Relation of Detergent HLB Number to Solubilization and Stabilization of D-Alanine Carboxypeptidase from Bacillus subtilis Membranes

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 70, No. 10, pp , October 1973 Relation of Detergent Number to Solubilization and Stabilization of D-Alanine Carboxypeptidase from Bacillus subtilis Membranes (hydrophobic lipophilic balance/nonionic detergents/succinate dehydrogenase/ phosphoacetylmuramyl-pentapeptide translocase) JAY N. UMBREIT* AND JACK L. STROMINGERt Biological Laboratories, Harvard University, Cambridge, Massachusetts 0213 Contributed by Jack L. Strominger, June 1, 1973 ABSTRACT The ability of various nonionic detergents to solubilize D-alanine carboxypeptidase and other membrane-bound enzymes was correlated to a physical property of the detergent, the number. Only a fairly narrow range of detergents were effective solubilizing agents. Purified carboxypeptidase required either a detergent or detergent plus a lipid fraction for stability. Only those detergents effective in solubilizing the enzyme were effective in stabilizing it. Among the most useful methods of solubilizing membrane enzymes is the use of nonionic detergents (1). Presumably the need for a detergent to solubilize a membrane protein is related to the protein's affinity for the lipoidal membrane phase. However, little is known about the mode of action of detergents in solubilizing proteins from membranes, nor is there any systematic approach for the selection and utilization of detergents. The present study of D-alanine carboxypeptidase from Bacillus subtilis (2) considers the relationship among the detergent, protein, and lipids. MATERIALS AND METHODS D-Alanine carboxypeptidase was assayed as described (2). Detergents were the gift of Imperial Chemical Industries, America (formerly Atlas Chemical Co.) or Rohm-Haas, Inc. numbers (, hydrophobic lipophilic balance) and other physical data were obtained from the technical literature provided by those companies or from Schick (). Lipids were obtained from B. subtilis cells by extraction according to the method of Bligh and Dyer (3). RESULTS Solubilization of D-Alanine Carboxypeptidase and Other Membrane-Bound Enzymes. Various nonionic detergents were used to attempt the solubilization of D-alanine carboxypeptidase from B. subtilis. Attempts to correlate the amount of activity solubilized to some physical or chemical property of the detergent revealed a striking dependence on the number of the detergent (Fig. 1). The (or hydrophobic lipophilic balance) number (4) is a measure of the relative hydrophobicity of the detergent, the most hydrophobic having an number approaching 0, and the most hydrophilic having a number of about 20. In general only those detergents with numbers between 12.4 and 13.5 were * Present address: Department of Biology, McCollum-Pratt Institute, Johns Hopkins University, Baltimore, Md t To whom reprint requests should be addressed effective in solubilizing the carboxypeptidase. The amount of solubilization obtained failed to correlate with surface tension, Draves test (wetting), Ross-Miller foaming index, interfacial tension, or critical micelle concentration (Fig. 2). Within a series of detergents with a common hydrophobic portion, solubilization did correlate with the length of the ethylene oxide chain (the water-soluble, hydrophilic portion), as does the number. Most important, the degree of solubilization did not depend, to a first approximation, on the chemical nature of the hydrophobic portion of the detergent. The chemical composition of the detergents used are given in Table 1. Some scatter was observed in the correlation with the number. For example,.brij 30 was a notable exception to the ft) I0 w z M. 0. (I) z 0. u A II s 11A I7x X14 2 I I I I I H L B Fig. 1. Solubilization of carboxypeptidase by different detergents. A sample of 250 Ml of membranes of B. subtilis in 100 mm Tris HCl (ph.6) was incubated for 30 min at 40 with 200 IAI of a 2% solution of detergent and then centrifuged (45,000 rpm, A321 rotor, International B-60 Centrifuge, 60 min). A 1-1Al aliquot of the supernatant solution was assayed. The use of about 1% detergent provided saturating concentrations of detergent (see ref. 2); addition of any of the detergents did not alter the total activity of the enzyme by more than 10%. Detergents used were indicated by a number assigned to the detergent in Table 1. 0, Triton; X, Tween; A, Atlas "G"; *, Brij.

