The synthesis of a photolabile detergent and its use in the isolation and characterization of protein

ANALYTICAL

BIOCHEMISTRY

The Synthesis

119, 304-3 12 ( 1982)

of a Photolabile Detergent and Its Use in the Isolation and Characterization of Protein

W. W. EPSTEIN,*

D. S. JONES,*

E. BRuENGER,t

Departments of *Chemistry and tbiochemistry,

AND H. C. RILLINGt

University of Utah, Salt Lake City, Utah 84112

Received June 16, 1981 A new ionic detergent sodium 4-( 3,3-dimethyl- 1-oxotridecyl)benzenesulfonate similar to sodium dodecyl sulfate has been synthesized which is photodegradable to sodium p-acetylbenzenesulfonate and a simple olefin mixture upon irradiation in aqueous solution with light 300 nm and above in wavelength. This photodegradable detergent can be used to solubilize many proteins and provide information as to the molecular weight and subunit composition of proteins by electrophoresis. The removal of this detergent by photolysis results in no apparent damage to protein.

Detergents have been extremely valuable surfactants for biochemists during the past several decades. They have been used for solubilizing membranes, thereby permitting the resolution of the individual components, including proteins, thus facilitating their isolation and characterization (l-3). Another, and very popular, use for detergents, such as SDS,’ has been the electrophoresis of proteins for the determination of their molecular weight and subunit composition (4). If further characterization of a protein is desired after it has been subjected to one of these procedures, it is usually necessary to remove the detergent quantitatively. Unfortunately, in many cases it is not possible to remove all of the detergent without denaturing the protein, and it is common to find several moles of detergent bound per mole of protein even after the most rigorous stripping procedures (3,4,6). Thus, although de-

tergents are useful for analytical procedures, their use in preparative work is limited. The physical properties of detergents which make them useful are the same properties that make them difficult to remove from proteins. The hydrophobic tail of the detergent will form stable complexes with hydrophobic regions of proteins. The strength of this interaction can be enhanced by ionic bonding between the ionic head of the detergent and ionic amino acids. The polar head group of nonionic detergents may also interact strongly with polar regions of polypeptides. In addition, when detergent micelles are dispersed in water, they may engulf or incorporate proteins by dissolving the peptide or portions of it in the hydrophobic interior. Another difficulty is that mixtures of proteins and micelles can be formed which are difficult to resolve by conventional techniques such as centrifugation or gel filtration. Quantitative removal of detergents from proteins usually entails extraction with solvents such as chloroform and methanol by ion-pair procedures (7) with resultant loss of biological activity of the protein. One way to overcome the difficulties brought about by the physical and chemical

’ Abbreviations used: SDS, sodium dodecyl sulfate; CMC, critical micelle concentration; HPLC, highpressure liquid chromatography; DNP, dinitrophenol; DMF, dimethylformamide; Tris, tris(hydroxymethyl)aminomethane; TLC, thin-layer chromatography. 0003-2697/82/020304-09%02.00/O Copyright 8 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

304

PHOTOLABILE

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SYNTHESIS

properties of detergents would be to sever the hydrophobic from the hydrophyllic portion of the detergent. If this could be accomplished, the hydrophobic part would become water insoluble and could be readily extracted into organic solvents, while the hydrophyllic end would no longer aggregate into micelles, nor would it interact strongly with the protein. As a consequence, the polar group could be removed by dialysis or gel filtration. Unfortunately, most covalent bonds require unacceptably harsh conditions for their cleavage. One exception is the disulfide bond which can be broken by disulfide interchange reactions and detergents containing this linkage have been prepared (8). However, because most proteins contain cysteine, they could become covalently linked to one half or the other of the detergent, thus defeating the purpose of having a readily cleavable detergent. Another approach, the subject of this paper, would be to insert a photolabile linkage in the detergent and to cleave the detergent with light. To be useful, this photolabile group should [ I] be reasonably stable under ordinary laboratory light conditions; [2] not produce long-lived radicals which could react with the macromolecules of interest; and [ 31 show an almost quantitative cleavage with little if any rearrangement simply leading to a different detergent molecule. Of the various known photochemical bondbreaking processes, the Norrish Type II cleavage of alkyl phenyl ketones appeared

