Anal. Chem.
1998, 70, 3505-3509
Quantitative Determination of Biological Sulfhydryl
Groups by Postcolumn Derivatization and
Elucidation of Microheterogeneity of Serum
Albumins
Tadashi Yasuhara† and Kiyoshi Nokihara*,‡
Junior College of Tokyo University of Agriculture, Sakuragaoka 1-1-1, Setagayaku, Tokyo 156, Japan, and Shimadzu
Scientific Research Inc., Kanda-Nishikicho 1-3, Chiyodaku, Tokyo 101, Japan
A quantitative analytical system for biological sulfhydryl
compounds has been developed using an ion-pair reagent
with isocratic elution and an on-line postcolumn deriva-
tization with Ellman-type reagents. As human or bovine
serum albumin has 35 cysteinyl residues, one cysteinyl
residue exists as a free sulfhydryl moiety, and this gives
rise to the microheterogeneity in serum albumin. Here
we report for the first time the quantitative characteriza-
tion of the microheterogeneity of serum albumin. Cys-
teine was found to be the major molecule attached to the
sulfydryl group of the serum albumins. Although glu-
tathione could not be detected, the Cys-Gly element of
glutathione was found. Freshly prepared human serum
albumin from healthy volunteers contained 0.46 nmol of
Cys/mL of serum, 0.24 nmol of Cys-Gly/mL of serum,
and very small amounts of glutathione (0.02 nmol/mL).
Cysteine residues in proteins and peptides play important roles
in stabilizing the conformation of molecules through disulfide
linkages in the process of protein folding and also occur as reactive
sulfydryl groups that contribute to oxidation/reduction processes
in vivo. Endogenous sulfhydryl compounds having small molec-
ular weight bind to cysteinyl residue(s) of proteins or peptides
and are considered to play a role in the folding process via
disulfide interchange that can give rise to microheterogeneity of
these cysteinyl residues in proteins. A sensitive and reproducible
quantitative analytical system for small molecular sulfhydryl
compounds is required in order to elucidate these biological
pathways as well as to characterize the microheterogeneity.
Human serum albumin (HSA)1 is a globular monomeric protein
with a molecular weight of 66 500 and contains 35 cysteinyl
* To whom correspondence should be addressed. Fax: +81-3-3219-5729.
E-mail: noki@ssr.shimadzu.co.jp.
† Tokyo University of Agriculture.
‡ Shimadzu Scientific Research Inc.
(1) Abbreviations used are as follows: BSA, bovine serum albumin; CE, capillary
electrophoresis; GSH, reduced form of glutathione; GSSG, oxidized form
of glutathione; HSA, human serum albumin; i.d., internal diameter; MALDI,
matrix-assisted laser desorption/ionization; MES, 2-(N-morpholino)ethane-
sulfonic acid; MS, mass spectrometry; RP-HPLC, reversed-phase high-
performance liquid chromatography; TOF, time-of-flight.
S0003-2700(98)00263-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/11/1998
residues, of which 34 residues participate in 17 disulfide bridges.2
Indeed, plasma HSA contains 17 disulfides and one free cysteine
at position 34 that is believed to be a mixture of a mercapto form
and a non-mercapto form which has a modified cysteinyl residue.
The sulfhydryl content was reported as 0.6-0.7 sulfhydryl/mol.3
Although Brown has reported the non-mercapto HSA as a mixed
disulfide with cysteine and glutathione,2 no experimental details
have been given. Recently, Ikegaya et al. have completely
determined the 17 disulfide forms of recombinant HSA and
indicated that HSA produced by yeast had a single cysteinyl
residue at position 34, which is a free sulfhydryl moiety.4 This
moiety is probably the site that causes microheterogeneity in
plasma-derived HSA, as this has a similar sulfhydryl content. Small
sulfhydryl compounds such as cysteine and glutathione might be
attached to the free sulfydryl group on Cys-34 of HSA.5 Although
this phenomenon is considered to be a part of the biological
function of HSA, the biological and clinical significance of
mercapto HSA has not yet been clarified. Sulfhydryl content
should be related to disease and the change of sulfhydryl amounts
of plasma HSA before and after renal filtration has been reported.6
However, only the sulfhydryl content was determined, and the
types of molecules bound to the sulfhydryl group were not
determined.
The present paper describes a rapid, facile, and reproducible
quantitative analytical method for biological sulfhydryl group
determination by on-line postcolumn derivatization and its ap-
plication to serum albumins that reveal their microheterogeneity.
