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Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high-performance liquid chromatography
ANAl.YTICAL
BIIOCHEMISTRY
114, 383-387
(1981)
Analysis of Biological Thiols: Derivatization with Monobromobimane and Separation by Reverse-Phase High-Performance Liquid Chromatography GERALD Department
L. NEWTON,
of Chemistry.
RANDEL
University
DORIAN,
of California,
Received
January
AND
San Diego,
ROBERT La Jolla,
C. FAHEY California
92093
5, 1981
A method is described for determination of biological thiols at the picomole level based upon conversion of thiols to fluorescent derivatives by reaction with monobromobimane and separation of the derivatives by reverse-phase high-performance liquid chromatography. Thiols separated by the procedure include N-acetylcysteine, coenzyme A, coenzyme M, cysteamine, cysteine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, y-glutamylcysteine, homocy:steine, hydrogen sulfide, 2-mercaptoethanol, mercaptopyrimidine, methanethiol, pantetheine, 4’-phosphopantetheine. thiosulfate, and 2-thiouracil. Since monobromobimane has little fluorescence and reacts very selectively with thiols to produce fluorescent derivatives, crude extracts can be derivatized and analyzed without prepurification of the thiols, the entire process requiring only 1 to 2 h. The technique is illustrated by determination of the thiol levels in red blood cells.
In a recent paper we described a general method for the determination of biological thiols at the picomole level ( 1). The method
BrCH2
‘CH2X
was based upon derivatization of the thiol with monobromotrimethylammoniobimane (qBBr)’ and monobromobimane (mBBr),
RSCH2
CH2X
X=H,mBBr X:@N(CH3)3,‘@Br
followed by separation of the derivatives by cation-exchange chromatography and detection by fluorometry. Combined analysis of the qBBr and mBBr derivatives provided unique determination of 17 of the 18 thiols examined. The principal limitation of the method is the time required for sample analysis, 334 h for each of the bromobimane derivatives. In the present paper we decribe a separation of the mBBr derivatives of these thiols by reverse-phase high-performance liquid chromatography which permits the 383
full analysis to be performed in less than 40 min. MATERIALS
AND
METHODS
Materials. All chemicals and biochemicals were as previously described (1) unless ’ Abbreviations used: qBBr, monobromotrimethylammoniobimane (3,7-dimethyl-4-bromomethyl+trimethylammoniomethyl1,5 - diazabicyclo[3.3.0]octa - 3.6 diene-2,8-dione); mBBr, monobromobimane (3,7-dimethyl - 4 bromomethyl 6 - methyl - I .5 - diazabicyclo [3.3.0]octa 3,6-diene-2,8-dione); hplc, high-performance liquid chromatography. 0003-2697/81/100383-05$02.00/O Copyright iG 1981 by Academic Press. Inc All rights of reproducuon in any form reserved
384
NEWTON,
DORIAN.
AND
FAHEY
El
% B 100 ,’ //’ 80 60
40
I
!
20
30 mln
FIG. I. Reverse-phase high-performance liquid chromatogram of a mixture See Table I for key to abbreviations. Loads were: Cys and CC, 63 pmol; CoA, 170 pmol; 4’PP. 25 pmol; TU. 376 pmol; all others, 38 pmol.
