Anal. Chem. 1985, 57, 1931-1937
1931
Isolation and Characterization of Cysteine-Containing Regions of Proteins Using 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and High-Performance Liquid Chromatography Toshimasa Toyo’oka and Kazuhiro Imai* Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1Hongo, Bunkyo-ku, Tokyo 113,J a p a n
Egg albumln, a model for thloltontalnlng protelns, was reacted wlth 4-( amlnoeulfonyl)-7-fluoro-2,l,3-benroxadlazole (ABD-F), a fluorogenlc reagent for thiols. I n the presence of 0.5% SDS at pH 8.0 and 60 O C for 1 h, all the cyaelne r W u m (no. 367,382,30,and 11) were speclflcally labeled. I n HM absence of SDS at pH 8.0 and 40 O C for 1 h, however, selective labelng of no. 387 cyaelne resklue occurred. These speclflc and Selective labellngs were conflrmed by spectroptwtometrlc compsrlson wRh SABD-hamocystehre and acid hydrolyds of the ABD-egg albumlns and by Isolation and purlflcatlon on reversedphase HPLC (monitored at fluorescence, 510 nm) and ABD-labeled peptlde fragments obtalned after aohymotrypllc digestion and thelr amlno ackl sequence analysis with a gas-phase sequencer. Appllcatlon of thls method for detection and lsolatlon of protelns and peptldes lo dlscursed.
The determination of the total amino acid sequence of an enzyme is important for the elucidation of its reaction mechanism. The partial sequence analysis is, however, also effective for knowing the environment of the active center of the enzyme, such as the cysteine-containing region of SH enzymes (1-5). Dansylaziridine, maleimide, bimane, fluorescein isothiocyanate, etc. have been used for the selective detection of cysteine residues in proteins (6-10). However, there have been few reported methods for the isolation and characterization of peptide fragments containing cysteine residues (11-14). Chang e t al. used 4-(dimethylamino)azobenzene-4’-iodoacetamide(DABIA) and 4-(dimethylamino)azobenzene-4’-N-maleimide (DABMA) for reaction with the cysteine residues of immunoglobulin light chain and isolated the labeled cysteine fragments by high-performance liquid chromatography (HPLC) for amino acid sequencing (I1,12). However, DABMA gave multiple adducta and DABIA was not easy to handle for obtaining the desired fragments (11). 4- (Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) (151, a fluorogenic reagent for the sensitive detection of low molecular weight thiols, seemed ideal for the above purpose because it reacts specifically with thiols to give one adduct under mild conditions (pH 8.0 for 10 min) (Figure 1)and the adduct is stable for a week at pH 8.0 and 5 “C. The reagent’s good solubility in water (10 mM) is also advantageous for reaction with native proteins. In this paper, we investigate whether ABD-F reacts exclusively with the four cysteine residues (16,lT)of egg albumin (mol w t 46 000, comprising 385 residues) in spite of the complex environmental matrix of the protein and whether it discriminates the cysteine(s) located around the hydrophilic region from the cysteine(s) located near the hydrophobic and/or sterically hindered regions. The isolation of the ABD-labeled peptide fragments by HPLC is also described. 0003-2700/S5/0357-1931$01.50/0