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ANALYSIS

to be most suitable for our purposes for a number of reasons (9- 12). The cleavage process which proceeds through a 1,4-diradical intermediate is quite efficiently affected by radiation at wavelengths harmless to proteins. Cyclization of the diradical to form a cyclobutanol, is the only known side reaction; but that can be virtually eliminated by the presence of a quaternary carbon beta to the ketone (13). The feasibility of affecting p cleavage in a micellar environment has been demonstrated for the case where the alkyl phenyl ketone moiety is at the hydrophobic end of a surfactant ( 14- 16) and these reactions have already been used to probe micellar environment ( 17). Taking into account the above information and available knowledge about surfactants like SDS, we designed 1 as a target molecule which would act as a detergent and on photolysis undergo /3 cleavage to form the water-soluble compound, sodium p-acetylbenzenesulfonate (2) and an olefin mixture of 3 and 4, none of which would be surface active (Scheme I). EXPERIMENTAL

Melting points were determined with a Mel-Temp apparatus in open capillaries and are uncorrected. Infrared spectra were recorded on a Beckman Acculab 3 spectrometer. ‘H-NMR spectra were recorded on Varian 360 and 390 spectrometers with chemical shifts reported in 6 units downfield from internal tetramethylsilane. Ultraviolet SO; Na+

I h-i

3. 0

4. SCHEME

305

I.

2.

306

EPSTEIN

ET AL.

spectra for CMC determinations were recorded on a Beckman Model 24 Spectrophotometer. HPLC was performed on a Waters ALC 202 instrument with a 560 X IO-mm-o.d. Cl8 Porosil column using a differential 280-nm uv detector or a differential refractometer; Gas-liquid chromatography was performed on a Varian Aerograph A-90-P with a 30-ft X 3/8-in. Carbowax 20 M column and a Varian Aerograph Model 1200 using a lo-ft X l/8-in. Carbowax 20 M column. A Rayonet photochemical reactor with 8 RPR-3000A lamps was used for the photolysis experiments.

Decyl magnesium bromide. To a mixture of 24.3 g (1 mol) of Mg turnings and 750 ml of dry ethyl ether in a three-necked 2liter flask equipped with a mechanical stirrer, condenser, and a 250-ml addition funnel under Nz atmosphere was added 2 10 ml (2 11 g, 1 mol) of 1-bromodecane (Aldrich) (initiated with I2 and 1,2-dibromoethane) at a rate sufficient to maintain reflux. Upon completion the filtered solution was determined to be 0.78 M by the sec.-butanol/ 1,l O-phenanthrolene method. Ethyl I -carboethoxy-3,3-dimethyltridecanoate (6). To a suspension of 15 g (80 mmol) CuI powder (MCB) in a solution of 80 g (400 mol) of 5 (prepared by the method of Cope and Hancock) (18) in 500 ml anhydrous ether in a dried 1500-ml threenecked flask fitted with a thermometer, me-

I. Synthesis The synthesis of sodium 4-( 3,3-dimethyl1-oxotridecyl)benzenesulfonate is outlined in Scheme II. ,COOE’

\

H

L

COOD 5.

cootzt A!$ R+

RscooH

COOB 7

6. F ! = C,&

-

I SCHEME II. Outline of synthesis of Compound 1. Reagents: (a) RMgBr, CuI; (b) NaOH, H,O; (c) HCI; (d) SOCI,; (e) C,H50H; (0 AI% WO,; S (g) NaH, DMF, Clll -N(Me),; C (h) 230°C neat; (i) HZ02, HCOOH, basic workup. 17%

