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Derivatization of Cysteine and Cystine for Fluorescence Amino Acid

Analysis with the O-phthaldialdehyde/2-mercaptoethanol Reagent

Article  in  Journal of Biological Chemistry · August 1979

DOI: 10.1016/S0021-9258(18)50355-0 · Source: PubMed

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THE JOURNAL OF BIOL.OGICAL. CHEMISTRY
Vol. 254, No. 14, Issue of July 25, pp. 6248-6251, 1979
Printed in U.S. A.

Derivatization of Cysteine and Cystine for Fluorescence Amino Acid

Analysis with the &Phthaldiald&hyde/Z-Mercaptoethanol Reagent*
(Received for publication, November 21, 1978, and in revised form, January 30, 1979)

Kang S. Lee+ and Dennis G. Drescher$,g

From the Laboratory of Neuro-otolaryngology, National Institute of Neurological and Communicative Disorders and
Stroke, National Institutes of Health, Bethesda, Maryland 20205

Previous reports (Drescher, D. G., and Lee, K. S. methods have yet been detailed for derivatizing these amino
(1978) Anal. Biochem. 84, 559-569; Lee, K. S., and acids to yield high fluorescence adducts specifically for OPA/
Drescher, D. G. (1978) Int. J. Biochem. 9,457-467) have 2-ME analysis. In a previous study (6), we determined the
shown that high performance liquid chromatographic relative fluorescence of OPA/Z-ME adducts of six common
analysis of amino acids with the o-phthaldialdehyde/2- cysteine derivatives and showed that the adducts of cysteic
mercaptoethanol reagent (OPA/P-ME) is one of the acid and S-3-sulfopropylcysteine have 30- to 40-fold greater
most sensitive procedures currently available for micro fluorescence than the “half-cystine” adduct. In the present
amino acid analysis. In the present paper, methods are study we, therefore, compare the suitability for fluorescence
presented for the modification of cysteine and cystine amino acid analysis of sulfopropylation (12) and performate
in proteins for micro amino acid analysis using OPA/2-
oxidation (13, 14), the reactions which form S-3-sulfopropyl-
ME. Cysteine and cystine, which both show low fluo-
cysteine and cysteic acid, respectively. We have chosen egg
rescence with OPA/P-ME, are converted to cysteic acid

