ANALYTICAL BIOCHEMISTRY 212, 154-159 (1993) Phase Separation of Nonionic Detergents by Salt Addition and Its Application to Membrane Proteins Beate Fricke Institute for Biochemistry, Department of Medicine, Martin Luther University, 0-4010 Halle, Germany Received October 13, 1992 By adding salts (sodium chloride, ammonium sulfate), possible to induce phase separation in membrane- solubilisates containing Triton X-100 or Noni- det P-40 at temperatures between 0 and 20°C. Other nonionic detergents of the Brij, Lubrol, and Tween se- ries can also be used for this procedure. The salt con- centration required for induction of phase separation is dependent on the hydrophobicity of the detergent used. For detergents of the Triton series it seems that deter- gents with lower hydrophilic lipophilic balance num- bers need lower salt concentrations to separate the phases than those with larger hydrophilic parts. Ammo- nium sulfate precipitation as an initial purification step for membrane proteins should be avoided in the pres- ence of the nonionic detergents tested. Instead of this procedure, phase separation induced by sodium chlo- ride or ammonium sulfate can be recommended, as was proved for membrane-bound proteases of Pseudomonas aeruginosa and Bacillus cereus and for bacteriorhodop- Detergents of the octylphenolpolyoxyethylene type (trade names: Triton or Nonidet)" are among the most used detergents for solubilization procedures of eukary- otic and prokaryotic membranes (1-3). Because of their excellent solubilizing and nondenaturating properties and their moderate price, they are important tools in membrane biochemistry. But only Triton X-114 is able ‘octylphenolpolyethylen- ‘elycolether; Brij 35, dodecylpolyoxyethyleneglycoletheryy; Tween 20, polyoayethylene,,-sorbitane-monolaurate; Tween 80, polyoxyeth- polyoxvethyleney- ylene-sorbitene-monooleate; Lubrol Y7AL7, lauryl” ether; sulfobetaine SB-12-N-dodecy! propane-sulfonate; CCP, casein-cleaving protease; TCA, trichloroacetic acid; ICP, intulin-claaving protease; CMC, evitical mi- celle concentration; PEG, polyethylene glycol. 154 to form separate phases at room temperature (4, 5); other Triton detergents possess higher cloud points. For example, Triton X-100 has its phase transition point at temperatures above 65°C (6)—a temperature which would lead to thermal inactivation of some membrane proteins. During the purification of membrane proteins, the phase separation with Triton X-114 often replaces the ammonium sulfate precipitation step, because this procedure makes it possible to efficiently separate hy drophobic proteins from contaminating hydrophili proteins with an additional concentrating effect (4, 5). Unfortunately, an additional detergent exchange is of- ten necessary if the detergent used for solubilization and further purification is not identical to Triton X-114. ‘A phenomenon which had not been described previ- ously appeared during solubilization experiments for halobacterial membrane proteases (7) in the presence of high sodium chloride concentrations. Detergents such as Triton X-100 and Nonidet P-40 were found insufli- ciently soluble with buffers containing high sodium chlo- ride concentrations (20-25% w/v NaCl). At first, the solutions became turbid, and then after standing for some hours the detergents separated from the aqueous solution and formed a clear detergent phase at room temperature. Similar effects were caused by ammonium sulfate with other nonionic detergents. In this article, the interactions of different nonionic as well as ionic detergents with salts were examined at different salt and detergent concentrations. The aim was to develop a phase separation method with some other nonionic detergents, similar to the phase separa- tion with Triton X-114. An additional aim was to inves tigate whether ammonium sulfate precipitation is prac- ticable for membrane protein purification in the presence of detergents. MATERIALS AND METHODS Chemicals ‘Triton X-100, Triton X-405, Tween 20, and Tween 80 were obtained from Ferak (Berlin, Germany). Nonidet 0003-26878 $5.00 Copyright © 1903 by Acndemie Prev ne [Lights of eprodction in any form reservedP-40 was purchased from Sigma (Deisenhofen, Ger- many). Lubrol 17A17, Brij 35, sulfobetain