2 299 Biochemistry: Umbreit and Strominger TABLE 1. Chemical composition of the nonionic detergents Hydrophile Polyoxyethylene, Assigned no. Trade name no Other Lipophile no. Brij t 1, 2 30, 35 4, 23 Lauryl alcohol 9.7, , 4 52, 56 2, 10 Cetyl alcohol 5.3, , 6 76, 7 10, 20 Stearyl alcohol 12.4, , 96, 9 10, 20 Oleyl alcohol 12.4, 15.3 Atlas Gt 9, , , 50 Sorbitol Hexaoleate 10.2, Not given Sorbitol Lanolin Monopalmitate Monolaurate 12. Tweent 14, 15 20, 21 20, 4 Sorbitan Monolaurate 16.7, Sorbitan Monopalmitate , 1 60, 61 20, 4 Sorbitan Monostearate 14.9, , 20 0, 1 20, 5 Sorbitan Monooleate 15.0, Sorbitan Trioleate 11.0 Spant Sorbitan Monolaurate.6 Tritont 23, 24, 25 X-35, X-45, X-114 3, 5, 7.5 Sorbitan Octylphenyl 7., 10.4, , 27, 2 X-100, X-102, X , 12.5, 16 Octylphenyl 13.5, 14.6, , 30- X-305, X , 40 Octylphenyl 17.3, , 32 N-101, N , 5 Nonylphenyl 13.4, X-67 (Alkylpolyether alcohol) X-155 (Alkylarylpolyether alcohol) , 36 CF-10, CF-21 (Alkylaryl polyether) CF-54 (Modified polyether adduct) DF-12 (Modified polyethoxylated alcohol) 39 CF-32 (Amine polyglycol condensate) 40 QS-15 (Sodium salt of amphoteric surfactant) Nonionic detergents used in this study had a general formula: hydrophile-lipophile. Polyethylene oxide (O-CHr-CHz-)nOH and/or another hydrophile provided the hydrophilic portion of the detergent. Detergents were classified according to the lipophilic portion. Different detergents with the same lipophilic portion differ in polyethylene content. A number was arbitrarily assigned to each detergent in order to represent it conveniently in the figures. This number is given in the left-hand column. Where more than one detergent is on the same line, they have the same lipophile and the assigned number and other properties are in sequence. * Number of ethylene oxide units. t From General Characteristics of Atlas Surfactants, Atlas Chemical Industries, In., Wilmington, Del. t From Rohm and Haas Surfactants (Technical bulletin), Rohm and Haas Co., Philadelphia, Pa. TABLE 2. Stability of Triton-dpleted D-alanine carboxypeptidase Activity, cpm Plus Plus 0. 1% 0.1% Triton Without Triton plus crude Treatment additions X-100 lipid Control (undepleted enzyme) Control (depleted enzyme) 30 min at 370, (ph 7.0) min at 450 (ph 5.5) hrs at 40 (ph.6) % dioxane M urea general rule. It is important to recall that these detergents are technical grade, commercial products and contain a family of chain lengths centered around a given number. In addition the distribution may vary slightly from bottle to bottle. Since all the studies were performed at high concentrations of detergent, the concentration of the components at the extremes of the chain length distribution may be high enough, in some cases, to affect solubilization. This could account for most of the scatter observed. Two other enzymes found in bacterial membranes, phosphoacetylmuramyl pentapeptide translocase (5) and succinate dehydrogenase, were also solubilized by use of a less extensive series of detergents. Again the correlation between solubilization and numbers was remarkable (Fig. 3). Of these three enzymes, only D-alanine carboxypeptidase has been purified. The observation that three apparently unrelated enzymes require detergents with the same number for solubilization suggests that the property may be quite general. Indeed, polyacrylamide gel electrophoresis in