Downloaded from www.jbc.org by guest, on July 9, 2011

with performic acid directly, or to S-3-sulfopropylcys- white lysozyme (containing 4 cystines) and human carbonic
anhydrase C (containing 1 cysteine) for these studies. In doing
teine with 1,3-propane sultone after reduction of cys-
tine with tri-n-butylphosphine. Cysteic acid and S-3- so, we establish a general, highly sensitive method for com-
sulfopropylcysteine form highly fluorescent adducts plete micro amino acid analysis of proteins using OPA/P-ME.
with OPA/S-ME. The formation of S-3-sulfopropylcys-
MATERIALS AND METHODS
teine in proteins and the subsequent hydrolysis of the
proteins with methanesulfonic acid are particularly Materials-Reagents of the highest purity available were used for
useful for complete amino acid analysis at the picomole amino acid analyses (5,6). All other chemicals were of reagent grade
level using a single sample. or better. Purified carbonic anhydrase C from human erythrocytes
was obtained from Worthington. Egg white lysozyme (3 times crys-
tallized, dialyzed, lyophilized), OPA, and cysteic acid were from
Sigma, and 2-ME was from Calbiochem. Amino acid standards, 4N-
methanesulfonic acid containing 0.2% tryptamine, constant boiling
Since Roth (1) first reported that amino acids form highly
HCl (Sequanal grade), S-3-sulfopropylcysteine, Brij-35 (30% solution),
fluorescent adducts in basic aqueous solutions with o-phthal- and thiodiglycol (25% solution) were from Pierce Chemical Co., Rock-
dialdehyde and 2-mercaptoethanol, amino acid analysis using ford, IL. Hydrogen peroxide (30%) was from Fisher, and tri-n-butyl-
this fluorometric reagent has gained acceptance as an alter- phosphine and 1,3-propane sultone were obtained from Aldrich
native to amino acid analysis using ninhydrin (2-6). The Chemical Co. Inc., Milwaukee, WI. Ignition tubes (Corning 9860, 14
reaction of OPA’ and 2-ME with amino acids is shown in Fig. x 100 mm) were from Corning Glass Works, Corning, NY. The tubes
1. The OPA reagent, used in conjunction with high perform- were prewashed in a solution of concentrated H2S04 and HNOs (9:l)
before use.
ance liquid chromatography, is at least 10 to 100 times more Performic Acid Oxidation-The method of performate oxidation
sensitive than ninhydrin for the detection of most amino acids described by Hirs (13, 14) was modified in this study for use with
(6). Although OPA/B-ME does not form fluorescent adducts microgram amounts of protein. One hundred ~1 of a 10 pM solution of
with proline and forms only weakly fluorescent adducts with egg white lysozyme or human carbonic anhydrase C (1 nmol of either
lysine, proline is accurately detected after oxidation with protein) was placed in an ignition tube, and the solution was frozen
chloramine-T (5, lo), and lysine is detected as an adduct of on drv ice and 1voDhilized. Performic acid was DreDared bv adding 0.1
ml or 30% hydrogen peroxide to 1.9 ml of s8% formic acid. ?‘he
intermediate fluorescence after adding the detergent Brij to solution was allowed to stand in a closed container at 25°C for 2 h.
the reaction mixture (2, 11). Cysteine and cystine both form Then the mixture was cooled to 0°C and used immediately. One
adducts of low fluorescence with the OPA reagent,’ but no hundred ~1 of performic acid was added to the ignition tube containing
the protein, and the mixture was allowed to stand at 0°C for 2% h.
* The costs of publication of this article were defrayed in part by The reaction was terminated by adding 2 ml of cold deionized water,
the payment of page charges. This article must therefore be hereby and the solution was frozen on dry ice and lvophilized.
marked “advertisement” in accordance with 18 U.S.C. Section 1734 Sulfopropylation-Either carbonic anhydrase or lysozyme was
solely to indicate this fact. treated with TBP and PS. TBP reduces cvstine to cvsteine (15-17).
$ Present address, Laboratory of Bio-otology, Department of Oto- and PS alkylates cysteine to S-3-sulfopropylcysteine-(12), and these
laryngology, Wayne State University School of Medicine, Detroit, MI reactions can be performed simultaneously. The method of Riiegg
48201. and Rudinger (12) was modified for microanalysis as follows. One
5 To whom all correspondence should be addressed at the Labo- nmol of protein that had been lyophilized in an ignition tube was
ratory of Bio-otology. dissolved in 0.1 ml of aqueous 0.5 N NaHC03 mixed with n-propanol
I The abbreviations used are: OPA, o-phthaldialdehyde; 2-ME, 2- (l:l, v/v). Ten ~1 of 50 mM TBP in 100% n-propanol and 6 ~1 of 240
mercaptoethanol; MSA, 4N-methanesulfonic acid containing 0.2%
tryptamine; PS, 3-hydroxypropanesulfonic acid y-sultone (1,3-pro- which are present in pure cysteine or in cysteine formed from cystine
pane sultone); TBP, tri-n-butylphosphine. in the presence of 2-ME compete intramolecularly with 2-ME for
‘The reason for the low fluorescence of OPA/B-ME adducts of position 1 in the isoindole (Fig. 1, and S. S. Simons, Jr., personal
cysteine and cystine is not known. It is possible that sulfhydryl groups communication).

