ANAIY,ICA,
BIOCHFMISIKY 92.444-446
Determination
of Protein-Bound Dodecyl by Gas Chromatography
MATTI
Sodium
dodecyl
The
presence
mination. sample
SARASTE
sulfate
group is hydrolyzed. an SE-30 column.
(1979)
has been
determined
and the liberated Dodecanol is clearly
of proteins
AND TIMO
or phospholipids
Using a conventional can be detected.
serum
used: SDS. albumin.
sodium
0003.2697/79/020444-03$02.00/O Copyright All
right\
c’ of
1979
by
reproduction
Academic in any
PET\.
lnc
form
rexrvcd
dodecyl
sulfate:
444
method.
The
is determined by gas chromatography from the common fatty acid methyl
in the sample
gas chromatograph.
Determination of sodium dodecyl sulfate (SDS)’ in samples containing protein or phospholipid is a cumbersome task when the radioactive detergent is not available. This is true especially when a membrane protein that is solubilized and purified by the aid of SDS, is prepared for reconstitution experiments, or when renaturation of polypeptides denaturated in SDS is carried out (1). The procedures developed for measurement of low concentrations of SDS are based on complexation of the detergent with methylene blue (2-4) or with fuchsin (5) and on spectrophotometric analysis of the colored complex extracted into chloroform. However, these methods were originally developed for protein- and lipid-free systems (2,5), although the presence of protein is reported not to interfere with the determination at a low protein to SDS concentration ratio (4), or when a high volume ratio of chloroform to water is used (3). Since we have found difficulties in the determination of SDS in the presence of hydrophobic proteins by the spectrophotometric methods, we introduce here a simple gas chromatographic procedure for ’ Abbreviations BSA, bovine
K. KORHONEN
by a gas chromatographic
dodecanol resolved
Sulfate
seems 0.5
to have
no effect
to I .O /~g of dodecyl
sulfate using esters.
on the detersulfate
in the
qualitative detection as well as quantitative determination of SDS. This method is based on hydrolysis of the sulfate group in acidic methanol and on determination of the subsequently liberated dodecanol by gas chromatography. METHODS Commercial SDS (specially purified, BDH Chemicals Ltd.) was used without further purification. In agreement with an earlier report (6). it was found to be of high purity with respect to the alkyl chain content, in contrast to some other commercial preparations tested, which contained up to 40% tetradecyl chains. Bovine serum albumin (BSA) was purchased from Sigma Chemical Company and crude soybean phospholipids (Asolectin) from Nutritional Biochemicals Corporation. Cytochrome c oxidase was purified from bovine heart mitochondria according to the method of Kuboyama cut(I/. (7). Cholic acid, purchased from E. Merck Company, was crystallized once from ethanol. For determination of SDS, the samples were dissolved or dialyzed into 50 mM Tris-acetate buffer (pH 8.0). and lyophilized in hydrolysis tubes. SDS was hydro-
GAS
CHROMATOGRAPHY
OF PROTEIN-BOUND
SDS
445
raphy. This compound was identified as free dodecanol by mass spectrometry (data not shown). Dodecanol can be easily distinguished from the methyl esters of common fatty acids using the SE-30 column. The fatty acids which could interfere with the determination are IO-methylundecanoic. 9methylundecanoic, P-hydroxydecanoic, and cis-5-dodecanoic acids (see Fig. 1). These are not found in the red cell (9) or in mitochondria (lo), and they are rather uncommon in microorganisms (11). Table I compares SDS determinations nC carried out by previous spectrophotometric FIG. 1. Retention times of dodecanol and standard methods and by gas chromatographic fatty acid methyl esters in SE-30 column. Logarithms procedure. The former methods, involving of retention time are plotted against the number of complexation of SDS with methylene blue in carbons in the fatty acids (nC). The symbols for neutral solution (3) or with fuchsin in acidic standard fatty acids are: (+). cr-hydroxy-: (x). psolution (5) and extraction of the complex hydroxy-; (O), normal saturated: (0). monoenoic; (0). iso-; and (A). anteiso-fatty acid. The arrow indicates into chloroform, gave in our hands unrelithe retention time of dodecanol ( I .70 min). able results in the presence of phospholipids. In both analyses phospholipids lyzed in acidic methanol by a conventional precipitated in the water-chloroform sysmethod used for methylation of fatty acids tem. The presence of cholic acid did not (8). Dodecanol. together with methylated interfere with the determination of SDS by fatty acids, if present in the sample, was any of the methods tested. When bovine extracted into chloroform. Two milliliters of serum albumin or a hydrophobic membrane acidic methanol solution was first extracted protein (the mitochondrial cytochrome c oxwith 4 ml of chloroform plus 1.5 ml Iof water, idase) was present in the sample. the fuchsin and the upper phase was further wa:shed two method gave results that were too low. The times with 2 ml of chloroform. The preferential binding of SDS to protein is chloroform solutions were combined, and probably the source of interference in the the solvent was evaporated with Nz gas at protein-containing samples. The methylene 20°C. The residue was dissolved in 50 ~1 of blue method was not affected by bovine cold hexane, treated for 1 min in a sonicating serum albumin in the sample and only bath (Brunsonic 32). and analyzed in a slightly affected by cytochrome c oxidase. Perkin-Elmer F 11 gas chromatograph (3 m The gas chromatographic measurements x 2-mm SE-30 column, temperature 185°C. gave reliable results in all cases tested. The high standard deviations of the gas chromatN, carrier gas, flame ionization detector). The standard fatty acids were methylated by ographic analyses are due to the multistep procedure and they can be controlled by the same procedure. Dodecanol was quantitated from the peak area (height of the peak accurate handling of samples. times half-width). The major advantage of the gas chromatographic method, as compared with the specRESULTS AND DISCUSSION trophotometric ones, is that phospholipids Hydrolysis of SDS (purchased from or proteins do not interfere with the Yet it is inexpensive and BDH) produced only one component with determination. sensitive enough for most purposes. The retention time of 1.7 min in gas chromatog-
446
SARASTE
AND TABLE
KORHONEN I
Nanomoles Sample” I. 2. 3. 4.