3 Nonionic Detergents and Membrane-bound Enzymes lp a. C-, 4-25 *.26 i * _05 0 * 2-13' IS - 6 * i *03 27 a9 * 0 ' * ) D :) SURFACE TENSION OF 1% SOLUTIONS CRlTICAL MICELLE CONCENTRATIOK )N INTERFACIAL TENSION OF A 1% (DYNES/cm) (grams/1ooml) SOLUTION (DYNES/MI) AT 25, ATREOL #9 (MINERAL OIL)against H P _ Q *27 c 3 2 ~ ROSS-MILES INITIAL FOAM HEIGHT DRAVES TEST FOR 25 SEC AT 120 (0.1% SOLUTION), mm WETTING, PERCENT ACTIVE INGREDI ENT Fig. 2. Relationship between solubilization of D-alanine carboxypeptidase and physical properties of nonionic detergents. The data were replotted from Fig. 1 or were obtained with different detergents in a manner identical to that described in Fig. 1. Values for the physical properties were obtained from the technical literature provided by the manufacturer or from Schick (). When different values were found for the same detergent, both values were plotted. Because some of the physical parameters were not available for all detergents, some detergents used ih this figure do not appear in Fig. 1, and visa versa. For any given lipophile, e.g., octylphenyl, the physical properties are not independent variables of the ethylene oxide content, or number. The detergents used were indicated by a number assigned to the detergent in Table 1. sodium dodecyl sulfate of membranes solubilized by Triton X-100 showed the same number of protein bands as whole membranes. X T o25 Stimulation of D-Alanine Carboxypeptidase Activity by Detergent. Purified D-alanine carboxypeptidase (2) could be absorbed to calcium phosphate gel and eluted with ammonium sulfate. If the gel was washed and eluted with buffers lacking Triton X-100, the eluted activity, depleted of detergent, was stimulated % by addition of exogenous Triton X-100 (Fig. 4). Different detergents stimulated the enzyme to different extents, and, as in solubilization, the ability to stimulate the detergent-depleted enzyme could be correlated to the number (Fig. 5). The correlations for stimulation (Fig. 5) and solubilization (Fig. 1) were nearly coincidental. The slight shift (equivalent to less than one ethylene oxide unit in a homologous series) of the stimulation curve to higher Fig. 3. Solubilization of membrane-bound enzymes. The procedure was the same as described in Fig. 1, except that membranes frots M. luteus were used. (A) The translocase, which catalyzes the synthesis of C55-isoprenyl-pyrophosphate-MurNAcpentapeptide from UDP-MurNAc-pentapeptide was assayed as described (5) in the presence of lipids. The detergents used were indicated by a number assigned to the detergent in Table 1. Tween 21, detergent number 15, failed to give a homogeneous solution, and some detergent was centrifuged into a pellet. These factors might have prevented appreciable solubilization of enzymic activity. (B) Succinate dehydrogenase activity, expressed as per cent of untreated membranes, was assayed by ferricyanide reduction (6). The detergents used were indicated by a number assigned to the detergent in Table 1. I I I. I. Ko,0 0O I PERCENT TRITON X-100 FIG. 4. Effect of addition of Triton X-100 to partially depleted carboxypeptidase. Enzyme eluted from calcium phosphate gel in the absence of Triton was assayed as described in Methods.