6248
 Fluorescence Amino Acid Analysis of Cysteine and Cystine 6249
TABLE I
Amino acid compositions of egg white lysozyme and human
carbonic anhydrase C
Compositions were determined by amino acid analysis using o-
phthaldialdehyde/2-mercaptoethanol. Data are reported as moles of
OPA Z-ME a -AMINO AClD residue per mol of protein (mean + S.D.). Hydrolysis was performed
POSTULATED
ADDUCT for 24 h for all amino acids (columns 1 to 4), including threonine and
serine. Each sample injected for analysis corresponded to 0.24 pg of
FIG. 1. Reaction of o-phthaldialdehyde (OPA) and P-mercaptoeth-
nrotein.
anol (2-ME) with a-amino acids to yield fluorescent adducts with the
postulated structure. The adducta have excitation and emission spec- A. Lysozyme
tra similar to those of 1-alkylthio-2-alkylisoindoles (7-9). 2. 3. 4.
1. Treated Treated Treated 5.
Untreated” with per- with TBP with TBP Ac-
mM PS in 60% n-propanol (both solutions prepared immediately (M8.W formic acidh and PS and PS” tua1
(MSN WI’3 (MSA)
before use) were added, and the reaction mixture was kept under NP
at 25°C for 2 h. The reaction was terminated by adding 50 ~1 of Asnartic acid 20.4 + 0.3 22.0 + 0.4 21.1 + 0.3 21.5 * 0.3 21
constant boiling HCl, and the mixture was diluted with 0.5 ml of Threonine 6.5 + 0.2 6.7 k 0.1 6.6 -r- 0.1 7.1 + 0.1
deionized water, frozen, and lyophilized. Lyophihzation, instead of Serine 8.9 + 0.1 9.2 f 0.3 9.1 f 0.2 9.4 + 0.3 10
rotary evaporation as reported by Ruegg and Rudinger (12), was Glutamic acid 5.2 -C 0.1 5.5 IL 0.1 5.4 -c 0.1 5.5 rt 0.2
performed because the small amounts of protein were apparently Proline 2.2 r 0.1 2.7 + 0.3 2.5 + 0.4 2.4 k 0.4
labile to rotary evaporation, evidenced by amino acid analyses of Glycine 12.3 + 0.2 12.2 f 0.1 11.7 + 0.3 12.5 -c 0.2
decreased accuracy (data not shown). Because PS is a suspected Alanine 12.3 -t 0.3 12.1 f 0.2 11.8 f 0.3 12.0 +- 0.1
carcinogen (18,19) the following precautions were taken. Three Pyrex Half-cystine’ N.D. 7.2 rf; 0.1 9.0 * 0.5 8.5 f 0.2
vapor traps (Corning 7729, 28 x 200 mm) cooled with the dry ice in Valine 5.9 + 0.1 5.8 f 0.1 5.7 + 0.1 5.7 + 0.2
acetone were connected in series between the lyophihzation flask and Methionine 2.1 + 0.0 2.1 f 0.1” 1.7 + 0.1 1.7 + 0.1
the lyophihzer. The sample tube, insulated with a piece of Styrofoam, Isoleucine 5.6 + 0.1 5.5 f 0.1 5.7 + 0.2 5.4 + 0.2
was placed inside the flask. One ml of 10% NaOH was placed inside Leucine 8.4 -c 0.0 8.4 f 0.1 8.4 f 0.2 8.0 f 0.2
each trap. Thus the PS was condensed and hydrolyzed to noncarcin- Tyrosine 3.3 -t 0.1 1.8 f 0.2 2.9 + 0.3 3.5 + 0.2