EXPERIMENTAL SECTION
Materials. Plasma HSA (HSA, Pentex) was purchased from
Miles Inc. Diagnostics Division (Kankakee, IL). Different prepa-
rations of bovine serum albumin (BSA) manufactured by Intergen
(2) Brown, J. R. In Albumin Structure, Function and Uses; Rosenoer, V. M.,
Murray, O., Marcus, A. R., Eds.; Pergamon Press: New York, 1977; pp 27-
51.
(3) Anderson, L.-O. In Plasma Proteins; Blombock, B., Hansen, L. P., Eds.; Wiley-
Interscience: New York, 1979; pp 43-54.
(4) Ikegaya, K.; Hirose, M.; Ohmura, T.; Nokihara, K. Anal. Chem. 1997, 69,
1986-1991.
(5) Janatova, J.; Fuller, J. K.; Hunter, M. J. J. Biol. Chem. 1968, 243, 3612-
3622.
(6) Nishimura, K.; Harada, K.; Nakayama, M.; Sugii, A.; Uji, Y.; Okabe, H. J.
Anal. Biosci. 1992, 15, 200-205.
Analytical Chemistry, Vol. 70, No. 16, August 15, 1998 3505

Figure 1. Schematic drawing of a system for characterization of
sulfhydryl compounds by on-line postcolumn derivatization. Dual
pump (1, 2), injector or autosampler (3), column oven (4), separation
column (5), coil reactor (6), detector (7), eluent (8), reactant (9), and
integrator (10).
(Purchase, NY) were generous gifts from Kokusai-Shiyaku Co.
Ltd. (Kobe, Japan). BSA was also purchased from Sigma (St.
Louis, MO). HSA and BSA were used as received. 5,5′-Dithiobis-
(2-nitrobenzoic acid) (DTNB) from Sigma, trifluoroacetic acid
(TFA), acetonitrile, dithiothreitol (DTT), H3PO4, NaH2PO4‚2H2O,
ethylenediamine tetraacetate (EDTA), NaOH, from Wako Pure
Chemicals (Osaka, Japan), the reduced form of glutathione (GSH)
and cysteinylglycine from Sigma, γ-glutamylcysteine from Kohjin
(Tokyo, Japan), and tri-n-butylamine from Tokyo Kasei (Tokyo,
Japan) were used without further purification. Human plasma was
obtained from 30 healthy volunteers between 21 and 23 years old.
MilliQ (Millipore, Bedford, MA) water was used.
Sulfhydryl Determination. Sulfhydryl groups were deter-
mined according to the method of Ellman.7 Reduction of proteins
was carried out with 0.5% DTT in 0.2 M NaH2PO4, 2 mM EDTA
(pH 8.0). Residual DTT was not removed from the reduction
mixture.
Instruments. Reversed-phase high-performance liquid chro-
matography (RP-HPLC) was carried out using a model CCPM
dual pump (Tosoh, Tokyo, Japan) for eluent and reactant transport,
respectively, a Rheodyne or an autosampler, model AS8010
(Tosoh), for sample injection, and a detector, model UV8010
(Tosoh), for monitoring. The materials were separated on a TSK-
ODS80TM column (4.6 mm i.d. × 250 mm, Tosoh). The above
column and a coil reactor constructed with Teflon tubing (0.2 mm
i.d. × 2 m) were placed in a column oven, model CO-8010 (Tosoh),
and analysis was carried out at 40 °C under isocratic conditions.
The system is indicated schematically in Figure 1. A model
SC8020 (Tosoh) was used for system control.
Sample Preparation and Analysis. Albumin was diluted to
20 nmol/100 µL with 0.1 M acetic acid, of which 10 µL was further
diluted with water to 100 µL and reduced with 100 µL of 0.5%
DTT in 0.2 M NaH2PO4 and 4.2 mM EDTA (pH 8.0) under a
(7) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
3506 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 2. Standard separation profiles of various biological sulf-
hydryl compounds (500 pmol each). Cysteinylglycine (1), cysteine
(2), homocysteine (3), GSH (4), γ-glutamylcysteine (5), mercaptoet-
hanol (6), mercaptoacetic acid (7), mercaptopropionic acid (8),
N-acetylcysteine (9), DTT (10). Column, TSK-ODS80TM (4.6 mm i.d.