PHOTOLABILE

DETERGENT

SYNTHESIS

chanical stirrer, a l-liter addition funnel, and an ice/acetone bath under a dry N2 atmosphere was added 512 ml (400 mmol) of the decyl magnesium bromide solution. The addition was regulated so that the temperature never rose above 0°C. The reaction mixture was allowed to slowly warm to 25°C and stir for an additional 4 h. The reaction mixture was poured onto 500 ml of 10% HCl and crushed ice, transferred to a separatory funnel, and shaken. The ether layer was washed successively with 500 ml of a 10% Na2C03 solution, 500 ml HzO, 500 ml sat. NaCl solution, dried (MgSO& concentrated in vucuo, and distilled thru a short-path column to give after a forerun 69 g (60%) of a liquid bp 140-152°C (0.5 mm Hg) of essentially pure material (TLC, 10: 1, hexane:ethyl acetate on silica gel, R,= 0.32). An analytical sample was prepared by HPLC with pure acetonitrile as solvent. ‘H-NMR (CDClJ 6 0.7-1.4(m, 33H), 3.2(s, lH), 4.l(q, 4H). IR (neat) 1750, 1730, 1140, 1040 cm-‘. Anal. Calcd for CZOH3804: C, 70.13; H, 11.18. Found: C, 70.25; H, 11.28. 3,3-Dimethyltridecanoic acid (7). A mixture of 65 g (190 mmol) of 6 and 400 ml of a 5% NaOH solution was refluxed until there was one phase (24 h.) Coned H,SO, was added to the cooled solution at a rate such that the temperature did not rise above 20°C until the solution was acidic (pH 2). The white solid which precipitated was extracted into 500 ml of ether. The ether was removed and the residue heated with 500 ml of 17% HCl at reflux for 100 h until CO* evolution ceased. The mixture was cooled and extracted with 100 ml ether, the ether layer was dried (MgS04) and concentrated in vacU0 to give 43.9 g (95%) of an orange oil. The sodium salt was prepared by titration with NaOH and isolated by salting out with saturated NaCl solution followed by crystallization from ether to give a material, mp 228-232°C. The acid (7) was regenerated (HCl) and molecularly distilled under high vacuum to give a colorless oil: ‘H-NMR (CDC13) 6 0.82(t, 3H), 0.99(s, 6H), 1.25(bd

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307

s, 18H), 2.20(s, 2H), 10.90(s, 1H); IR (neat) 3400-2200,1735,1700, and N.E. 246 + 3.7. Anal. Calcd for Cr5H3,,02: C, 74.32; H, 12.48. Found: C, 74.32; H, 12.06. 3,3-Dimethyitridecanoyl chloride (8). A mixture of 9.4 g (39 mmol) of 7 and 4.6 ml (59 mmol) of SOCIZ was stirred for 12 h at 25°C and heated to 80°C for 1 h. Excess SOC12 was removed by distillation and the product distilled through a lo-cm Vigreux column to give 8.7 g (85%) of material, bp 115°C (0.5 mm Hg), ‘H-NMR (CDC&) 6 0.8O(t, 3H), l.OO(s, 6H), 1.2O(bd s, 18H), 2.78(s, 2H); IR (neat) 1800 cm-‘. Phenyl 3,3-dimethyltridecanoate (9). To a flask equipped with a magnetic stirrer containing 3.39 g (36 mmol) of phenol was added 7.04 g (27 mmol) of 8. When gas evolution (HCl) ceased, the mixture was heated at 1OO’C for 1 h. The reaction mixture was cooled and stirred overnight. The product was isolated by chromatography on a 4 X 1 l-cm column of silica gel using 1: 1, CH,Cl,/ hexane as eluant, giving 8.3 g (97%) of 9. The analytical sample was purified by molecular distillation under high vacuum; ‘HNMR (CDC&) 6 0.87(t, 3H), l.lO(s, 6H), 1.29(bd s, 18H), 2.42(s, 2H), 6.90-7.45(m, 5H); IR (neat) 1760, 730, 695 cm-‘. Anal. Calcd for CZ1HJ402: C, 79.19; H, 10.76. Found: C, 79.55, H, 10.55. I- (4 - Hydroxyphenyl) - 3,3 - dimethyl - I tridecanone (10). To a solution of 16.0 g (50

mmol) of 9 in 16 ml of nitrobenzene (distilled from P205) in a dry 50-ml threenecked flask equipped with a mechanical stirrer, condenser, and solid adding flask in an ice bath under N2 atmosphere was added 13.6 g (100 mmol) of AIClj in portions over 20 min. The mixture was heated (42-44°C) for 120 h and poured over a mixture of 150 ml 10% HCl and crushed ice. The mixture was extracted into ether and the ether phase extracted with 150 ml of a solution of 3 g NaOH in a 3:1, H,O:ethanol mixture. The aqueous ethanol layer was washed twice with ether and then acidified with concentrated HCl. The oil which separated was dissolved in ether and washed with H20, decolorized

