 1 month of storage at 0 degrees C in the absence of detergents. At low concentrations of detergents, the enzyme is also stable for several days, but irreversible inactivation occurs rapidly as the detergent concentration is further increased. This inactivation begins at well-defined threshold concentrations for each detergent, and these concentrations generally occur in the order of the detergent critical micelle concentrations. Increasing the concentration of the electric organ membranes causes a linear increase in the inactivation threshold concentrations of Lubrol WX, deoxycholate, and cholate. The onset of inactivation evidently occurs when the mole fraction of detergent associated with the membrane lipids reaches a critical value in the narrow range of 0.2-0.4, in contrast to the large differences in the bulk concentrations of these detergents. The eel Na,K-ATPase is more sensitive to detergents than the sheep kidney enzyme.     

Variations in the activity of microsomal palmitoyl-CoA hydrolase in mixed micelle solutions of palmitoyl-CoA and non-ionic detergents of the triton X series

The kinetics of palmitoyl-CoA hydrolase were influenced by both the availability of the substrate and formation of micelles. At palmitoyl-CoA concentrations below the critical micelle concentration, addition of non-ionic detergent increased the activity until the critical micelle concentration of the mixed micelles was reached. At palmitoyl-CoA concentrations above the critical micelle concentration, inhibitor of the activity was observed, but addition of detergents of the Triton X series reversed the inhibition. Maximum palmitoyl-CoA hydrolase activity was found when the ratios (w/v) of palmitoyl-CoA: Triton X-100 and palmitoyl-CoA: Triton X-405 were approximately 0.35 and 0.05, respectively. At these above the mixed critical micelle concentration. The results indicate that monomer palmitoyl-CoA is the substrate and that monomer forms of the non-ionic detergents of the Triton X series activate the enzyme. Isolated microsomal lipids activated the microsomal palmitoyl-CoA hydrolase, suggesting that a hydrophobic environment is advantageous for interaction between enzyme and substrate in vivo. The maximum activity in the presence of mixed micelles is discussed in relation to a model where mixed micelles are regarded as artificial membranes to which the enzyme may adhere in an equilibrium with the monomer substrate and detergent in the monomer form. It is suggested that intracellular membranes may resemble mixed micelles in equilibrium with detergent-active substrates such as palmitoyl-CoA.     

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

Vesicle to micelle transitions of egg phosphatidylcholine liposomes induced by nonionic surfactants, poly(oxyethylene) cetyl ethers.

Vesicle to micelle transitions of sonicated liposomes of egg yolk phosphatidylcholine (EPC) induced by a homologous series of nonionic surfactants, poly(oxyethylene) cetyl ethers [POE(n) cetyl ether], were investigated by using the method of turbidity titrations. The turbidities of the mixed dispersions of sonicated vesicles and surfactant were systematically measured as a function of the surfactant added for a wide range of lipid concentrations (from 0.51 to 6.35 mM EPC). From the titration curves, two threshold points representing onset and complete solubilization of liposomal membranes were determined as a probe for the effect of the length of ethylene oxide (EO) moiety on the phase behavior of ternary system of POE(n) cetyl ethers-EPC-excess water. Patterns of turbidity curves and the surfactant concentrations at two threshold points as well as widths of region between two transitions, where lamellar sheets and mixed micelles may coexist, mainly depended on the length of EO head group. With changing the lengths, solubilization of liposomes and phase diagram showed optimal behavior. That is, in the middle range of EO numbers, it resulted in narrowest coexistence region between onset and complete solubilization. Assuming the equilibrium partitioning model, critical effective molar ratios of surfactant to lipid, Rsat, free surfactant concentrations, Dw, and the partition coefficient of surfactant between bilayer and aqueous phase, K, in surfactant-saturated liposomes were quantitatively determined as a function of EO number. Effective ratios, Rsol, and free surfactant concentration in mixed micelles were also determined. In addition, the effects of CMC and HLB of surfactants on the solubilization of liposome were discussed.     

5-Dodecanoylaminofluorescein as a probe for the determination of critical micelle concentration of detergents using fluorescence anisotropy

A method for determining the critical micelle concentration (CMC) of various detergents based on fluorescence polarization (anisotropy) of the lipophilic probe 5-dodecanoylaminofluorescein is presented. Nonionic, cationic, anionic, and steroid-based detergents can all be evaluated by this method and the determined CMC values of selected detergents agree well with those reported in the literature. In addition, we report the CMC of domiphen bromide, whose CMC value has not previously been described. In the case of ionic detergents, the method described is particularly sensitive at discerning changes in the CMC with increasing ionic strength of the medium and can discriminate detergent CMCs in 5 mM versus 25 mM buffering components. The described fluorescence polarization technique allows very low (submicromolar) concentrations of probe to be employed, thus minimizing the perturbation of micelle formation by 5-dodecanoylaminofluorescein insertion.     