× 250 mm); monitored at 412 nm; operation temperature, 40 °C;
elution solution (isocratic conditions with a flow rate of 0.8 mL/min),
0.1% tri-n-butylamine, 50 mM phosphate buffer (pH 2.3); reactant
(transferred at a flow rate of 1.0 mL/min to the coil reactor), 0.025%
DNTB, 2 mM EDTA, 0.1 M phosphate buffer (pH 7.6).
stream of nitrogen at room temperature in the dark with stirring
for 60 min. The resulting mixture (20 µL) was injected into the
HPLC. Ethanol (400 µL) was added to the human plasma (100
µL) and allowed to stand at 0 °C overnight. Precipitate obtained
by centrifugation (10 000 rpm at 0 °C for 20 min) was suspended
in 80% ethanol (1 mL) and recentrifuged as above. The resulting
precipitate was again suspended in 70% ethanol and centrifuged
as above. This protein was suspended in water (0.5 mL), and
the supernatant after centrifugation as above was used as the
albumin fraction. Next, 0.5% DTT in 0.2 M NaH2PO4 and 4.2 mM
EDTA (pH 8.0) was added to 100 µL of this fraction contained in
a Eppendorf tube (500 µL) under a stream of nitrogen and allowed
to stand at room temperature for 1 h. The resulting mixture (20
µL) was injected into the HPLC.
Analysis was carried out with 0.1% (v/v) tri-n-butylamine, 50
mM phosphate buffer, pH 2.30, as an eluent under isocratic
conditions at a flow rate of 0.8 mL/min and monitored at 412 nm.
Reactant consisting of 0.025% DNTB, 2 mM EDTA, and 0.1 M
phosphate buffer (pH 7.6) was transferred at a flow rate of 1.0
mL/min to the above coil reactor, maintained at 40 °C.
The column could be used for more than 50 analyses by using
an autosampler and can easily be regenerated by washing with
0.1% TFA and 0.1% TFA acetonitrile in the conventional manner
of the gradient elution.
RESULTS AND DISCUSSION
A quantitative analytical system for biological small molecular
sulfhydryl compounds was constructed by on-line postcolumn
derivatization with Ellman-type reagents. The system is illustrated
in Figure 1, consisting of a HPLC pump operating with isocratic
elution, an injector, and a column for RP-HPLC, followed by a coil
reactor, contained in an oven, and a UV detector. Ellman reagent-
containing reactant for postcolumn labeling was continuously
injected into the coil. By the use of tri-n-butylamine solution as
an ion-pair reagent, biological sulfhydryl compounds such as
cysteinylglycine, cysteine, homocysteine, GSH, γ-glutamylcysteine,
 Table 1. Characterization of Sulfhydryl Compounds in
Various Commercial Serum Albumin Preparations
(mole %)

sulfhydryl
albumin (lot no.)a Cys(SH)-Gly Cys(SH) contents
(1) HSA (42) 9.0 29.9 60
(2) BSA (M64606) 6.1 9.5 70
(3) BSA, Cohn Fr. V, 7.7 14.8 66
pH 5 (P7812)
(4) BSA, Cohn Fr. V, 6.3 14.5 71
pH 7 (N77069)
(5) BSA, Modified 6.0 11.9 61
Cohn Fr. V (P21075)
(6) BSA, pH 7 11.1 19.9 55
(RT93606)
(7) BSA (RT12801) 10.6 18.4 55
(8) BSA, standard, 9.9 21.8 53
pH 5 (M22204)
(9) BSA, standard, 10.0 21.8 60
pH 7 (R40102)
(10) BSA (PT88705) 7.8 14.9 56
Figure 3. Calibration curves of sulfydryl compounds related to GSH. (11) BSA (L58807) 10.7 20.3 56
(12) BSA (P65806) 11.3 19.4 59
(13) BSA (84H0341) 6.3 17.1 59
mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, (14) BSA (75H0894) 4.8 12.5 56
(15) BSA (34H0275) 4.8 14.2 59
N-acetylcysteine, and DTT could be efficiently and quantitatively (16) BSA (14H0246) 6.0 15.4 61
analyzed within 25 min. The flow rates of elution and of the
a Key: (1) Pentex, Miles; (2-5) Intergen, manufactured by the cold
reactant were changed to give optimal resolution and retention
ethanol process; (2) microbiological grade; (6-12) Intergen, manu-
times. A standard separation profile of these biological sulfhydryl factured by the heat shock process; (6) biotechnology grade; (7)
compounds is shown in Figure 2. GSH and its components could endotoxin reduced biotechnology grade; (10) fatty-acid-free biotech-
nology grade; (11) clinical reagent grade; (12) protease-free powder;
be determined within 10 min. Figure 3 indicates the calibration (13-16) Sigma.