4 3000 Biochemistry: Umbreit and Strominger Proc. Nat. Acad. Sci. USA 70 (197) P- lipids Triton 10 k J t - so x 6- B I) 1.0- X (?~~~ /\ r\ f\ A FIG. 5. Effect of different detergents on partially depleted carboxypeptidase. The enzyme eluted from calcium phosphate gel in the absence of Triton X-100 was assayed as described in Methods with addition of detergents at a final concentration of 0.1% (w/v). The detergents used were indicated by a number assigned to the detergent in Table 1. numbers could have been due to the chain-length heterogeneity of the detergent samples, or to the difference in ph and temperature between assay of enzymatic activity and soltubilization. Role of Detergent and Lipid in Enzymic Activity. Stimulation by detergent could be due to the increased stability of the purified enzyme in the presence of detergent. The increased stability was illustrated by incubation at 450, where addition of detergent (Triton X-100) decreased the rate of inactivation (Fig. 6). The requirement of a detergent for stability suggested that lipids might play that role physiologically. A crude chloroform-methanol extract of the bacteria was even more effective than detergent aione in stabilizing the enzyme (Fig. 6). Stabilization by detergent or detergent and lipid occurred under several different conditions (Table 2), and inactivation of the depleted enzyme could occur at 40 overnight. The If) AT ZoI FRACTION NUMBER FIG. 7. Gel filtration of the carboxypeptidase at ph 5.5 in the absence of lipid or detergent (A) or in the presence of lipid (B). (A) An aliquot of 10 MAl of enzyme eluted from calcium phosphate gel with 7% (NH4)2SO4 in 1% Triton X-100 was applied to a 5-ml column of Sephadex G-100 equilibrated in 100 mm cacodylate buffer (ph 5.5) without added Triton X-100 and eluted with the same buffer. 60 Fractions of were collected over 3 hr. Assays were performed on 15 Mul of each fraction in the presence of 0.1% Triton X-100. The activity obtained is shown by the dashed line. (B) In order to form the enzyme-lipid complex, a lipid extract containing 750,ug of organic phosphate was dried under reduced pressure, suspended in 100 u1 of 0.5 A1I cacodylate buffer (ph 5.5) containing 1% Triton X-100, and sonicated for 30 min. To this 10 ul of enzyme was added; the mixture was then sonicated for 1 min at 4. The suspension was added to a column identical to that in A and elution was performed as described above. Thin-layer chromatography of the fractions indicated that all the detectable phospholipid was in the void volume of the column with the activity and all the detectable Triton X-100 was in the column volume. 10 Mul of every other fraction were spotted on a silica gel G thin-layer plate and chromatographed in chloroform-methanol-water-glacial acetic acid 125:37:2:10. The phospholipids (RF 0.17, 0.4, 0.57) and Triton X-100 (RF 1.0) were detected by iodine vapor. Activity, solid line. enzyme in the presence of detergent was stable for over months at 4. This evidence suggested that the detergent played a role in the purified enzyme equivalent to the role of lipid in the membrane. There appeared to be no specificity for the lipid used, since many fractions from a silicic acid 60 x x 0. I _ W 0.6.I I MINUTES AT 450 FIG. 6. Effect of Triton X-100 and lipids on stability of partially depleted carboxypeptidase. The enzyme was incubated in 10 Ml of 0.5 M cacodylate buffer (ph 5.5) ior up to 30 mini at 450 in the absence of added Triton X-100 (0), in the presence of 0.1% Triton X-100 (X), or in the presence of 0.1% Triton and a crude lipid extract (containing 75 nmol of phosphate) (.). After a given time, substrate was added and the assay was continued as described in Methods. Lipid alone could not be assayed because it was not soluble in buffer without detergent. I , i ~~~~X/ FIG.. Sensitivity of the lipid-carboxypeptidase complex to propicillin. The apparent rate of inactivation by 0.02 mg/ml of propicillin was measured for the purified enzyme in Triton X-100 (X), the crude membrane-bound enzyme (.), and the enzyme-lipid complex eluted in the void volume of a G-200 Sephadex column (0). The method used was the same as that in the preceding paper (7). sec