otherwise indicated. Samples of human red blood cells were kindly provided by Dr. Thomas Vedvick. Preparation of samples. Standard thiol derivatives and red blood cell thiol derivatives were prepared as described previously (1). The samples were diluted in 0.2 N sodium citrate, pH 2.2, so that injection on either the cation-exchange column or the hplc column was possible. It should be noted that changing the sample diluent has some effect upon the derivative retention times. High-performance liquid chromatography. Separations were carried out on an Altex Model 332 gradient liquid chromatograph equipped with a Model 42 1 controller, a Gilson Spectra/G10 filter fluorometer with standard flow cell and o-phthalaldehyde filters, and a Shimadzu CR 1A printer-plotter. An Altex Ultrasphere-ODS (C-18) 5-ym column (4.6 X 150 mm), Part No. 256-06, was used at ambient temperature and a flow rate of 1.5 ml/min. The buffer system was a minor modification of that described by Bhown et al. (2)-buffer A: 100 ml methanol (Hplc grade, Fisher), 2.5 ml acetic acid (reagent grade, Mallincrodt), diluted to one liter with glass distilled water; buffer B: 900 ml methanol, 2.5 ml acetic acid, diluted to 1 liter with water. Buffers were adjusted to pH 3.9 with 50% NaOH and filtered through an 0.2~pm Millipore filter. The elution pro-
of thiol-mBBr derlvativcs. TS. Erg, NAC. CyA, and
file was as follows: O-1 0 min, 8% B isocratic; lo-20 min, 8-40% B, linear gradient; 20-25 min, 40% B, isocratic; 25530 min, 40--90% B, linear gradient; 30-32 min, 90-100% B, linear gradient (column regeneration). RESULTS The initial objective was to obtain a complete separation of the mBBr derivatives prepared previously (1) using a single-elution protocol on a reverse-phase hplc column. Acetonitrile, methanol, water, and acetic acid, adjusted to various pH values, were tested as mobile phases but the methanolwater-acetic acid system of Bhown et al. (2) proved most effective. A satisfactory separation of the derivatives was obtained using a combination of isocratic and linear gradient elution as shown in Fig. 1. A few pairs of derivatives are not fully resolved and the peak for the cysteamine derivative is rather broad, but the separation is more than satisfactory for most purposes. Attempts to improve the resolution of the early portion of the chromatography have not been successful and a longer column may be required for this purpose. The peaks identified as hydrolysis products in Fig. 1 are observed when mBBr is allowed to react with water in the absence of added thiol. It was found that the sensitivity could be
PICOMOLE
DETERMINATION
OF TABLE
RFTENTION
Thiol N-Acetylcysteine Coenzyme A Coenzymme M Cysteamine Cysteine Cysteinylplycine Ergothioneine Ethanethiol Glutathione -&lutamylcysteine Homocysteine Hydrogen sulfide Hydrolysis 2-Mercaptoethanol Mercaptopyrimidine Methanethiol Pantetheine 4’-Phosphopantetheine Thiosulfa1.e 2-Thiouracil
TIMES
~i\in
RELATIVE
BIOLOGICAL
I
FL UORESCF.NCE
FOR Ttitot-mBBr
Symbol (Figs. I and 2)
Retention (min)
NAC CoA CoM CYA CYS CG Erg Et GSH YGC HC HS H 2ME MPY Me P 4’PP TS TU
Il.8 20.9 6.7 12.4 3.7 5.2 9.0 29.2 6.1 4.5 7.2 24.6 10.2, 18.3, 19.5 25.1 24.3 22.2 17.7 4.3 19.8
increased by a factor of 10 over that shown in Fig.1 without an unacceptable increase in baseline noise so that it is possible to quantitate rather low levels of thiols, e.g., 2 pmol of cysteine or 20 pmol of ergothioneine. Retention times and relative fluorescence yields are given in Table 1. The latter generally parallel those obtained by cation-exchange chromatography ( 1) but tend toward higher values at higher methanol content owing to higher fluoresence yields in methanol relative to water. As in the previous study (1) red blood cells were chosen to test the utility of the method. Cells were lysed directly in a solution of mBBr and the extract was deproteinized with HCl--sodium methanesulfonate ( 1). After dilution in running buffer the chromatogram shown in Fig.2 was obtained. The main thiol component is GSH (2.9 mM in red blood cells) with ergothioneine ( 100 HIM) also being present at a substantial level. Cysteine is present only at a trace level (I
385
THIOLS
All other are attributed and 18.5 min) izing reagents sults are in the viously ( 1,3).