EXPERIMENTAL SECTION Materials. 4Fluoro-2,1,3-benzoxadiazolewas prepared by the method of Nunno et al. (18). 4-(Aminosu1fony1)-7-fluoro-2,1,3benzoxadiazole (ABD-F) and ABD adduct of homocysteine (SABD-homocysteine) were synthesized and purified as described previously (15). Egg albumin (crystallized five times) was a commercial product of Chemalog (New Jersey, USA). a-Chymotrypsin (67 u/mg, three times crystallized and salt free) was obtained from Worthington Biochemicals (Freehold, NJ). Cysteine hydrochloride (Ajinomoto, Tokyo, Japan) and homocysteine (Sigma,St. Louis, MO) were used. Guanidine hydrochloride, urea, trifluoroaceticacid (TFA), and sodium lauryl sulfate (SDS) were purchased from Nakarai Chemicals (Kyoto, Japan). Ethylenediaminetetraacetic acid disodium salt (EDTA 2Na) was from Kanto Chemicals (Tokyo, Japan). All other chemicals were of analytical-reagent grade and were used without further purification. Deionized, distilled water was used. Apparatus. A ‘HNMR spectrum was recorded on a JEOL Model FX-100 spectrometer at 100 MHz using tetramethylsilane (Me4Si) as an internal standard (abbreviation: s, singlet; d, doublet; m, multiplet). Ultraviolet (UV) spectra were measured with a UVIDEC 505 (JASCO, Tokyo, Japan) and a Shimadzu SPD-2A spectrophotometric detector was used with a 12-pL flow cell as an HPLC detector. A Hitachi 650-10s fluorescence spectrophotometer was used with a 1-cm quartz cell for manual method or an 18-pL flow cell for HPLC detection. UV and fluorescence measurements were operated without spectral correction. A Waters high-performance liquid chromatograph, equipped with a U6K universal injector and a 6000A pump was used. For gradient elution, another 6000A pump was connected and used under control by a 660 solvent programmer (Waters Associates). NOVA PAK ODS (150 X 3.9mm, id., 5 pm, Waters), pBondapak C18 (300 X 3.9 mm, i.d., 8-10 pm, Waters), and Zorbax-CN (250 Y. 4.6 mm, i.d., 5 pm, Du Pont) columns for HPLC were used at ambient temperature. The flow rate was 1.0 ml/min. The eluting solvent was filtered through a type HA filter (0.45 pm, Millipore, Bedford, MA) and degassed just prior to use. Temperatures for the labeling reaction and for acid hydrolysis were controlled by a BT-21 water bath (Yamato, Tokyo, Japan) or a TP-IC air bath for the GC-4B gas chromatograph (Shimadzu, Kyoto, Japan), respectively. A Model 470A gas-phase protein sequencer (Applied Biosystems) was used for the amino acid sequence analysis of the peptides. Phenylthiohydantoin (PTH) amino acids were separated and detected by an SP 8100 HPLC system (Spectra-Physics) with a UV detector. Synthesis of ABD Adduct of Cysteine (S-ABD-Cysteine). To 185 mg (0.85 mmol) of ABD-Fin 10 mL of CH,CN was added 30 mL of cysteine hydrochloride (157 mg, 1.0 mmol) in 0.1 M borate (pH 8.0, Na+). The solution was adjusted to pH 8.0 with 1N NaOH. After being heated for 20 min at 50 OC, the reaction mixture was cooled on ice water and neutralized with 1N HCl. The neutral solution was evaporated under reduced preeaure. One hundred milliliters of MeOH was added to the residue and the suspension was filtered through a type HA filter (0.45 pm, Millipore). The filtrate was evaporated in vacuo and chromatographed on a Bit-Gel P-2 column (200-400 mesh, 60 X 2 cm,eluent H20). The fluorescent fractions correspondingto S-ABD-cysteine were collected,evaporated, and recrystallized from H20 (yellow needles, yield 20%): mp 174-176 OC (uncorrected); NMR (in 0 I985 American Chemical Soclety
1932
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
&N\o'N
G N ," o
RSH
SO2 NIi2
SO2 NIi2
ABD-F
ABD-SR
Figure 1. Structure of ABD-F and Its reaction with thiols.