curve of sulfydryl compounds generated from glutathione. The
present method allows determinations in the range from 10 pmol
to 1 nmol. Hence, the difference slopes for the compounds
seemed to reflect the reactivity in the postcolumn derivatization. instance, when a buffer of pH 2.75 was used, cysteinylglycine
GSH metabolism is one of the most important biological eluted at 3.5 min and DTT at 25 min. More than 97% recovery of
pathways. Meister reviewed the formation of sulfhydryl com- each sulfhydryl compound used in this study was achieved, as
pounds from GSH by γ-glutamyltransferase and aminopeptidase calculated from the UV absorption.
M or dipeptidase to cysteinylglycine and cysteine.8 Thus, the Recently, Russell and Rabenstein reported a sensitive analysis
analysis of GSH components can provide a tool for the elucidation of biological thiols and disulfides by capillary electrophoresis
of the biological processing. It can be predicted that GSH bound (CE).9 As CE requires several nanomole per liter (20 µL) samples,
to sulfhydryl groups of proteins may partially decompose and the minimum amount of sample necessary for analysis is similar
cause heterogeneity in the material. in both methods. However, the present method has several
Postcolumn derivatization offers the advantage that various advantages. In the CE method, peaks derived from reagents are
labeling reagents can be used and collection of intact target also detected, while in the present method peaks are observed
material in preparative amounts is much easier; this material can only for sulfhydryl compounds. In the CE method, derivatization
then be subjected to nuclear magnetic resonance spectroscopy (approximately 20 min) must be carried out prior to the analysis,
and/or mass spectrometry (MS). When 2,2′-dithiodipyridine was while here this is not necessary; hence, the analysis is more simple
used as labeling reagent and monitored at 343 nm, only half the and rapid. The sensitivity in the CE method is sample dependent,
sensitivity was achieved (data not shown). Labeling with the while here a similar sensitivity is found for all target compounds;
fluorescent reagents can also be employed and may be expected
therefore, it is suitable for following the time course of GSH.
to give a higher sensitivity, although it is not practical because of
Although the CE method is not relevant for the analysis of
the high cost in running continuous infusion of expensive reagents
sulfhydryl compounds bound through disulfide linkages, the
to the coil reactor. The present sensitivity of DTNB with
present method can be used for such compounds after release
monitoring at 412 nm is a practical compromise and shows
from proteins by reduction with DTT, etc. Finally, HPLC is
sufficient sensitivity with a low background. In addition, the
superior with respect to preparative separation.
stability of the DTNB solution was adequate for routine analysis
Both plasma and recombinant HSA have one cysteinyl residue
using an autosampler. The present system allows isocratic
at position 34 with a free sulfhydryl moiety, although sulfhydryl
separation with a high reproducibility and a stable baseline during
determination according to Ellman7 was 0.6-0.7,4 and this gives
analytical runs. The pH value of the eluent in Figure 2 is 2.30.
Efficient analyses could be conducted between 2.0 and 3.5, rise to the microheterogeneity of HSA. The present method was
although the pH of the eluent influences the retention time; for applied to reveal this heterogeneity in the different serum albumin

(8) Meister, A. Trends Biochem. Sci. 1981, 6, 231-234. (9) Russell, J.; Rabenstein, D. L. Anal. Biochem. 1996, 242, 136-144.

Analytical Chemistry, Vol. 70, No. 16, August 15, 1998 3507
 Table 2. Sulfhydryl Compounds in the Freshly
Prepared HSA Fraction of 30 Healthy Volunteers
(nmol/mL Serum)

no. Cys(SH)-Gly Cys(SH) GSH γGlu-Cys(SH)

1 0.82 0.87 0.21 0.16
2 0.27 0.42 0.03
3 0.24 0.42 0.03
4 0.27 0.40
5 0.24 0.38 0.03
6 0.31 0.45 0.03
7 0.22 0.36
8 0.26 0.40
9 0.22 0.36
10 0.24 0.47 0.03
11 0.22 0.45 0.03
12 0.29 0.47 0.06
13 0.24 0.49 0.03
14 0.22 0.44 0.03
15 0.24 0.56
16 0.24 0.51
17 0.24 0.47
18 0.20 0.44
19 0.22 0.49
20 0.22 0.40
21 0.24 0.44
22 0.22 0.42
23 0.18 0.40
24 0.20 0.42
25 0.18 0.49 0.03
26 0.13 0.40
27 0.16 0.42 0.03
28 0.18 0.45
29 0.16 0.51
Figure 4. Profiles of two analytical examples. Background was 30 0.16 0.44
subtracted. (a) BSA (3) in Table 1; (b) HSA (1) in Table 1. HPLC av 0.24 0.46 0.02
conditions are the same as in Figure 2. The large peak eluted after
20 min is DTT used in the sample preparation.