PM).
DERIVATIVES
time
Relative fluoresence 0.5 0.4 I.1 0.8 I .o 0.7 0.08 I .8 ( I .OO) 0.8 0.8 0.9
18.6 I.1 I .2 I .o I.1 0.8 0.2 0.1
peaks in the chromatogram to hydrolysis products (9.8 or artifacts of the deprotein(1.7 and 19.8 min). The rerange of values reported pre-
DlSCUSSlON With the present use of reverse-phase hplc to separate mBBrrthio1 derivatives our approach to analysis of biological thiols is com-
:
h
1j
Fit;. 2. Reverse-phase high-performance liquid matogram of the mBBr derivatized. deproteinized tract from red blood cells.
chroex-
386
NEWTON.DORIAN,ANDFAHEY
plete. The main limitation of the earlier method (1) was the time required to prepare both the mBBr and qBBr derivatives and to separate them by ion-exchange chromatography. With reverse-phase hplc it proved possibleto separate the complete list of thiols as their mBBr derivatives, thereby circumventing the need to use qBBr derivatives as well. Since the mBBr derivatives are more stable and more fluorescent than the qBBr derivatives this is a decided advantage in itself, as well as a time-saving feature. With hplc analysis the total time for preparation and analysis of a sample is only l-2 h, the time per sample being even lessif many samples are processed simultaneously. A second difficulty found using cation-exchange chromatography was that the acidic thiol derivatives eluted shortly after the breakthrough volume and were therefore not well resolved. Thus, the thiosulfate and coenzyme M derivatives with qBBr were not separated and most of the mBBr derivatives of acidic thiols coeluted. In the present separation (Fig. 1) the mBBr derivatives of acidic compounds (thiosulfate, coenzyme M, Nacetylcysteine, 4’-phosphopantetheine, and coenzyme A) are fully resolved and distributed throughout the chromatogram. Of course, even with the improved resolution of the hplc procedure there remains the possibility that two different thiol derivatives will coelute. It is therefore useful to be able to check assignments using cation-exchange chromatography of the mBBr derivative. Should doubt still remain the qBBr derivative can be prepared and examined by cation-exchange chromatography or by thinlayer chromatography and electrophoresis as described previously (4). With this combination of techniques available there should be little possibility for mistake in the identification of a specific biological thiol. However, for routine determinations where identity of the components has been established the hplc procedure is by far the most efficient. Two recent papers have described meth-
ods for analysis of biological thiols based upon high-performance liquid chromatography. Reeve et al. (5) utilized Ellman’s reagent to convert cysteine, GSH, and yglutamylcysteine to the mixed disulfides of 5-thio-2-nitrobenzoic acid and separated the latter by reverse-phase high-performance liquid chromatography. Peaks were detected spectrophotometrically at 280 nm providing nanomole sensitivity for the determination of these thiols. The present method has several advantages over this approach. First, not all biological thiols form mixed disulfides with Ellman’s reagent (3); thus, ergothioneine does not react and dithiols are oxidized to cyclic disulfides. Second, various peptides and other nonthiol biological compounds absorb at 280 nm and can potentially interfere with the analysis whereas background peaks resulting from fluorescent nonthiol compounds are much less common. Third, fluorescent detection of the mBBr derivatives is more sensitive by some two orders of magnitude. Finally, it has been possible to separate the full range of biological thiols using the mBBr derivatives whereas it is not clear that this is possible using derivatives prepared with Ellman’s reagent. The other recent adaptation of high-performance liquid chromatography to analysis of thiols is the study by Reed et al. (6). They utilized initial carboxymethylation of the thiol group to block thioldisulfide exchange reactions. Free amino groups were then 2,4dinitrophenylated to provide a chromophoric group. The derivatives were separated on a 3-aminopropylsilane derivatized silica column and detected spectrophotometrically with nanomole sensitivity. Since all compounds having free amino groups are detected the method is less selective than the one reported in this work. Also, thiols lacking free amino groups (ergothioneine, coenzymes A and M, 4’-phosphopantetheine, methanethiol, etc.) cannot be measured with this approach. On the other hand their method is capable of measuring disulfides
PICOMOLE
DETERMINATION
and other o.xidized forms of sulfur amino acids whereas the present method is limited to the detection of thiols or of disulfides following reduction to thiols. In the latter case the disulfide ‘components can be detected but information as to what specific disulfides or mixed disulfides were present prior to reduction is lost. Thus, the method of Reed et al. (6) appears to be the best available for determining specific disulfides and mixed disultides. ACKNOWLEDGMENTS This research was supported by the USPHS under Grant GM 22122 from the National Institute of General Medical Sciences and Grant CA 25009 from the National Cancer Institute. We thank Dave Smith of Cole
OF
BIOLOGICAL
387
THIOLS
Scientific for making in these studies.