Downloaded from www.jbc.org by guest, on July 9, 2011

ogenic products. Phenylalanine 3.0 -C 0.1 3.1 f 0.1 2.9 -+ 0.1 3.0 * 0.3
Preparation of Samples for Amino Acid Analysis-One-nmol Histidine 1.4 + 0.4 1.8 + 0.2 1.6 + 0.3 1.5 f 0.4
protein samples were hydrolyzed with 100 ~1 of MSA at 115’C for 24 Tryptophan 5.8 + 0.1 N.D. N.D. 5.0 -c 0.2 6
h according to the method of Simpson et al. (20) modified by Lee and Lysine 6.5 + 0.1 6.1 t- 0.1 6.2 + 0.3 5.9 +- 0.3 6
Drescher (6). After hydrolysis, the samples were partially neutralized Arginine 11.3 + 0.2 10.8 + 0.2 11.0 & 0.3 10.6 f 0.3 11
with 100 aI of 3.5 N NaOH. Some samples were hydrolyzed with 100
B. Carbonic anhydrase
$ of constant boiling HCl at 110°C for 22 h (12) instead of with MSA,
and these samples were partially neutralized with 100 pl of 5.25 N Treated Treated Treated
NaOH (21). When necessary, neutralized samples were appropriately Untreated with per- with TBP with TBP AC-
diluted with the first buffer used for amino acid analysis. Analyses (MSA) formic acid and PS and PS ma1
were performed with an Aminco AminaIyzer high performance liquid (MSA) WCU (MS.4
chromatography system (American Instrument Co., Silver Spring, Aspartic acid 28.2 + 0.9 29.5 f 1.0 28.1 + 1.0 29.6 + 0.9 29
MD) using OPA/Z-ME (5, 6). Threonine 11.5 + 0.2 11.5 f 0.1 11.8 + 0.2 12.7 + 0.2 12
Serine 16.6 -t 0.3 16.4 f 0.6 17.3 + 0.3 18.0 f 0.3 18
RESULTS Glutamic acid 24.2 + 0.1 24.4 f 0.5 24.5 + 0.2 25.7 f 0.1 24
Proline 17.2 f 1.5 17.8 -+ 0.8 17.7 + 1.2 16.4 f 1.9 17
Table I shows the amino acid compositions of (A) egg white Glycine 22.3 rt 0.3 22.0 * 0.7 21.7 -t 0.3 22.8 + 0.3 22
lysozyme and (B) human carbonic anhydrase C, determined Alanine 13.9 f 0.2 13.5 f 0.4 13.9 + 0.2 14.0 -r- 0.2 13
for four different treatments (columns 1 to 4) namely: 1) no Half-cystine’ N.D. 1.3 + 0.1 1.5 -+ 0.1 1.3 + 0.1
treatment of the protein, followed by hydrolysis of the protein Valine 16.7 + 0.2 16.4 f 0.9 16.4 f 0.3 15.8 + 0.7 17
in MSA; 2) per-formic acid oxidation and hydrolysis in MSA; Methionine 1.2 -c 0.0 1.0 -c 0.1s 0.9 + 0.1 0.9 +- 0.0
Isoleucine 8.8 + 1.0 8.1 f 0.4 8.5 f. 0.1 7.7 + 0.3 9
3) reduction with TBP, sulfopropylation with PS, and hy-
Leucine 27.2 + 0.4 27.2 + 0.9 27.6 + 0.4 25.2 f. 0.6 26
drolysis in HC1; and 4) reduction with TBP, sulfopropylation Tyrosine 8.4 f 0.4 5.2 it 0.3 7.5 + 0.3 8.6 + 1.3 8
with PS, and hydrolysis in MSA. The actual amino acid Phenylalanine 11.9 -t 0.4 11.0 f 1.6 11.5 rfr 0.3 12.4 f 0.7 12
compositions, determined from sequence data (22, 23), are Histidine 11.1 -t 0.5 13.0 + 1.0 12.1 + 0.3 12.5 f 0.7 12
shown in column 5. For the untreated proteins (column l), Tryptophan 6.4 + 0.1 N.D. N.D. 5.5 -r- 0.2
cysteine and cystine were difficult to detect because of the Lysine 25.3 -t 0.8 23.9 + 1.0 23.8 + 0.3 22.9 + 0.6 24
Arginine 7.3 + 0.1 7.3 + 0.3 7.2 f 0.1 7.2 + 0.1
known acid lability of these amino acids (24) and the low
fluorescence of the cystine-OPA/Z-ME adduct (5, 6), and, a Column 1, A and B, shows the analysis of unmodified lysozyme
therefore, were not determined. However, for proteins treated and carbonic anhydrase, respectively, performed as described under
“Materials and Methods.” “ MSA” refers to hydrolysis in 4N-meth-
with performic acid (column 2), cysteine and cystine were anesulfonic acid containing 0.2% tryptamine. For lysozyme, the num-