308

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with charcoal and dried (MgS04). After concentration in vucuo a viscous brown liquid (9.8 1 g, 61%) resulted which was pure enough for the next step. An analytical sample was prepared by medium-pressure chromatography on a silica gel column using 5:2, hexane/ethyl acetate as eluant followed by molecular distillation under high vacuum to yield a light yellow oil; ‘H-NMR (CDC&) 6 0.85(t, 3H), 1.02(s, 6H), l.lO-1.55(bd s, 18H), 2.80(s, 2H), 6.90(d, 2H), 7.89(d, 2H); IR (neat) 3600-2500, 1640,850 cm-‘. Anal. Calcd for CZ1H3402: C, 79.19; H, 10.76. Found: C, 79.32; H, 10.55. N,N-Dimethylthionocarbamate of 10 (11). To 1.66 g (34 mmol) of a 50% NaH in an oil dispersion (Ventron) under a Nz atmosphere in a three-necked loo-ml flask equipped with a magnetic stirrer and an addition funnel was added dropwise a deoxygenated solution of 9.8 g (3 1 mmol) of 10 in 25 ml of DMF. The mixture was stirred at 25°C for 1.5 h, cooled in an ice bath, and 4.64 g (37 mmol) of N,N-dimethylthiocarbamyl chloride (Aldrich) in 35 ml DMF added slowly. The reaction mixture was warmed to 25°C stirred overnight, and poured into 150 ml of 5% NaOH solution. The mixture was extracted twice with lOOml portions of a 5: 1, benzene/pentane solution. The organic layer was washed with HZ0 until the aqueous layer was clear (three times), saturated NaCl solution, dried (MgS04), and concentrated in vacua. The product was crystallized from pentane (-78°C) and recrystallized to give 8.75 g (70%) of 11 as yellow crystals, mp 47.548.5; ‘H-NMR (CDC13) 6 0.88 (t, 3H), 1.05(s, 6H), 1.29(bd s, 18H), 2.82(s, 2H), 3.37(d, 6H), 7.11(d, 2H), 7.95(d, 2H); IR (nujol) 3050, 1670, 1600 cm-‘. Anal. Calcd for C24H3gNOzS: C, 71.06; H, 9.69; N, 3.45; S, 7.90. Found: C, 71.07; H, 9.46; N, 3.79; S, 7.94. N,N-Dimethylthiocarbamate of 10 (12). In a N2-purged flask 7.54 g (19 mmol) of 11 was heated at 230-235°C for 40 min to give a viscous brown oil which was sufficiently

ET AL.

pure for the next step; ‘H-NMR (CDQ) 6 0.85(t, 3H), 1.05(s, 6H), l.lO-1.48(bd s, 18H),2.73(s, 2H), 3.00(s, 6H), 7.40(d, 2H), 7.77(d, 2H); IR (neat) 1672, 1590 cm-‘. The analytical sample was prepared by chromatography on silica gel using CHzClz as eluant to give a clear oil which was characterized as a 2,4-DNP derivative, mp 136139°C and analyzed by high-resolution mass spectroscopy. Anal. Calcd for C24H39N02S: 405.2692. Found: 405.2677. Sodium 4-(3,3-dimethyl-oxotridecyl)benzenesulfonate (1). A mixture of 6.7 g (17 mmol) of crude 12 and 100 ml formic acid was cooled to 0°C and 25 ml of 30% H,O, was added over 10 min. The mixture was stirred at 0°C for 2 h and at 25°C for l/2 h. Solid Na2S03 was added until iodide test paper gave a weak positive test for peroxide. The formic acid was removed in vac~o and 125 ml of 5% NaOH solution was added. The mixture was cooled to 0°C the solid removed by filtration and recrystallized from 15:1, hexane/ethanol to give 3.6 g (57% from 11) of 1; ‘H-NMR (DzO) F 0.82(bd s, 9H), 1.24(bd s, 18H), 2.70(s, 2H), 7.72(bd s, 4H); IR (KBr) 1665, 1575, 1465, 1395, 1365, 1315, 1135, 1050, 1015, 660 cm-‘; UV max ( H20) 248 nm (c 38,000), shoulder 282 ( 1400), shoulder 318 (160). Anal. Calcd for C21H3304SNa: C, 62.35; H, 8.22; S, 7.93. Found: C, 62.20; H, 8.40; S, 7.81.

The CMC was measured by a modification of the dye method of Corrin and Harkins ( 19-21) using 5 X lop6 M acridine orange in 0.1 M NaN03 and found to be 8 f

1 x

10-5M.

II.