5 column used for lipid separation protected against thermal inactivation at 450. Interaction of Lipid and Purifed Enzyme. At low ph, solubilization of D-alanine carboxypeptidase from membrane by Triton X-100 was poor (2), suggesting that at low ph the protein-detergent interaction was weak. Gel filtration of the purified enzyme in the absence of Triton X-100 at ph 5.5 (Fig. 7, curve A) led to a large decrease in activity (in contrast to gel filtration at higher ph, see Fig. 13 in ref. 2) and considerable inclusion of the remaining activity into the gel. Since the active form of the protein in the presence of detergent was a detergent-protein micelle of large molecular weight excluded from the gel, this behavior on gel filtration in the absence of detergent was interpreted as a breakdown of the aggregate and subsequent inactivation. When a crude choloroform-methanol extract of B. subtilis was emulsified with the purified enzyme (which still contained Triton X- 100) before gel filtration, the activity was retained and found in a large-molecular-weight aggregate with the lipid (Fig. 7, curve B). However, the residual detergent was now included in the gel, i.e., the enzyme had been transferred from a detergent micelle to a lipid micelle. 50% of this activity could be sedimented at 120,000 X g in an hour, whereas the purified enzyme in detergent could not be sedimented under these conditions. The enzyme-lipid aggregate formed in this way had a sensitivity to j3-lactam antibiotics similar to the membranebound enzyme in contrast to lower sensitivity for the purified enzyme in detergent (Fig. ). DISCUSSION Detailed investigation of reactions catalyzed by membranebound enzymes and their control, and of the interaction between membrane and enzyme has been hampered by the difficulty of obtaining membrane enzymes in pure form. Nonionic detergents may provide a more or less general approach to solubilization of membrane components and provide a wide diversity of types of chemicals that can be used. Nonionic detergents can be classified by their number (4). Those detergents with an number about were effective in solubilizing proteins from membranes. This was demonstrated for three different enzymes, D-alanine carboxypeptidase of B. subtilis, and phosphoacetylmuramylpentapeptide translocase and succinate dehydrogenase, both from M. luteus. However, it seems possible that other more Nonionic Detergents and Membrane-bound Enzymes 3001 hydrophobic or more hydrophilic membrane enzymes or other proteins would be solubilized optimally by nonionic detergents in a different range. There might be two different ways a detergent could act in solubilization of a membrane protein. It might dissolve the membrane lipid, leaving the protein free in solution; it might dissolve the protein itself; or both might occur; i.e., protein and/or lipid might be bound to detergent in such a way as to render them soluble. In the present case D-alanine carboxypeptidase existed in a detergent micelle (2), and interaction of detergent and protein was demonstrated. Furthermore, the range of numbers required for a detergent to interact with and stabilize the enzyme was very similar to the range required to solubilize the protein. The detergent micelle could be thought of as providing an environment like the original lipid environment of the membrane. The enzyme could be inserted into the detergent micelle from the true membrane by a process akin to partition between solvents. In some cases, the detergent might not be a sufficiently "close fit" to the natural lipid environment required by the enzyme to maintain conformational integrity. Such behavior would be observed as an inactivation of the enzyme by detergents known to be effective solubilizers. This phenomenon occurred with the translocase (5), and in this case, the lipids have to be returned to the enzyme in order for the enzyme to be assayed. Presumably related to this is the observation that whereas D-alanine carboxypeptidase shows no specificity for the lipids used in stabilization, the translocase had more stringent requirements. In order to purify such an enzyme, lipids might have to be emulsified with the buffers. Supported by research grants from USPHS (AI and AM ) and NSF (GB-29747X). This is paper XXXV in the series Biosynthesis of the Peptidoglycan of Bacterial Cell Walls. 1. Tzagoloff, A. & Penefsky, H. S. (1971) in AMethods in Enzymology, ed. Jakoby, W. B. (Academic Press, New York), Vcl. 22, pp Umbriet, J. N. & Strominger, J. L. (1972) J. Biol. Chem. 24, in press. 3. Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, Griffin, W. G. (1949) J. Soc. Cosmet. Chem. 1, Umbreit, J. N. & Strominger, J. L. (1972) Proc. Nat. Acad. Sci. USA 69, Datta, A. & Franklin, R. M. (1969) Virology 39, Umbreit, J. & Strominger, J. L. J. Biol. Chem. 24, in press.. Schick, M. J.; ed. (1967) Nonionic Surfactants (Marcel Dekker, New York).