The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octyl beta-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70-80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

Liposomal encapsulation of yeast alcohol dehydrogenase with cofactor for stabilization of the enzyme structure and activity

Yeast alcohol dehydrogenase (YADH) with its cofactor nicotinamide adenine dinucleotide (NAD+) could be stably encapsulated in liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine). The YADH- and NAD+-containing liposomes (YADH-NADL) were 100 nm in mean diameter. The liposomal YADH and NAD+ concentrations were 2.3 mg/mL and 3.9 mM, respectively. A synergistic effect of the liposomal encapsulation and the presence of NAD+ was examined on the thermal stability of YADH at 45 and 50 degrees C. The enzyme stability of the YADH-NADL was compared to the stabilities of the liposomal YADH (YADHL) containing 3.3 mg/mL YADH without NAD+ as well as the free YADH with and without NAD+. Free YADH was increasingly deactivated during its incubation at 45 degrees C for 2 h with decrease of the enzyme concentration from 3.3 to 0.01 mg/mL because of the dissociation of tetrameric YADH into its subunits. At that temperature, the coexistence of free NAD+ at 3.9 mM improved the stability of free YADH at 2.3 mg/mL through forming their thermostable complex, although the stabilization effect of NAD+ was lowered at 50 degrees C. The turbidity measurements for the above free YADH solution with and without NAD+ revealed that the change in the enzyme tertiary structure was much more pronounced at 50 degrees C than at 45 degrees C even in the presence of NAD+. This suggests that YADH was readily deactivated in free solution due to a decrease in the inherent affinity of YADH with NAD+. On the other hand, both liposomal enzyme systems, YADH-NADL and YADHL, showed stabilities at both 45 and 50 degrees C much higher than those of the above free enzyme systems, YADH/NAD+ and YADH. These results imply that the liposome membranes stabilized the enzyme tertiary and thus quaternary structures. Furthermore, the enzyme activity of the YADH-NADL showed a stability higher than that of the YADHL with a more remarkable effect of NAD+ at 50 degrees C than at 45 degrees C. This was considered to be because even at 50 degrees C the stabilization effect of lipid membranes on the tertiary and quaternary structures of the liposomal YADH allowed the enzyme to form its thermostable complex with NAD+ in liposomes.     

Solubilization and reconstitution of membrane proteins of Escherichia coli using alkanoyl-N-methylglucamides

Alkanoyl-N-methylglucamides, nonionic detergents, were utilized to solubilize membrane proteins of Escherichia coli and were used to reconstitute them into liposomes. First, critical micelle concentrations (CMC) of nonanoyl-N-methylglucamide and decanoyl-N-methylglucamide were determined to be 25 mM and 7 mM, respectively, by photometric assay. Then solubilization and reconstitution of the melibiose transport carrier were performed using these detergents at concentrations above the CMC. Melibiose counterflow activity was observed with the proteoliposomes reconstituted from the extracted proteins and phospholipids. The proton-translocating ATPase complex (F1-F0) was also solubilized with these detergents. These results indicate that nonanoyl- and decanoyl-N-methylglucamide are useful detergents for solubilization and reconstitution of membrane proteins.     

Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge

Upon mixing detergent solutions with the neutral fluorescent molecule 1,6-diphenyl-1,3,5-hexatriene a large increase in fluorescence is observed if detergent exceeds the critical micelle concentration. This property has been used to determine the critical micelle concentration of anionic, uncharged, zwitterionic, and cationic detergents. Regardless of detergent charge, the critical micelle concentrations obtained agree with the values obtained by other methods. This fluorescence assay is both sensitive and rapid, and should provide a simple and general method for determination of critical micelle concentration of any detergent.     