accurately determined because of the relatively high fluores- ber of determinations, N, equals 6; for carbonic anhydrase, N = 5.
cence of the cysteic acid-OPA/2-ME adduct (6). The methi- * Column 2 shows the analysis of performate-oxidized proteins (see
onine sulfone resulting from per-formic acid oxidation of me- text for details). For lysozyme, N = 4; for carbonic anhydrase, N = 4.
thionine (13,14) was also determined using OPA/Z-ME. How- ’ Column 3 shows the analysis of protein treated with tri-n-butyl-
ever, with performic acid oxidation, tryptophan was com- phosphine (TBP) and 1,3-propane sultone (PS). “HCl” refers to
hydrolysis in constant boiling HCl. For lysozyme, N = 8; for carbonic
pletely lost and tyrosine was partially destroyed.3 When the anhydrase, N = 3.
proteins were treated with TBP and PS and hydrolyzed in d Column 4 shows the analysis of proteins treated as in column 3,
HCl according to the modified method of Ruegg and Rudinger except that hydrolysis was performed with MSA. For lysozyme, N =
(12) (column 3, and “Materials and Methods”), the recoveries 5; for carbonic anhydrase N = 4.
of all of the amino acids were good, except that tryptophan ’ Column 5 shows theoretical amino acid compositions for egg white
lysozyme (22) and human carbonic anhydrase C (23).
3 Incomplete recovery of tyrosine has been shown to be due to /Not determined (N.D.) for untreated proteins; determined as
halides such as chloride (13, 14,25). Halide impurities in the reagents cysteic acid in column 2, and as S3sulfopropylcysteine in columns 3
are probably important at the low concentrations of protein employed and 4.
in the present study (M. Doscher, personal communication). g Determined as methionine sulfone after performate oxidation.
6250 Fluorescence Amino Acid Analysis of Cysteine and Cystine
FORMATE
PH 325 ,
FORMATE
PI+425
BORATE
pH10.6
material is adequate for compositional studies of proteins and
I
/
peptides. This is particularly noteworthy, because with flu-
‘00 A orescamine and ninhydrin, even under ideal conditions, anal-
s yses are 5 to 10 times less sensitive than with OPA/2-ME (2,
6).
However, the analysis of unaltered cysteine and cystine
with OPA/S-ME presents certain problems. Cysteine and
cystine yield OPA/B-ME adducts that are much less fluores-
cent than most of the other amino acid adducts (the “half-
cystine” adduct is about 40-fold less fluorescent on an equi-
molar basis), thus making the determination of cystine and
cysteine difficult (1, 6). The problem is compounded by the
low abundance of cysteine and cystine in proteins and by the
fact that cystine and alanine show peak resolution that is
extraordinarily pH dependent.5 Such characteristics make it
important to develop a method for specifically modifying
cysteine and cystine in proteins prior to OPA/2-ME amino
acid analysis.
In the present paper, we have compared the suitability of
four methods for performing complete micro amino acid anal-
yses using OPA/B-ME (Table I). Analysis of unmodified pro-
teins (column 1) yields values that are close to the actual
values (column 5), with the exception of cystine. Performate
ot- oxidation (column 2) suffers drawbacks because of the destruc-
tion of tryptophan and tyrosine. Sulfopropylation with HCl