100 100 100 100
nmol of SDS nmol of SDS nmol of SDS nmol of SDS
+ + + +
1 1 I I
mg mg mg mg
A” of of of of
Asolectin cholate BSA cytochrome
B”
--Cl
t’ oxidase
97.1 2 5.6 38.2 z 4.6 16.1 -t I.1
of SDS” c
-11 103.9 102.6 92.9
t 3.7 -+ 2.4 i- 5.2
96.9 89.3 99.6 96.2
2 7.4 i- 15.4 2 15.2 + 6.9
” Results of the analyses of SDS by the fuchsin method (5) in column A, by the methylene blue method (2). as modified in (4). in column B. and by the gas chromatographic method in column C. The SD of five parallel determinations are indicated in the table. ’ The first three samples were prepared by dissolving a weighed amount of crude soybean phospholipids (Asolectin), sodium cholate. or BSA into SDS standard. The fourth sample was prepared by mixing a cytochrome c oxidase solution (50 mM Tris-acetate. pH 8.0. 0.5% sodium cholate) with the SDS standard. I’ The blanks which contained 1 mg of BSA. cholate. or cytochrome (’ oxidase but no SDS gave negligible absorbances. ” Determinations could not be carried out, because of the turbidity in chloroform solution caused by precipitated phospholipids.
resolution limit of the method is in the same range as with the spectrophotometric procedures (4,5). In a conventional gas chromatographic analysis approximately 0.5 to 1.0 pg (1.7-3.5 nmol) of SDS can be detected, and if capillary column or electron capture detection is used, resolution may be enhanced by two to three orders of magnitude. ACKNOWLEDGMENTS We are indebted to Dr. Jorma Karkkainen for performing the mass spectrometric analysis. T.K.K. was supported by the Emil Aaltonen Foundation.
REFERENCES I. Weber, K.. and Kuter, 246, 4504-4509.
D. J. ( 1971) ./. B;,J/. C/rem.
2. Ray, A., Reynolds. J. A., Polet, H.. and Steinhardt. J. (1966) Biochrmi.s/ry 5, 2606-2616. 3. Reynolds. J. A.. and Tanford, C. (1970) Prr~. R;c~t. Ac,trtl. SC i. USA 66, 1002-1007. 4. Hayashi. K. ( 1975) Am/. Bi~~~hcrn. 67. 503-506. 5 Waite. J. H., and Wang, C.-Y. (1976) Anrrl. Bioc~hrm. 70, 279-280. 6. Birdi, K. S. (1976) And Bioc~hcm. 74, 620-622. 7. Kuboyama. M.. Yong. F. C.. and King. T. E. (1972) ./. Biol. C11enr. 247, 6375-6383. 8. Renkonen. 0. (1965) J. Amc,r. Oil Clam. SCW. 42, 298-304. 9. Pennell, R. B. (1964) in The Red Blood Cell (Bishop, C., and Surgenor, D. M.. eds.). pp. 48-52. Academic Press. New York. IO. Parkes. J. G.. and Thompson. W. (1970) Bl,~c~lrirn. Biopkys. Actcl 196. l62- 169. Il. O’Leary. W. M. (1973) in Handbook of Microbiology, Vol. II: Microbial Composition (Laskin. A. I., and Lechevalier, H. 4.. eds.), pp. ?41327. CRC Press. Cleveland. Ohio.