Molecular Weight Determination of Proteins by Electrophoresis in Gels Containing 1

Red blood cell ghosts were prepared by the method of Dodge et al. (22). Gel tubes ( 16 cm) were filled to almost half of their

PHOTOLABILE

DETERGENT

SYNTHESIS

length with the following gel mixture: 4.17 ml 30% acrylamide (w/v), 1 ml N,N-bismethyleneacrylamide; 3.75 ml M Tris-HCl, pH 8.8; 10 mg 1 2.5 ~1 N,N,N,N’-tetramethylethylenediamine; 25 ~1 10% ammonium persulfate; and Hz0 to a volume of 10 ml. After pouring, the gels were overlaid with HI0 and allowed to polymerize. The proteins were dissolved in buffer containing 60 mM Tris, pH 6.8, 20% sucrose, 1% 1, 5% mercaptoethanol, and 0.001% bromophenol blue. After heating in a boiling water bath for 10 min, an aliquot of the protein-containing solution was placed on top of the polymerized gels. Electrode buffer to which 10% (w/v) sucrose and 0.1% 1 had been added was carefully layered above the sample. Finally the tubes were topped off with electrode buffer which was 25 mM Tris, 192 mM glycine, at pH 8.3, and the upper reservoir also filled with electrode buffer. Electrophoresis was at 3 mA/gel until the tracking dye came to the end of the tubes. The gels were removed from the tubes and the protein bands visualized by staining with amido black. The gels were destained electrophoretically. When electrophoresis in the presence of 1 was compared to that in the presence of SDS, the gels were run side by side. In these experiments 1 was replaced with the same amount (w/v) of SDS. III.

Isolation of Proteins from Gels Containing 1 for Amino Acid Analysis and Peptide Mapping

Preparative vertical slab gels were poured and polymerized for analytical tube gels. Approximately 5 mg of egg white lysozyme (Sigma) was applied to the gel and the electrophoresis performed with a Bio-Rad vertical slab gel apparatus, Model 220, at 35 mA until the indicator dye had reached the lower end of the gel. The gel was removed and stained in 0.2% Coomassie blue G-250 dissolved in metha-

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309

nol:water:acetic acid (9:9:2) for 2-4 h and gels were destained by diffusion in methanol:water:acetic acid (10:83:7). The resulting protein band was cut, exposed to a UVL21 light source at 1.5 cm distance overnight and then extracted (after hydration) into 60% formic acid utilizing a glass homogenizer with a motor-driven Teflon pestle (23). Most of the acrylamide particles were removed by centrifugation and the pellet was washed twice with 60% formic acid. The extracts were combined and concentrated to 22 ml by flash evaporation and passed over a Sephadex G-25 column (1 X 55 cm) in 60% formic acid. The protein which eluted ahead of the dye was located by absorbancy at 280 nm. The formic acid was again removed by evaporation and the recovered protein used for amino acid analysis on a Beckman 120 C amino acid analyzer and for tryptic peptide mapping by the method of Gracy (24). RESULTS

AND DISCUSSION

The synthesis of 1 is outlined in Scheme II. The starting material, diethyl isopropylidene malonate (5) was prepared according to the procedure of Cope and Hancock (18) and in a conjugate addition process decyl magnesium bromide in the presence of cuprous iodide converted 5 into ethyl-l-carbothoxy-3,3-dimethyltridecanoate, 6, in 60% yield. Hydrolysis and decarboxylation gave a 95% crude yield of 3,3-dimethyltridecanoic acid, 7. Compound 7 in turn was converted to its phenyl ester, 9, via the acid chloride, 8, in 82% overall yield for the two steps. Phenyl-3,3-dimethyltridecanoate was allowed to undergo the Fries rearrangement in nitrobenzene with 2 eq of AlC13 to maximize the para derivative (25-29) to yield 1-(p-hydroxyphenyl)-3,3-dimethyl-ltridecanone, 10, in 61% yield. Sulfur was introduced via the Newman-Kwart rearrangement (30) of the O-arylthionocarbamate, 11, made from 10, and N,N-dimethylthiocarbamoyl chloride. The crude