SB-12, so- dium cholate, and sodium deoxycholate were delivered by Serva (Heidelberg, Germany). Purple membranes of Halobacterium halobium were a generous gift from Prof. D. Oesterheldt (Max-Planck-Institut Martinsried, Ger- many). Bacteriorhodopsin was solubilized and '“C-acetylated as described recently (7). All other chemicals were of analytical grade. Methods Determination of phase separation. All detergents used were dissolved in distilled water in a concentration of 100 g/liter. Detergent solutions were mixed with various volumes of an ammonium sulfate (400 g/liter) or sodium chloride stock solution (250 g/liter) and dis- tilled water to obtain salt dilution series, always con- taining 2% detergent. Formed clouding was measured at 600 nm immedi- ately after vigorous mixing against blanks consisting of the same percentage detergent in distilled water. An i creased optical density was the first sign for a beginning phase separation. About 12 h after mixing or a centrifu- gation time of 20 min (20,000g) the detergent and the aqueous phases were completely separated. For Triton X-100 and Nonidet P-40 the detergent concentration in the lower phase was spectrophometri- cally determined at 280 nm (Diode Array System DU 7500, Beckman, Fullerton, CA) for a concentration range of 0.05-2% (w/v) detergent/assay. After centrifu- gation the plastic tubes were punctured from the bot- tom and the lower phase was carefully removed, Deter- gent concentrations were determined from a calibration curve. Phase partition with “C-acetylated bacteriorhodop- sin. “C-Bacteriorhodopsin (50 xl, corresponding to 200,000 dpm) was diluted with 2 ml NaCl (25% w/v) and 2 ml 4% (w/v) Nonidet P-40 or 2 ml ammonium sulfate (concentrations 16 oF 24% w/v) and 2 ml 4% (w/v) Non- idet P-40. The samples were shaken for 5 min. After standing overnight at room temperature the phases were completely separated. The phase volume was de- termined and aliquotes were mixed with a scintillator for measurement in a TriCarb (LC-6000, Beckman). Cultivation of bacteria. Pseudomonas aeruginosa strain PAO 1 was obtained from the strain collection of the Institute for Biochemistry, University, Leipzig, Ger- many). Bacteria were cultivated on a complex medium, consisting of 10 g yeast extract (Difco, Detroit, MI), 5 g (NH),PO,, 22 g KH; PO,, 10.1 g NasHPO, x 12H,0, 0.2 ¢ MgSO, x 7H,0, and 0.2 g CaCl, per liter medium (pH 7.0) in a fermentor (Biostat S, Braun, Melsungen, Germany) at 37°C until the end of the exponential growth, :TERGENTS BY SALTS 155 Bacillus cereus was isolated from soil probes and clas- sified by Dr. Verbarg (German collection of microorgan- isms, Braunschweig, FRG). This strain was grown under similar cultivation conditions, but at a tempera- ture of 32°C. Freshly harvested bacterial cells were washed with an excess of sodium chloride solution (5 g/liter) and sus pended in Tris-HCI buffer (buffer A, 0.05 M, pH 7.5) in a cell concentration of about 100 mg wet wt/ml. Cells of P. aeruginosa were disintegrated by stirring with an equal volume glass beads (d = 0.10-0.11 mm) as described re- cently (8). Cells of B. cereus were disrupted by ultrasonic treatment for 30 min (30x 1 min with 1 min interme- diate cooling in an ice bath). Cells remaining intact were removed by centrifugation (4000g, 15 min). Cell homogenate was separated in the particular (crude cell envelopes) and the soluble cell components (cytosol and periplasm) (2 h, 60,000g), resuspended, and washed again under the same conditions. Crude cell en- velopes were stored at ~20°C. Solubilization of bacterial membranes. Cell enve- lopes were homogenized in an equal volume of Nonidet P-40 or Triton X-100 (8 g/100 ml, dissolved in buffer A) and shaken (180 rpm) at room temperature. The sedi- ment was separated from the supernatant by centrifuga- tion (1h, 60000g). Phase separation in the Nonidet P-40 solubilisates was induced by addition of solid sodium chloride up toa concentration of 15% (w/v). Phase partition of Triton X-100 solubilisates was caused by mixing with anammo- nium sulfate solution (40% w/v) to a final concentration of 10% ammonium sulfate in the solubilisate. After cen: trifugation (20 min, 20,000g) the upper phase was taken from the top. The excess of detergent or salt was