preparations. Characterization of sulfydryl compounds in the

various commercial preparations (mole %) is listed in Table 1. As
examples, the chromatograms of two analytical runs are shown
in Figure 4. Albumin amounts were calculated from UV absorp-
tion at 279 nm,10 and sulfhydryl content was determined according
to the method of Ellman.7 Although details of the downstream
processing of commercially manufactured serum albumin were
not disclosed, different protocols were employed for different
purposes. Intergen’s BSA is manufactured in two distinct pro-
cesses, by the cold ethanol precipitation and by the heat shock
process. However, there were no significant differences in the
present analytical results. In all commercial preparations, neither Figure 5. Profile of a representative chromatogram. Background
GSH nor γ-glutamylcysteine was found. The other biological was subtracted. HPLC conditions are the same as in Figure 2.
sulfhydryl derivatives, not related to GSH, such as homocysteine,
mercaptoethanol, mercaptoacetic acid, mercaptopropionic acid, less than that of HSA, and the difference seemed to be due to
and N-acetylcysteine, were also not found. Hence, cysteine, dimer.
molecular mass 120.9 Da, was confirmed by MALDI-TOF-MS in Sulfhydryl compounds in freshly prepared HSA fractions from
eluate without derivatization, although Cys-Gly could not be the sera of 30 healthy volunteers were also determined. The HSA
identified by MS, since the eluate contained a high concentration fraction was separated by ethanol precipitation of plasma and was
of salts. Consequently, the commercial preparations of BSA carried out after uptake of blood without long-term storage. The
contain 55-70% sulfhydryl molecules, and the major substituted results are summarized in Table 2. Figure 5 is a representative
material is 10-22% cysteine and 6-10% cysteinylglycine. The rest profile. In the microheterogeneity of freshly prepared HSA,
is considered to be dimer as reported by Janatova et al.5 On the sulfhydryl compounds bound to HSA are individual-specific,
other hand, HSA is 29.9% cysteine, 9.0% cysteinylglycine and although cysteine was (0.46 nmol/mL of serum) the major
contained almost no dimer. The amount of cysteine in BSA was substituent, together with cysteinyl glycine (0.24 nmol/mL). GSH,
(10) Peters, T. Jr. In Plasma Proteins; Putnam, F. W., Ed.; Academic Press: New which did not occur in purified commercial HSAs, was found in
York, 1975; Vol. 1, p 147. very small amounts (0.02 nmol/mL), but no γ-glutamyl cysteine
3508 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
was present. Sulfhydryl contents should be related to the status
of some diseases,6 and hence characterization of such molecules
is interesting and may be performed with the present system.
CONCLUSION
We have developed a rapid, sensitive, and reproducible
quantitative analytical system for biological small molecular
sulfhydryl compounds using an on-line postcolumn derivatization
with Ellman-type reagents. With this method, sulfhydryl com-
pounds bound to cysteinyl residue(s) of proteins or peptides can
be easily determined. The system has been applied to serum
albumins, and we have succeeded for the first time in quantita-
tively characterizing their heterogeneity. Hence, with regard to
the sulfhydryl moiety, significant microheterogeneity was dem-
onstrated. Cysteine was found to be the major molecule attached
to the sulfydryl group of the cysteinyl residue of serum albumin.
Although glutathione could not be detected, peptidyl elements of
the glutathione molecule were found. The system provides a
useful method for quality control not only for plasma-derived
proteins but also for recombinant proteins. The characterization
and investigation of the relationship between diseases and sulf-
hydryl compounds will also be of clinical interest.
ACKNOWLEDGMENT
A part of the present study was supported by the Sunbor grant.
We thank Dr. V. Wray, Gesellschaft für Biotechnologische
Forschung, Braunschweig, Germany, for discussion and linguistic
assistance with the manuscript.
Received for review March 9, 1998. Accepted May 29,
1998.
AC9802630
Analytical Chemistry, Vol. 70, No. 16, August 15, 1998 3509