BIOCHFMISIKY 92.444-446
Determination
of Protein-Bound Dodecyl by Gas Chromatography
MATTI
Sodium
dodecyl
The
presence
mination. sample
SARASTE
sulfate
group is hydrolyzed. an SE-30 column.
(1979)
has been
determined
and the liberated Dodecanol is clearly
of proteins
AND TIMO
or phospholipids
Using a conventional can be detected.
serum
used: SDS. albumin.
sodium
0003.2697/79/020444-03$02.00/O Copyright All
right\
c’ of
1979
by
reproduction
Academic in any
PET\.
lnc
form
rexrvcd
dodecyl
sulfate:
444
method.
The
is determined by gas chromatography from the common fatty acid methyl
in the sample
gas chromatograph.
Determination of sodium dodecyl sulfate (SDS)’ in samples containing protein or phospholipid is a cumbersome task when the radioactive detergent is not available. This is true especially when a membrane protein that is solubilized and purified by the aid of SDS, is prepared for reconstitution experiments, or when renaturation of polypeptides denaturated in SDS is carried out (1). The procedures developed for measurement of low concentrations of SDS are based on complexation of the detergent with methylene blue (2-4) or with fuchsin (5) and on spectrophotometric analysis of the colored complex extracted into chloroform. However, these methods were originally developed for protein- and lipid-free systems (2,5), although the presence of protein is reported not to interfere with the determination at a low protein to SDS concentration ratio (4), or when a high volume ratio of chloroform to water is used (3). Since we have found difficulties in the determination of SDS in the presence of hydrophobic proteins by the spectrophotometric methods, we introduce here a simple gas chromatographic procedure for ’ Abbreviations BSA, bovine
K. KORHONEN
by a gas chromatographic
dodecanol resolved
Sulfate
seems 0.5
to have
no effect
to I .O /~g of dodecyl
sulfate using esters.
on the detersulfate
in the
qualitative detection as well as quantitative determination of SDS. This method is based on hydrolysis of the sulfate group in acidic methanol and on determination of the subsequently liberated dodecanol by gas chromatography. METHODS Commercial SDS (specially purified, BDH Chemicals Ltd.) was used without further purification. In agreement with an earlier report (6). it was found to be of high purity with respect to the alkyl chain content, in contrast to some other commercial preparations tested, which contained up to 40% tetradecyl chains. Bovine serum albumin (BSA) was purchased from Sigma Chemical Company and crude soybean phospholipids (Asolectin) from Nutritional Biochemicals Corporation. Cytochrome c oxidase was purified from bovine heart mitochondria according to the method of Kuboyama cut(I/. (7). Cholic acid, purchased from E. Merck Company, was crystallized once from ethanol. For determination of SDS, the samples were dissolved or dialyzed into 50 mM Tris-acetate buffer (pH 8.0). and lyophilized in hydrolysis tubes. SDS was hydro-
GAS
CHROMATOGRAPHY
OF PROTEIN-BOUND
SDS
445
raphy. This compound was identified as free dodecanol by mass spectrometry (data not shown). Dodecanol can be easily distinguished from the methyl esters of common fatty acids using the SE-30 column. The fatty acids which could interfere with the determination are IO-methylundecanoic. 9methylundecanoic, P-hydroxydecanoic, and cis-5-dodecanoic acids (see Fig. 1). These are not found in the red cell (9) or in mitochondria (lo), and they are rather uncommon in microorganisms (11). Table I compares SDS determinations nC carried out by previous spectrophotometric FIG. 1. Retention times of dodecanol and standard methods and by gas chromatographic fatty acid methyl esters in SE-30 column. Logarithms procedure. The former methods, involving of retention time are plotted against the number of complexation of SDS with methylene blue in carbons in the fatty acids (nC). The symbols for neutral solution (3) or with fuchsin in acidic standard fatty acids are: (+). cr-hydroxy-: (x). psolution (5) and extraction of the complex hydroxy-; (O), normal saturated: (0). monoenoic; (0). iso-; and (A). anteiso-fatty acid. The arrow indicates into chloroform, gave in our hands unrelithe retention time of dodecanol ( I .70 min). able results in the presence of phospholipids. In both analyses phospholipids lyzed in acidic methanol by a conventional precipitated in the water-chloroform sysmethod used for methylation of fatty acids tem. The presence of cholic acid did not (8). Dodecanol. together with methylated interfere with the determination of SDS by fatty acids, if present in the sample, was any of the methods tested. When bovine extracted into chloroform. Two milliliters of serum albumin or a hydrophobic membrane acidic methanol solution was first extracted protein (the mitochondrial cytochrome c oxwith 4 ml of chloroform plus 1.5 ml Iof water, idase) was present in the sample. the fuchsin and the upper phase was further wa:shed two method gave results that were too low. The times with 2 ml of chloroform. The preferential binding of SDS to protein is chloroform solutions were combined, and probably the source of interference in the the solvent was evaporated with Nz gas at protein-containing samples. The methylene 20°C. The residue was dissolved in 50 ~1 of blue method was not affected by bovine cold hexane, treated for 1 min in a sonicating serum albumin in the sample and only bath (Brunsonic 32). and analyzed in a slightly affected by cytochrome c oxidase. Perkin-Elmer F 11 gas chromatograph (3 m The gas chromatographic measurements x 2-mm SE-30 column, temperature 185°C. gave reliable results in all cases tested. The high standard deviations of the gas chromatN, carrier gas, flame ionization detector). The standard fatty acids were methylated by ographic analyses are due to the multistep procedure and they can be controlled by the same procedure. Dodecanol was quantitated from the peak area (height of the peak accurate handling of samples. times half-width). The major advantage of the gas chromatographic method, as compared with the specRESULTS AND DISCUSSION trophotometric ones, is that phospholipids Hydrolysis of SDS (purchased from or proteins do not interfere with the Yet it is inexpensive and BDH) produced only one component with determination. sensitive enough for most purposes. The retention time of 1.7 min in gas chromatog-
446
SARASTE
AND TABLE
KORHONEN I
Nanomoles Sample” I. 2. 3. 4.