available
the hplc equipment
used
REFERENCES I. f:ahey, R. C.. Newton. G. L., Dorian. R.. and Kosower. E. M. (1981) Anal Biochem 111, 357 365 2. Bhown, A., Mole, J. E., Weissinger, A., and Bennett, J. C. (1978) J. Chromofogr. 148. 532-535. 3. Carlsson, J., Kierstan, M. P. J.. and Brocklehurst. K. (1974) Biochem. J. 139, 237-242. 4. Fahcy, R. C. Newton, G. L.. Dorian, R.. and Kosower, E. M. (1980) .4nal. Biochem. 107, I-IO. 5. Reeve, J.. Kuhlenkamp, J., and Kaplowitz, N. (1980) J. Chromarogr. 194, 424-428. 6. Reed, D. J., Babson. J. R., Beatty. P. W., Brodie, A. E.. Ellis. W. W., and Potter. D. W. (1980) .4nal. Biochem. 106, 55-62.
BIIOCHEMISTRY
114, 383-387
(1981)
Analysis of Biological Thiols: Derivatization with Monobromobimane and Separation by Reverse-Phase High-Performance Liquid Chromatography GERALD Department
L. NEWTON,
of Chemistry.
RANDEL
University
DORIAN,
of California,
Received
January
AND
San Diego,
ROBERT La Jolla,
C. FAHEY California
92093
5, 1981
A method is described for determination of biological thiols at the picomole level based upon conversion of thiols to fluorescent derivatives by reaction with monobromobimane and separation of the derivatives by reverse-phase high-performance liquid chromatography. Thiols separated by the procedure include N-acetylcysteine, coenzyme A, coenzyme M, cysteamine, cysteine, cysteinylglycine, ergothioneine, ethanethiol, glutathione, y-glutamylcysteine, homocy:steine, hydrogen sulfide, 2-mercaptoethanol, mercaptopyrimidine, methanethiol, pantetheine, 4’-phosphopantetheine. thiosulfate, and 2-thiouracil. Since monobromobimane has little fluorescence and reacts very selectively with thiols to produce fluorescent derivatives, crude extracts can be derivatized and analyzed without prepurification of the thiols, the entire process requiring only 1 to 2 h. The technique is illustrated by determination of the thiol levels in red blood cells.