Me2SO-d6)6 7.93 (1H, d, J a b = 7.6 Hz, Ha), 7.61 (1H, d, J a b = 7.6 Hz, Hb), 7.71-7.94 (2 H, HJ, 3.32-3.87 (6 H, m, Anal. Calcd for CBH10N405S2"20: C, 32.14; H, 3.60; N, 16.66. Found: C, 31.95; H, 3.52; N, 16.89. U V ,A, (H20)= 375 nm; t375nm= 8160. Fluorescence in HzO: A,, = 378 nm, A,, = 502 nm. The structure of the S-ABD-cysteine was confirmed on the basis of the above analytical data and also by comparison with the NMR data of aromatic protons at the ortho position of ammonium 7-methoxy-2,1,3-benzoxadiazole-4-sulfonate (SBD-OMe) (19) or N-methylaniline. d
cool e
f
SCHz-CH-NH;
@ o;:;; SOz"2c
S-ABD-Cysteine
Stability of ABD-F in Alkaline Medium. One milliliter of 0.5 mM ABD-F in 0.1 M buffer of various pHs (pH 8.0, 9.0 and 10.0) containing 1 mM EDTA 2Na was placed in a 5-mL glass tube. The tube was capped and heated at 60 "C over 120 min. At fixed time intervals, the tube was taken out and cooled on ice water. A 20-pL portion of the solution was injected into a NOVA PAK ODS (150 X 3.9 mm, id., 5 pm) column for HPLC. The eluting solvent was CH3CN-0.1% H3P04(pH 2.2) (1090). The eluate was monitored by absorption at 280 nm and the peak height method was used for the calculation of the remaining ABD-F. Stability of ABD-Adducts in 6 N HCl. The 0.2-mL portions of 1.65 mM ABD adducts (S-ABD-cysteine or S-ABD-homocysteine) in 6 N HCl were placed in a series of glass tubes. The tubes were flushed with dried nitrogen, sealed up, and heated at 110 "C over 24 h in the air bath. At fixed time intervals, a tube was taken out and 50 pL of solution in the tube was withdrawn and diluted ten times with 0.1 M NaH2P04 (pH 4.46). Fifteen microliters of the solution was injected into a NOVA PAK ODS (150 X 3.9 mm, i.d., 5 pm) column for HPLC. The eluting solvent was CH3CN-0.1% H3P04(pH 2.2) (595). The eluate was monitored by fluorescence at 500 nm with excitation at 380 nm. The remaining ABDadduct was calculated by the peak height method in comparison with the authentic S-ABD-cysteine or S-ABDhomocysteine. Reaction of Cysteine with ABD-F in 0.1 M Borate (pH 8.0, Na+) Containing SDS or Urea or Guanidine Hydrochloride. One milliliter of 1 mM ABD-F in 0.2 M borate (pH 8.0, Na+) containing 4 mM EDTA 2Na, and an equal volume of 10 pM cysteine in H20 containing 12 M urea or 10 M guanidine hydrochloride or 1% SDS were mixed in 10-mL glass tubes (final pH, 8.0). The tubes were capped immediately and heated at 40 "C. At certain time intervals, a tube was taken out and stored in a freezer (-20 "C) until analyzed. The reagent blank without cysteine was treated in the same manner. Fifteen microliters of the solution was injected into a NOVA PAK ODS (150 X 3.9 mm, i.d., 5 pm) column for HPLC and the eluate was monitored for S-ABD-cysteine by both fluorescence at 500 nm (excitation at 380 nm) and absorption at 280 nm. The eluting solvent was CH,CN-O.l% (PH 2.2) (5~95). Fluorescence Intensities of the Authentic S-ABD-Cysteine i n 0.1 M Borate (pH 8.0, Na+) and 0.1 M Borate (pH 8.0, Na+) Containing 0.5% SDS or 6 M Urea or 5 M Guanidine Hydrochloride. The authentic S-ABD-cysteine (8.3 pM) was dissolved in 0.1 M borate (pH 8.0, Na+) and 0.1 M borate containing 0.6% SDS or 6 M urea or 5 M guanidine hydrochloride. The fluorescence intensities and the maximal wavelengths of the solutions were measured. Reaction of Egg Albumin with ABD-F in 0.1 M Borate (pH 8.0, Na+) with or without 0.5% SDS. To a 10-mL glass tube were added 1.0 mL of ABD-F (1mM) in 0.1 M borate (pH
8.0, Na') containing 1% SDS and 1.0 mL of 2.0 pM egg albumin in 0.1 M borate (pH 8.0, Na+) containing 4 mM EDTA 2Na. The tube was vortex mixed, capped, and heated at various temperatures (40-60 "C) in the water bath. At fixed reaction time intervals, the tube was taken out and cooled on ice water. The fluorescence intensities were measured at ambient temperature with emission at 480 nm (excitation at 390 nm). The net fluorescence intensities were calculated by the sample fluorescence minus blank fluorescence (obtained from the reaction with ABD-F without egg albumin). The treatment of egg albumin with ABD-F in 0.1 M borate (pH 8.0, Na+) without SDS was