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0 20 40 60 80 loo hydrolysis (column 3) yields good values for all the amino
TIME (MINUTES) acids except tryptophan, and, therefore, a separate sample is
FIG. 2. Elution diagrams for OPA/B-ME amino acid analysis of required for the determination of this amino acid (6). How-
(A) egg white lysozyme and (B) human carbonic anhydrase C, after ever, when MSA instead of HCl is used for hydrolysis (column
treatment with tri-n-butylphosphine and 1,3-propane sultone (see 4), the sulfopropylation method yields good analyses of tryp-
text). SP-CYS denotes S-3.sulfopropylcysteine. Sulfopropylation and tophan (about 80%) and excellent analyses of the other amino
hydrolysis were performed using 1 nmol of protein. For each analysis, acids.’ In addition, only a single hydrolysis sample is required.
10 ~1 of a solution of hydrolyzed protein (0.24 pg) was used; for
Inglis and Liu (30), Inglis et al. (31), and Simpson et al. (20)
lysozyme, this is l/60 of the total sample, and for carbonic anhydrase,
l/120 of the total sample. The peak at 84 min is the sum of histidine have described methods for obtaining complete ninhydrin
and the “buffer change” (BC) peak (5). Time after sample introduc- amino acid analyses of proteins using single hydrolysates. A
tion is shown on the abscissa. Elution buffers are marked at the top central part of these procedures is the formation of S-sulfo-
of the diagram (6). cysteine from cystine after treatment of the protein hydroly-
sate with dithiothreitol and tetrathionate. However, these
was completely destroyed, presumably because of the HCl methods (20, 30, 31) are not suitable for OPA/2-ME analysis,
used for the hydrolysis (26). The most accurate values, i.e. because S-sulfocysteine yields an OPA/B-ME adduct that is
those closest to the actual amino acid compositions, were only 2.9 times as fluorescent as the “halfcystine” adduct (6).
obtained after treatment with TBP and PS and hydrolysis in The modified sulfopropylation method, as discussed above, is
MSA (column 4). well suited for OPA/2-ME analysis because S-3sulfopropyl-
Fig. 2 shows the elution diagrams for OPA/2-ME amino
acid analysis of (A) egg white lysozyme and (B) human 4 Unpublished data.
carbonic anhydrase C after treatment of the proteins with ’ In our system (6), cystine and alanine peaks overlap above pH
3.25 and are optimally separated at pH 3.23. This kind of pH depend-
TBP and PS and hydrolysis in MSA. In Fig. 2, it can be seen ence occurs in other cation exchange systems as well (27).
that S-3-sulfopropylcysteine is easily detected as the OPA/2- ‘When protein is not treated with TBP and PS, but is hydrolyzed
ME adduct because of its high fluorescence and its early in MSA (Table I, column I), tryptophan is recovered at 91 to 97%
elution position (8 min). For lysozyme (Fig. 2A), with 8 “half- yields. This 3 to 9% loss of tryptophan is due to MSA hydrolysis and
cystines” and 8 leucines, the S-3-sulfopropylcysteine peak is can be corrected kinetically (20,28). Protein that is treated with TBP
about the same size as the leucine peak, emphasizing the fact and PS and hydrolyzed in MSA (column 4) gives tryptophan that is
recovered at 79 to 83% yields. The difference in tryptophan recoveries
that the S3sulfopropylcysteine adduct with OPA/Z-ME has
between columns 1 and 4, 12 to 14%, is probably due to the TBP/PS
about the same fluorescence as the “high fluorescence” ad- reaction itself. Recovery of methionine in Table I appears to be
duct, leucine-OPA/B-ME (1, 5, 6). Fig. 2B shows that for somewhat low, by 10 to 15%, due to the TBP/PS reaction (compare
human carbonic anhydrase C, even 1 cysteine residue (5, 23) columns 3 and 4 with columns 1 and 2; see also Ref. 12, p. 450). The
can easily be determined by the method of sulfopropylation mechanisms for these losses are unknown, and the losses cannot
combined with OPA/B-ME amino acid analysis. easily be corrected kinetically. However, for practical purposes, the
losses are usually not a problem; methionine is commonly present in
low amounts in proteins and can, therefore, be assigned accurate
DISCUSSION
residue numbers despite small errors, and tryptophan is recovered in
High performance liquid chromatographic amino acid anal- yields that are as good as, or better than, yields obtained by other
ysis using the OPA reagent is probably the most sensitive hydrolysis techniques (26,29). It is suggested that constant correction
liquid chromatographic method yet devised for studies of factors of 10% for methionine and 15% for tryptophan be applied
when using the TBP/PS/MSA method. In addition, note that the 24-
amino acid composition of proteins and peptides. In our h values for threonine and serine in column 4, Table I, are close to
studies, only 0.24 pg of hydrolyzed protein per analyzer run the theoretical values. Values for the hydroxylic amino acids probably
was sufficient to detect most of the amino acids with high need not be corrected kinetically for MSA hydrolysis times of 24 h or
accuracy (Table I). In the limit, as little as 10 to 20 ng of less.
 Fluorescence Amino Acid Analysis of Cysteine and Cystine 6251