In a recent paper we described a general method for the determination of biological thiols at the picomole level ( 1). The method
BrCH2
‘CH2X
was based upon derivatization of the thiol with monobromotrimethylammoniobimane (qBBr)’ and monobromobimane (mBBr),
RSCH2
CH2X
X=H,mBBr X:@N(CH3)3,‘@Br
followed by separation of the derivatives by cation-exchange chromatography and detection by fluorometry. Combined analysis of the qBBr and mBBr derivatives provided unique determination of 17 of the 18 thiols examined. The principal limitation of the method is the time required for sample analysis, 334 h for each of the bromobimane derivatives. In the present paper we decribe a separation of the mBBr derivatives of these thiols by reverse-phase high-performance liquid chromatography which permits the 383
full analysis to be performed in less than 40 min. MATERIALS
AND
METHODS
Materials. All chemicals and biochemicals were as previously described (1) unless ’ Abbreviations used: qBBr, monobromotrimethylammoniobimane (3,7-dimethyl-4-bromomethyl+trimethylammoniomethyl1,5 - diazabicyclo[3.3.0]octa - 3.6 diene-2,8-dione); mBBr, monobromobimane (3,7-dimethyl - 4 bromomethyl 6 - methyl - I .5 - diazabicyclo [3.3.0]octa 3,6-diene-2,8-dione); hplc, high-performance liquid chromatography. 0003-2697/81/100383-05$02.00/O Copyright iG 1981 by Academic Press. Inc All rights of reproducuon in any form reserved
384
NEWTON,
DORIAN.
AND
FAHEY
El
% B 100 ,’ //’ 80 60
40
I
!
20
30 mln
FIG. I. Reverse-phase high-performance liquid chromatogram of a mixture See Table I for key to abbreviations. Loads were: Cys and CC, 63 pmol; CoA, 170 pmol; 4’PP. 25 pmol; TU. 376 pmol; all others, 38 pmol.
otherwise indicated. Samples of human red blood cells were kindly provided by Dr. Thomas Vedvick. Preparation of samples. Standard thiol derivatives and red blood cell thiol derivatives were prepared as described previously (1). The samples were diluted in 0.2 N sodium citrate, pH 2.2, so that injection on either the cation-exchange column or the hplc column was possible. It should be noted that changing the sample diluent has some effect upon the derivative retention times. High-performance liquid chromatography. Separations were carried out on an Altex Model 332 gradient liquid chromatograph equipped with a Model 42 1 controller, a Gilson Spectra/G10 filter fluorometer with standard flow cell and o-phthalaldehyde filters, and a Shimadzu CR 1A printer-plotter. An Altex Ultrasphere-ODS (C-18) 5-ym column (4.6 X 150 mm), Part No. 256-06, was used at ambient temperature and a flow rate of 1.5 ml/min. The buffer system was a minor modification of that described by Bhown et al. (2)-buffer A: 100 ml methanol (Hplc grade, Fisher), 2.5 ml acetic acid (reagent grade, Mallincrodt), diluted to one liter with glass distilled water; buffer B: 900 ml methanol, 2.5 ml acetic acid, diluted to 1 liter with water. Buffers were adjusted to pH 3.9 with 50% NaOH and filtered through an 0.2~pm Millipore filter. The elution pro-
of thiol-mBBr derlvativcs. TS. Erg, NAC. CyA, and
file was as follows: O-1 0 min, 8% B isocratic; lo-20 min, 8-40% B, linear gradient; 20-25 min, 40% B, isocratic; 25530 min, 40--90% B, linear gradient; 30-32 min, 90-100% B, linear gradient (column regeneration). RESULTS The initial objective was to obtain a complete separation of the mBBr derivatives prepared previously (1) using a single-elution protocol on a reverse-phase hplc column. Acetonitrile, methanol, water, and acetic acid, adjusted to various pH values, were tested as mobile phases but the methanolwater-acetic acid system of Bhown et al. (2) proved most effective. A satisfactory separation of the derivatives was obtained using a combination of isocratic and linear gradient elution as shown in Fig. 1. A few pairs of derivatives are not fully resolved and the peak for the cysteamine derivative is rather broad, but the separation is more than satisfactory for most purposes. Attempts to improve the resolution of the early portion of the chromatography have not been successful and a longer column may be required for this purpose. The peaks identified as hydrolysis products in Fig. 1 are observed when mBBr is allowed to react with water in the absence of added thiol. It was found that the sensitivity could be