100 100 100 100
nmol of SDS nmol of SDS nmol of SDS nmol of SDS
+ + + +
1 1 I I
mg mg mg mg
A” of of of of
Asolectin cholate BSA cytochrome
B”
--Cl
t’ oxidase
97.1 2 5.6 38.2 z 4.6 16.1 -t I.1
of SDS” c
-11 103.9 102.6 92.9
t 3.7 -+ 2.4 i- 5.2
96.9 89.3 99.6 96.2
2 7.4 i- 15.4 2 15.2 + 6.9
” Results of the analyses of SDS by the fuchsin method (5) in column A, by the methylene blue method (2). as modified in (4). in column B. and by the gas chromatographic method in column C. The SD of five parallel determinations are indicated in the table. ’ The first three samples were prepared by dissolving a weighed amount of crude soybean phospholipids (Asolectin), sodium cholate. or BSA into SDS standard. The fourth sample was prepared by mixing a cytochrome c oxidase solution (50 mM Tris-acetate. pH 8.0. 0.5% sodium cholate) with the SDS standard. I’ The blanks which contained 1 mg of BSA. cholate. or cytochrome (’ oxidase but no SDS gave negligible absorbances. ” Determinations could not be carried out, because of the turbidity in chloroform solution caused by precipitated phospholipids.
resolution limit of the method is in the same range as with the spectrophotometric procedures (4,5). In a conventional gas chromatographic analysis approximately 0.5 to 1.0 pg (1.7-3.5 nmol) of SDS can be detected, and if capillary column or electron capture detection is used, resolution may be enhanced by two to three orders of magnitude. ACKNOWLEDGMENTS We are indebted to Dr. Jorma Karkkainen for performing the mass spectrometric analysis. T.K.K. was supported by the Emil Aaltonen Foundation.
REFERENCES I. Weber, K.. and Kuter, 246, 4504-4509.
D. J. ( 1971) ./. B;,J/. C/rem.
2. Ray, A., Reynolds. J. A., Polet, H.. and Steinhardt. J. (1966) Biochrmi.s/ry 5, 2606-2616. 3. Reynolds. J. A.. and Tanford, C. (1970) Prr~. R;c~t. Ac,trtl. SC i. USA 66, 1002-1007. 4. Hayashi. K. ( 1975) Am/. Bi~~~hcrn. 67. 503-506. 5 Waite. J. H., and Wang, C.-Y. (1976) Anrrl. Bioc~hrm. 70, 279-280. 6. Birdi, K. S. (1976) And Bioc~hcm. 74, 620-622. 7. Kuboyama. M.. Yong. F. C.. and King. T. E. (1972) ./. Biol. C11enr. 247, 6375-6383. 8. Renkonen. 0. (1965) J. Amc,r. Oil Clam. SCW. 42, 298-304. 9. Pennell, R. B. (1964) in The Red Blood Cell (Bishop, C., and Surgenor, D. M.. eds.). pp. 48-52. Academic Press. New York. IO. Parkes. J. G.. and Thompson. W. (1970) Bl,~c~lrirn. Biopkys. Actcl 196. l62- 169. Il. O’Leary. W. M. (1973) in Handbook of Microbiology, Vol. II: Microbial Composition (Laskin. A. I., and Lechevalier, H. 4.. eds.), pp. ?41327. CRC Press. Cleveland. Ohio.