cvsteine yields an OPA/B-ME adduct that is about 40-fold 9. Simons, S. S., Jr., and Johnson, D. F. (1978) J. Org. Chem. 43,
more fluorescent than the “half-cystine” adduct. In addition, 2886-2891
10. St. John, P. A. (1976) in Aminco Reprint No. 541, American
the sulfopropylation method is straightforward; the reduction,
Instrument Company, Silver Spring, Maryland
alkylation, and MSA hydrolysis are performed in a single 11. Schwabe, C., and Catlin, J. C. (1974) Anal. Biochem. 61,302-304
hydrolysis tube. 12. Riiegg, U. T., and Rudinger, J. (1974) Int. J. Pept. Protein Res.
The power of the combined approach of sulfopropylation, 6,447-456
MSA hydrolysis, and OPA/B-ME amino acid analysis is well 13. Hirs, C. H. W. (1956) J. Biol. Chem. 219,611-621
illustrated for human carbonic anhydrase C (Fig. 2B). It is 14. Him, C. H. W. (1967) Methods Enzymol. 11, 197-199
difficult to obtain an accurate determination of 1 cysteine in 15. Ruegg, U. T., and Rudinger, J. (1974) Israel J. Chem. 12, 391-
401
259 amino acid residues (5), especially for small amounts of 16. Sweetman, B. J., and MacLaren, J. A. (1966) Aust. J. Chem. 19,
protein. The modified sulfopropylation method reported here 2347-2354
yields both an accurate determination of the single cysteine in 17. MacLaren, J. A., and Sweetman, B. J. (1966) Aust. J. Chem. 19,
human carbonic anhydrase C and accurate determinations of 2355-2360
all of the other amino acids in the protein (Table I, column 4). 18. Ulland, B., Finkelstein, M., Weisburger, E. K., Rice, J. M., and
It is suggested that the method for micro amino acid analysis Weisburger, J. H. (1971) Nature 230, 460-461
19. Van Duuren, B. L., Melchionne, S., Blair, R., Goldschmidt, B. M.,
using OPA/B-ME presented here and in previous papers (5,6) and Katz. C. (1971) J. N&Z. Cancer. Inst. 46. 143-149
has the potential for replacing ninhydrin amino acid analysis 20. Simpson, R: J., ‘Neuberger, M. R., and Liu, T.:Y. (1976) J. Biol.
as a common biochemical techniaue. Chem. 251, 1936-1940
21. Spitz, H. D. (1973) Anal. Biochem. 56,66-73
22. Canfield, R. E. (1963) J. Biol. Chem. 238,2698-2707
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4. ReiIand, J., and Benson, J. R. (1976) in Durrum

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98,7098-7099 30. Inglis, ‘A. S., and Liu; T.-Y. (1970) J. Biol. Chemy 245, 112-116
8. Simons, S. S., Jr., and Johnson, D. F. (1977) J. Chem. Sot. Chem. 31. Inglis, A. S., McMahon, D. T. W., Roxburgh, C. M., and Takay-
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