PICOMOLE
DETERMINATION
OF TABLE
RFTENTION
Thiol N-Acetylcysteine Coenzyme A Coenzymme M Cysteamine Cysteine Cysteinylplycine Ergothioneine Ethanethiol Glutathione -&lutamylcysteine Homocysteine Hydrogen sulfide Hydrolysis 2-Mercaptoethanol Mercaptopyrimidine Methanethiol Pantetheine 4’-Phosphopantetheine Thiosulfa1.e 2-Thiouracil
TIMES
~i\in
RELATIVE
BIOLOGICAL
I
FL UORESCF.NCE
FOR Ttitot-mBBr
Symbol (Figs. I and 2)
Retention (min)
NAC CoA CoM CYA CYS CG Erg Et GSH YGC HC HS H 2ME MPY Me P 4’PP TS TU
Il.8 20.9 6.7 12.4 3.7 5.2 9.0 29.2 6.1 4.5 7.2 24.6 10.2, 18.3, 19.5 25.1 24.3 22.2 17.7 4.3 19.8
increased by a factor of 10 over that shown in Fig.1 without an unacceptable increase in baseline noise so that it is possible to quantitate rather low levels of thiols, e.g., 2 pmol of cysteine or 20 pmol of ergothioneine. Retention times and relative fluorescence yields are given in Table 1. The latter generally parallel those obtained by cation-exchange chromatography ( 1) but tend toward higher values at higher methanol content owing to higher fluoresence yields in methanol relative to water. As in the previous study (1) red blood cells were chosen to test the utility of the method. Cells were lysed directly in a solution of mBBr and the extract was deproteinized with HCl--sodium methanesulfonate ( 1). After dilution in running buffer the chromatogram shown in Fig.2 was obtained. The main thiol component is GSH (2.9 mM in red blood cells) with ergothioneine ( 100 HIM) also being present at a substantial level. Cysteine is present only at a trace level (I
385
THIOLS
All other are attributed and 18.5 min) izing reagents sults are in the viously ( 1,3).
PM).
DERIVATIVES
time
Relative fluoresence 0.5 0.4 I.1 0.8 I .o 0.7 0.08 I .8 ( I .OO) 0.8 0.8 0.9
18.6 I.1 I .2 I .o I.1 0.8 0.2 0.1
peaks in the chromatogram to hydrolysis products (9.8 or artifacts of the deprotein(1.7 and 19.8 min). The rerange of values reported pre-
DlSCUSSlON With the present use of reverse-phase hplc to separate mBBrrthio1 derivatives our approach to analysis of biological thiols is com-
:
h
1j
Fit;. 2. Reverse-phase high-performance liquid matogram of the mBBr derivatized. deproteinized tract from red blood cells.
chroex-
386
NEWTON.DORIAN,ANDFAHEY
plete. The main limitation of the earlier method (1) was the time required to prepare both the mBBr and qBBr derivatives and to separate them by ion-exchange chromatography. With reverse-phase hplc it proved possibleto separate the complete list of thiols as their mBBr derivatives, thereby circumventing the need to use qBBr derivatives as well. Since the mBBr derivatives are more stable and more fluorescent than the qBBr derivatives this is a decided advantage in itself, as well as a time-saving feature. With hplc analysis the total time for preparation and analysis of a sample is only l-2 h, the time per sample being even lessif many samples are processed simultaneously. A second difficulty found using cation-exchange chromatography was that the acidic thiol derivatives eluted shortly after the breakthrough volume and were therefore not well resolved. Thus, the thiosulfate and coenzyme M derivatives with qBBr were not separated and most of the mBBr derivatives of acidic thiols coeluted. In the present separation (Fig. 1) the mBBr derivatives of acidic compounds (thiosulfate, coenzyme M, Nacetylcysteine, 4’-phosphopantetheine, and coenzyme A) are fully resolved and distributed throughout the chromatogram. Of course, even with the improved resolution of the hplc procedure there remains the possibility that two different thiol derivatives will coelute. It is therefore useful to be able to check assignments using cation-exchange chromatography of the mBBr derivative. Should doubt still remain the qBBr derivative can be prepared and examined by cation-exchange chromatography or by thinlayer chromatography and electrophoresis as described previously (4). With this combination of techniques available there should be little possibility for mistake in the identification of a specific biological thiol. However, for routine determinations where identity of the components has been established the hplc procedure is by far the most efficient. Two recent papers have described meth-