performed in the same manner as described above. Effect of ABD-F Concentration on the Reaction with Egg Albumin. A l-mL volume of 0.2 pM egg albumin in 0.1 M borate (pH 8.0 Na+) containing 4 mM EDTA 2Na and an equal volume of various concentrations of ABD-F (0.1-1.0 mM) in 0.1 M borate (pH 8.0, Na') containing 1% SDS was mixed in glass tubes. The tubes were capped immediately and heated at 50,"C. At certain time intervals, the tube was taken out and the net fluorescence intensities were measured by the same method mentioned above. Preparation of ABD-Egg Albumins (ABD1-, ABDLB-, ABD3.5(SDS)-, and ABD4(SDS)-Egg Albumin). To 10 mL of ABD-F (20 mg) in 0.1 M borate (pH 8.0, Na+) containing 2 mM EDTA 2Na with 0.5% SDS (or without SDS) was added 5 mg of egg albumin. The mixture was allowed to stand at a certain temperature (40-60 "C). To remove the excess amount of ABD-F and/or SDS, the solution was transferred into a cellulose tube (16 mm diameter) and was dialyzed at 3 f 1"C for 3 days against six changes of 6 L of deionized, distilled water. Then ABDl-egg albumin (sample treated without SDS at 40 "C for 1 h), ABD1.5-egg albumin (sample treated without SDS at 60 "C for 1h), ABD3.5(SDS)-egg albumin (sample treated with 0.5% SDS at 40 "C for 5 min), and ABD4(SDS)-eggalbumin (sample treated with 0.5 % SDS at 60 "C for 1h) were lyophilized. These samples were used for further analysis. Acid Hydrolysis of ABD-Egg Albumins. A few milligrams each of ABD-egg albumins dissolved in 0.4 mL of 6 N HC1 was added to the glass tube into which was bubbled the dried nitrogen to remove the air. The tube was sealed and allowed to stand at 110 "C for 24 h in the air bath. After the reaction, 10 pL of the solution was injected into a NOVA PAK ODS (150 X 3.9 mm, i.d., 5 pm) column for HPLC. The eluting solvent was CH3CN0.1% H3P04 (pH 2.2) (5:95) and the eluate fluorescence was monitored at 500 nm with excitation at 380 nm. Spectrophotometric Determination of the Labeled ABD Content in Egg Albumins Treated with ABD-F. ABD-egg albumins (ABD1-, ABD1.5-, ABD3.5(SDS)-, and ABD4(SDS)+gg albumin, about 1mg each) were dissolved individually in 2.0 mL of 0.025 M phosphate buffer (pH 6.86). The absorption spectra of the solutions were recorded on a UVIDEC 505 spectrophotometer. The amounts of the ABD contents of ABD-egg albumins were estimated based on their absorption at 280 nm and 385 nm. The molar absorptivities (e) used for those calculation were 33 800 at 280 nm for native egg albumin (20) and 3000 at 280 nm and 7800 at 385 nm for S-ABD-homocysteine. a-Chymotryptic Digestion of ABD-Egg Albumins and Isolation of ABD-Labeled Peptides. The experimental procedure is shown in Figure 2. Five hundred micrograms of ABD4 (SDS)-egg albumin, ABD3.5(SDS)-egg albumin, ABD1.5-egg albumin, and ABD1-egg albumin dissolved in 500 pL of 0.1 M borate (pH 8.0, Na+) containing 500 pg of a-chymotrypsin were digested at 37 "C for 24 h. After digestion, 5 p L of the solution was subjected to HPLC. HPLC conditions are as follows: column, pBondapak CI8 (300 X 3.9 mm, id., 8-10 pm); elution, linear gradient elution from CH&N-H20 (5:95) containing 0.1% TFA to CH3CN-H20 (8020) containing 0.1% TFA over 60 min (0100%);flow rate, 1.0 mL/min, fluorescence detection, A, 385 nm, A, 510 nm; UV detection, 210 nm. The ABD-labeled peptides separated by the above HPLC system were further purified on two types of columns, Zorbax-CN and pBondapak CIS The most suitable eluents were selected for each peptide by considering its separation from the interfering peaks with monitoring at 210 nm. Sequence Analysis of ABD-Labeled Peptides. The amino acid sequence of the ABD-labeled peptides isolated was determined by a Model 470A gas-phase protein sequencer (Applied
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
500 500
fg
1933
ABD-egg albumins in 0.5 m l of 0.1 M borate ( p H 8.0)
Pg d-chymotrypsin
1
37OC, 2 4 hr
0.1 ml, HPLC separation
r
co1umn:r-Bondapak C18(300x3.9mm, i.d.,8-10 m ) ; gradient e1ution:CH CN-H 0(5:95) containing 0.1 % TFA t o C H 3 C N - H 2 0 3 2 (80:20) containing 0.1 % TFA over 60 min ( 0 t o 100 % ) ; flow rate:l.O ml/min
i
peak 1
&
J.