Studies of the spontaneous transfer of retinol from the retinol:retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37 degrees C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol:retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

Studies of the spontaneous transfer of retinol from the retinol: retinol-binding protein complex to unilamellar liposomes

The transfer of retinol from its complex with the retinol-binding protein to cell surfaces was studied using unilamellar liposomes as a cell surface model. The transfer of retinol to liposomes at 37°C was rapid and reached an apparent equilibrium within 60 min. The amount of retinol transferred to the liposomes at equilibrium was directly proportional to the starting concentration of retinol: retinol-binding protein over a wide range of retinol:retinol-binding protein concentrations and also directly proportional to the concentration of liposomal phospholipid in the system, when the concentration of retinol:retinol-binding protein was held constant. The transfer increased slightly with temperature. Transfer was increased by a factor of 1.8 at pH 4.5 compared to pH around 7. Prealbumin in amounts sufficient to complex all retinol:retinol-binding protein, decreased retinol transfer to liposomes indicating that prealbumin increases the affinity of retinol-binding protein for retinol. Addition of apo retinol-binding protein to the system decreased the transfer of retinol to liposomes considerably probably through competition with the liposomes for retinol. In similarly designed experiments delipidated bovine serum albumin competed much less with liposomes for retinol. The results show that spontaneous transfer of retinol from the retinol:retinol-binding protein complex to liposomal membranes occurs in vitro and suggests that a similar transfer may occur in vivo from retinol:retinol-binding protein to cell surface membranes.     

Determination of the aggregation number of detergent micelles using steady-state fluorescence quenching.

The development of a simple, reliable method for determination of detergent micelle aggregation number that relies solely on measurement of steady-state fluorescence quenching is presented. The degree of steady-state fluorescence quenching of a micelle-solubilized fluorophore (pyrene) by a quencher that partitions greatly into the micelles (coumarin 153) is dependent on the micelle concentration, which can therefore be determined. The aggregation number is calculated as the micelle concentration/detergent monomer concentration (the total detergent concentration above the critical micelle concentration). For the determination to be accurate, the partition coefficient of the quencher into the micelle phase is determined and used to calculate the micellar concentration of quencher. Also, the quenching of pyrene by a coumarin 153 molecule within the same micelle must be complete, and this was confirmed by time-resolved fluorescence measurements. Aggregation numbers were determined for one cationic and several nonionic detergents and were found to be consistent with literature values. The approach presented is an improvement on a previous luminescence quenching technique (Turro, N.J., and A. Yekta. 1978. J. Am. Chem. Soc. 100:5951-5952) and can be used on cationic, anionic, and nonionic detergents with micelles ranging greatly in size and under varying conditions, such as detergent concentration, ionic strength, or temperature.     

Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effects of the concentration and type of detergent

We have established the generality of using detergents for facilitating the reactivation of 6 M guanidinium chloride-denatured rhodanese that was recently described for the nonionic detergent lauryl maltoside (LM) (Tandon, S., and Horowitz, P. (1986) J. Biol. Chem. 261, 15615-15618). We report here that not only LM but other nonionic as well as ionic and zwitterionic detergents also have favorable effects in reactivating the denatured enzyme. Not all detergents are useful, and the favorable effects occur over a limited concentration range. Above and below that range there is little or no effect. Zwittergents, which represent a homologous series with varying critical micelle concentrations (CMCs) are effective only above their CMCs. Induction phases occur in the progress curves of rhodanese refolded in the presence of the effective detergents, suggesting the presence of refolding intermediates that are apparently stabilized by detergent interactions. Gel filtration chromatography of rhodanese with and without LM suggests that even though the renaturation of the denatured enzyme requires detergent at concentrations above its CMC, the enzyme does not bind an amount of detergent equivalent to a micelle. It is suggested that renaturation of other proteins might also be assisted by inclusion of "nondenaturing" detergents, although the optimal conditions will have to be determined for each individual case.     

A detergent-activated tyrosinase from Xenopus laevis. II. Detergent activation and binding

Tyrosinase purified from Xenopus is enzymatically inactive in aqueous buffers but is activated for both of its substrates by exposure to a variety of anionic detergents. Cationic and nonionic detergents, as well as a variety of other agents are ineffective. This stimulation by detergents is observed at all stages of the purification (Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 12535-12541). Sodium dodecyl sulfate (NaDodSO4) is the most effective activator, and it was chosen for further characterization. Activation of both activities by NaDodSO4 is rapid and concentration dependent, resulting in maximal activity after 4 min at 1 mM NaDodSO4. NaDodSO4 treatment also results in both long and short term stabilization of the enzyme. The activation and stabilization are separable but stoichiometrically related. Both effects occur well below the critical micelle concentration suggesting that the interaction of NaDodSO4 monomers with the enzyme is involved in these processes. In support of this suggestion, the enzyme is shown to bind NaDodSO4 with high affinity, as determined by equilibrium dialysis. The isotherm for this binding correlates well with the requirement of NaDodSO4 for both activation and stabilization. All three effects are observable at 3 X 10(-5) M NaDodSO4 in the presence of 0.1 M sodium chloride. Activation and stabilization are maximal at 6 X 10(-4) M NaDodSO4, the critical micelle concentration of NaDodSO4 under these conditions.