ods for analysis of biological thiols based upon high-performance liquid chromatography. Reeve et al. (5) utilized Ellman’s reagent to convert cysteine, GSH, and yglutamylcysteine to the mixed disulfides of 5-thio-2-nitrobenzoic acid and separated the latter by reverse-phase high-performance liquid chromatography. Peaks were detected spectrophotometrically at 280 nm providing nanomole sensitivity for the determination of these thiols. The present method has several advantages over this approach. First, not all biological thiols form mixed disulfides with Ellman’s reagent (3); thus, ergothioneine does not react and dithiols are oxidized to cyclic disulfides. Second, various peptides and other nonthiol biological compounds absorb at 280 nm and can potentially interfere with the analysis whereas background peaks resulting from fluorescent nonthiol compounds are much less common. Third, fluorescent detection of the mBBr derivatives is more sensitive by some two orders of magnitude. Finally, it has been possible to separate the full range of biological thiols using the mBBr derivatives whereas it is not clear that this is possible using derivatives prepared with Ellman’s reagent. The other recent adaptation of high-performance liquid chromatography to analysis of thiols is the study by Reed et al. (6). They utilized initial carboxymethylation of the thiol group to block thioldisulfide exchange reactions. Free amino groups were then 2,4dinitrophenylated to provide a chromophoric group. The derivatives were separated on a 3-aminopropylsilane derivatized silica column and detected spectrophotometrically with nanomole sensitivity. Since all compounds having free amino groups are detected the method is less selective than the one reported in this work. Also, thiols lacking free amino groups (ergothioneine, coenzymes A and M, 4’-phosphopantetheine, methanethiol, etc.) cannot be measured with this approach. On the other hand their method is capable of measuring disulfides
PICOMOLE
DETERMINATION
and other o.xidized forms of sulfur amino acids whereas the present method is limited to the detection of thiols or of disulfides following reduction to thiols. In the latter case the disulfide ‘components can be detected but information as to what specific disulfides or mixed disulfides were present prior to reduction is lost. Thus, the method of Reed et al. (6) appears to be the best available for determining specific disulfides and mixed disultides. ACKNOWLEDGMENTS This research was supported by the USPHS under Grant GM 22122 from the National Institute of General Medical Sciences and Grant CA 25009 from the National Cancer Institute. We thank Dave Smith of Cole
OF
BIOLOGICAL
387
THIOLS
Scientific for making in these studies.
available
the hplc equipment
used
REFERENCES I. f:ahey, R. C.. Newton. G. L., Dorian. R.. and Kosower. E. M. (1981) Anal Biochem 111, 357 365 2. Bhown, A., Mole, J. E., Weissinger, A., and Bennett, J. C. (1978) J. Chromofogr. 148. 532-535. 3. Carlsson, J., Kierstan, M. P. J.. and Brocklehurst. K. (1974) Biochem. J. 139, 237-242. 4. Fahcy, R. C. Newton, G. L.. Dorian, R.. and Kosower, E. M. (1980) .4nal. Biochem. 107, I-IO. 5. Reeve, J.. Kuhlenkamp, J., and Kaplowitz, N. (1980) J. Chromarogr. 194, 424-428. 6. Reed, D. J., Babson. J. R., Beatty. P. W., Brodie, A. E.. Ellis. W. W., and Potter. D. W. (1980) .4nal. Biochem. 106, 55-62.