310

EPSTEIN

rearrangement product, 12,2 was directly oxidized to 1 by the method of Cooper and Paul (31) in an overall yield of 57% from 11. The CMC for 1 as determined photometrically with acridine orange as an indicator was 8 -t 1 X 10m5 M. This is one order of magnitude smaller than the CMC for SDS. The photolysis of an aqueous solution of 1 proceeded as expected giving essentially a quantitative conversion into 2 and a mixture of 3 and 4. Careful analysis of the water-soluble materials using a reversedphase HPLC system indicated no product other than 2. The water-insoluble olefin mixture was isolated by GLC and again there was no evidence of any other products. A ratio of 1.6:1 for the difficult-to-separate olefin mixture of 3 and 4, respectively, was found by NMR analysis. The electrophoretic procedure of Laemmli is commonly used for the determination of the molecular weights of polypeptides. To test 1 as a substitute for SDS, the procedure was modified simply by replacing SDS with 1. Because of the limited quantity of 1 available no detergent was used in the reservoir buffers. To prevent loss of 1 in the gel tube, the gel tubes were only half filled with 1 containing polyacrylamide which was then topped off with reservoir buffer containing 1 and 10% glycerol for stabilization against convection. Electrophoresis was at 3 mA/gel until the tracking dye was within 2mm of the end of the tube. After electrophoresis the gels were extruded and stained with amido black. The log of the molecular weight of the protein as a function of electrophoretic mobility is shown in Fig. 1 for 1 and SDS detergents. As can be seen, the two lines are slightly offset but have nearly identical slopes. The reason that the two lines do not coincide is probably that the individual protein molecules are coated to different extents 2 This compound consistently ysis beyond acceptable values purification.

gave combustion analregardless of method of

ET AL

.. .

RNDSe

FIG. 1. A plot of the log of molecular weight of various proteins as a function of their electrophoretic mobility. Gels containing 1, 0 __ 0; gels containing SDS, A---A.

with the different detergents. 1 with the bulky aromatic ring near the head group will probably pack fewer molecules into a given area. This would result in less charge per protein-detergent complex resulting in a slower migration which is what is observed. When the enzyme, prenyltransferase, was reduced and alkylated in the presence of 1 and then subjected to electrophoreses in polyacrylamide gels containing 1, a sharp single band of protein was observed. Similar experiments with SDS also gave a single band with a similar rate of migration. Thus, 1 like SDS facilitates the cleavage of prenyltransferase into its two identical subunits and like the standard proteins, the subunits migrate during electrophoresis as though a detergent-peptide aggregate exists. Consequently, 1 is suitable for separation of peptides under denaturing conditions where migration is proportioned to molecular weight. When red blood cell ghosts were treated with 1 in HZ0 a clear solution was obtained indicating that the proteins and lipids of this membrane preparation had been solubilized. When the solubilized material was subjected to electrophoresis, several important bands of protein appeared with rates of migration similar to more densely staining bands ob-

PHOTOLABILE

DETERGENT

SYNTHESIS

served on electrophoresis of the same preparation in the presence of SDS. However, the bands obtained in l-containing gels were more diffuse and the normally obtained but less intense bands were absent and too diffuse to be seen. In addition, substantial amounts (about half) of the protein failed to penetrate l-containing gels. Thus, 1 does not duplicate the detergent effect of SDS for all proteins. If the free radicals generated during photolysis of 1 were to react with protein rather than to complete the cleavage reaction, peptide and amino acid derivatives would be generated that could interfere with further analysis of the proteins or with the determination of their biological activity. To test for this possibility egg white lysozyme was electrophoresed in polyacrylamide gels containing 1 on a preparative scale. After staining and destaining the zone containing protein was excised and exposed to a 354-nm light to photolyze 1. The protein was then extracted into 60% formic acid and passed over a Sephadex column. One portion of the protein thus purified was digested with trypsin and another hydrolyzed in 6 N HCl. The two-dimensional map of these tryptic peptides was indistinguishable from the one obtained from the digestion of protein that had not been subjected to electrophoresis. Likewise the amino acid analysis of treated and untreated lysozymes was identical. These two procedures indicated that side reactions involving protein must be minor. It is known that the length and structure of the aliphatic chain of a detergent as well as the nature of its head group affects its CMC and detergent properties (32). Only a few detergents differing from SDS primarily in hydrocarbon chain length have been shown to function like SDS in making well-defined detergent-protein complexes. Consequently it may be necessary to test a family of detergents closely related to 1 to find a detergent that closely mimics the properties of SDS. Alternatively, 1 under different conditions of pH and ionic strength

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ANALYSIS

for electrophoresis may provide more efficient solubilization and resolution of protein (32). Nonetheless, 1 is an effective removable detergent and the chemistry involved in its removal is applicable to nearly all classes of ionic and nonionic detergents now in use. ACKNOWLEDGMENT This work was supported and GM 13140.

by NIH

Grants

GM

26245

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31. Cooper, J. E., and Paul, J. M. ( 1970) J. Org. Chem. 35, 2046-2048. 32. Tanford, C. (1973) The Hydrophobic Effect: Formation of Micelles and Biological Membranes, Wiley, New York.