peak 2
peak 3
peak 4 ;
column: Zorbax-CN (250x4.6mm, i.d.,
1
MeOH-H20(35:65) -0.1 % TFA
isocratic elution
1
I
I
I
I
I
MeOH-H20(35:65) -0.1 % TFA
MeOH-H20(60:40) -0.1 % TFA
MeOH-H20(60:40)
-0.1
%
TFA
column: r-Bondapak Cl8(300x3.9mm, i.d., 8-10pm); isocratic elution
I
CH3CN-H20(10:90) -0.1 % TFA
MeOH-H20(35:65) -0.1 % TFA
MeOH-H20(50:50) -0.1 % TFA
determination of amino acid sequences
CH3CN-H20(30:70)
-0.1
%
TFA
Figure 2. Isolation procedures of ABD-labeled peptides. ~~
Table I. Stability of ABD-F in Alkaline Medium" time, min 0
10 30 60 120
pH 8.0
pH 9.0
100 100 100.5 100.4 100.2
100 100.4 100.3
97.7 96.7
pH 10.0 100
99.7 99.5 97.5 95.4
"0.5 mM ABD-F in 0.1 M borate (pH 8.0, 9.0, and 10.0) containing 1 mM EDTA 2Na was reacted at 60 'C. A 20-pL portion of the solution was injected into a NOVA PAK ODS (150 X 3.9 mm, i.d., 5 Mm) column: eluent, CH,CN-O.l% H3P04 (pH 2.2) (1090): UV detection, 280 nm; flow rate, 1.0 mL/min.
Biosystems). The HPLC conditions were as follows: column, SEQ-4 (C8-silica,300 X 4.6 mm, i.d., 7 wm, Sensyu Scientific); flow rate, 1.0 mL/min; UV detection, 269 nm; eluent, (A) CH3CN,(B) 40 mM acetate buffer (pH 4.9, Na+), (C) H20. The following stepwise linear gradient elutions (AB:C, volume ratio) were performed initial condition (361054),from 0 to 3 min (42:1048), from 3 to 4 min (42:15:43), from 4 to 5 rnin (5015351, from 5 to 9 rnin (52:15:33), and from 9 to 18 rnin (52:15:33).
" 0 P P
?