re- moved by overnight dialysis against buffer A (0.1% w/v detergent) with several buffer exchanges. The protease activities of dialyzed solubilisates and upper and lower phases were determined. Determination of proteolytic activities. Leucyl amin- opeptidase was determined with leucy! hydrazide as substrate (9). Azocasein was used as substrate for the casein-cleaving proteinase (CCP) of B. cereus. TCA-s0- luble peptides of azocasein formed by the proteolytic cleavage were measured at 366 nm (10). ‘The insulin- cleaving proteases (ICP) of P. aeruginosa and B. cereus were determined with 'I-insulin as described recently (11). RESULTS. As result of mixing with sodium chloride, the Nonidet P-40 solution became turbid at NaCl concentrations beyond 10% (Fig. 1). Increased turbidity at 600 nm in comparison to the same detergent solution without salt always resulted in a phase separation. The volume of156 1a-——— ‘0D (600 nm) 0 5 10 15 20 25 NaCl (%) FIG. 1. Dependence of the clouding point of ortylphenol polyethy: Ienegiycol detergents (2% w/v) at room temperature (20°C) on the sodium chloride concentration (% w/v). (@) Nonidet P-40 (NP-40) (4) Triton X-100 (TX-100). (Z) Triton X-405 (TX-405). Measure: ‘ment must be performed 10 s after vigorous mizing to prevent phase the upper phase decreased dependent on the salt con- centration used, Beyond the critical micelle concentra- tion (CMC) the separation of detergent from the aqueous phase was a function of the salt concentration only. ‘The same phase separation behavior could be ob- served in a concentration range from 0.1-2% (w/v) Nonidet P-40 and at a constant NaCl concentration of 12.5% (w/v). The lower phases contained detergent amounts of only about 0.05% (w/v) independent of the detergent concentration used, also near the CMC (12). Phase separation of Triton X-100 solutions occurred at higher concentrations of sodium chloride (beyond 14%). Triton X-405 could not be forced to phase separa- tion by sodium chloride (Fig. 1). When sodium chloride ‘was replaced by ammonium sulfate, Triton X-405 solu- tions also showed a phase separation (Fig. 2). Lower salt concentrations were needed than for sodium chloride attempts. ‘The ammonium sulfate concentration should not ex- ceed 18% for Nonidet P-40 or 20% for Triton X-100 and ‘Triton X-405, because the detergents formed solid ag- gregates beyond these concentrations. The optical den- sity at 600 nm was further decreased. Separating the phases was not possible using the described techniques. Several other nonionic as well ionic detergents were subjected to the same tests. The results are summarized in Table 1. Brij 35, Lubrol 17A17, Tween 20, and Tween 80 showed bebavior similar to that of Triton X-405. Phase separation could only be induced by ammonium sulfate. The range of ammonium sulfate suitable for ap- BEATE FRICKE 1.6 Tx-100 1 12 (00 (600 nm) Tx-408 0 5 10 18 20 2 30 35 ‘Ammonium sulfate (%) FIG. 2. Dependence of the clouding point of octylphenol polyethy: leneglycol detergents (2% w/v) at room temperature (20°C) on the ‘ammonium sulfate concentration (% w/v). (m) Nonidet P-40 (NP-40) (4) Triton X-100 (TX-100). (1) Triton X-4085 (TX-405). plication of these detergents to phase separation of solu- bilized membrane proteins is shown in Table 1. ‘The investigations were also extended to the ionic de- tergents sodium deoxycholate and sodium cholate (Figs. 3 and 4). The stronger hydrophobic deoxycholate formed insoluble aggregates at lower salt concentra- tions than sodium cholate—a similar dependence be- tween the hydrophobicity and the salt concentration as for octylphenolpolyoxyethylen detergents (Fig. 1). The awitterionie detergent sulfobetain SB-12 showed an in- creased turbidity at higher ammonium sulfate concen- trations, but not a phase separation. TABLE 1 Salt Concentrations Usable for Phase Separation (Non- ionic Detergents) or Lowest Salt Concentration Causing De- tergent Precipitation (Ionic Detergents) in % (w/v) NaCl HLB numbert Nonionic detergents Nonidet P-40 10-25 131 ‘Triton X-100 16-25 9-23 135, ‘Triton X-405 16-28 179 ‘Tween 20 = 16-28 167 Tween 80 12419 150 Brij 95 36-19 169 Lubrol 17417 = 16-22 4 Tonic detergents Deoxycholate = 19 Cholate 8 6 Sulfobstain SB-12 2 * Values from the manufacturer.