0
50-
2 0
6
12
24 hr
Figure 3. Stability of ABD adducts in 6 N HCI at 110 OC: (0)SABD-cysteine; ( 0 )S-ABD-homocysteine; concentrations of ABD adducts, 1.65 mM each. HPLC conditions were as follows: column, NOVA PAK ODS (150 X 3.9 mm, Ld., 5 pm); eluent, CH,CN-0.1% H,PO, (pH 2.2) (595); fluorescence detection, A,, 380 nm, ,A, 500 nm; flow rate, 1.0 mL/min. I
RESULTS Stability of ABD-F i n Alkaline Medium. As shown in Table I, ABD-F at 60 "C and 0.1 M borate (pH 8.0, Na+) for 120 min was scarcely decomposed, and even at pH 10.0 the degradation was less than 5%. However, in the experiment hereafter, the reaction was performed at less than 60 OC and pH 10.0 for less than 120 min. Stability of ABD Adducts in 6 N HCl. As depicted in Figure 3, ABD adducts were gradually decomposed in 6 N HCl at 110 "C with the increment of the reaction time, but the decompositions were about 10% after heating at 110 "C for 24 h. Influence of D e n a t u r a t i o n Reagents (Urea and Guanidine Hydrochloride) o r Detergent (SDS) on the
'I0/
Y 10 20 30 min
0
Flgure 4. Time courses of the reaction of cysteine with ABD-F in the absence or presence of 0.5% SDS, 6 M urea, and 5 M guanidine hydrochloride: (0)pH 8.0; (A)6 M urea; (V)5 M guanidine hydrochloride; (0)0.5% SDS. The 5 wM cysteine and 500 pM ABDF were reacted at 40 "C and pH 8.0 (2 mM EDTA 2Na). HPLC conditions were as follows: column, NOVA PAK ODs (150 X 3.9 mm, i.d., 5 pm); eluent, CH,CN-0.1 % H3W4(pH 2.2) (5:95); injection amount, 15 pL; fluorescence detection, A, 380 nm, , A , 500 nrn; flow rate, 1.0 mL/mln.
1934
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
NFI
L&L
0
5
10
min
0
5
0
10
5
min
10
min
Figure 5. Chromatograms obtained from the reaction of cysteine with ABDF in the absence or presence of 0.5% SDS. Reaction procedure and HPLC conditions were the same as those of Figure 4 (a, S-ABDcysteine): A, reaction for 20 min in the absence of SDS; B, reaction for 20 min in the presence of 0.5% SDS; C, reaction for 20 min without cysteine for 20 min In the presence of 0.5% SDS.
Table 11. Maximal Fluorescence Wavelengths and Fluorescence Intensities of S-ABD-Cysteine in Denaturation Reagents and a Detergento A,.
pH 8.0 only 0.5% SDS 6 M urea 5 M guanidine HCI
nm
ex
em
RFIb
378 378 381 385
504 504 503 506
100 125 198 22
"8.3 fiM of S-ABD-cysteine was dissolved in pH 8.0 solution containing 0.5% SDS, 6 M urea, or 5 M guanidine HC1. *Fluorescence intensity in pH 8.0 solution was arbitrarily taken as
Flgure 6. Time courses of the net fluorescence intensities obtained from cysteine with ABD-F in the absence or presence of 0.5% SDS: (0)60 OC with 0.5% SDS; (V)50 OC with 0.5% SDS (0)40 OC with 0.5% SDS; (0)60 OC without SDS; (V)50 OC without SDS; (B) 40 OC without SDS. The 1 pM egg albumin and 500 pM ABD-F were reacted in 0.1 M borate (pH 8.0, 2 mM EDTA 2Na). Fluorescence detection was A, 390 nm and A, 480 nm.
4
NFI
I
100.
Formation of S-ABD-Cysteine. As shown in Figure 4, the reaction of cysteine with ABD-F in 0.1 M borate (pH 8.0, Na+) without denaturation reagents such as urea and guanidine hydrochloride and a detergent (SDS) was completed in 5 min. That was almost true for the reaction with the addition of the denaturation reagents to the buffer. In case of the reagent blank without cysteine, any formation of ABD adduct with the denaturation reagents or a detergent was observed judging from their corresponding chromatograms (Figure 5). Effects of 0.5% SDS, 6 M Urea, and 5 M Guanidine Hydrochloride (Final pH, 8.0 Each) on Fluorescence Intensities of 5-ABD-Cysteine. The addition of 6 M urea to the medium containing S-ABD-cysteine approximately doubled the fluorescence intensity (Table 11). The fluorescence intensity was lowered in the case of 5 M guanidine hydrochloride addition. This decrement seems not to be attributed to the decomposition of S-ABD-cysteine with guanidine but to the quenching effect of chloride ion on the fluorescence, since the original amount of S-ABD-cysteine remained, judging from the HPLC chromatogram. SDS had no effect on the intensity. Considering the above results the reaction buffer containing 0.5% SDS (final pH, 8.0) was used in the subsequent work. Optimization of the Labeling Reaction of Egg Albumin with ABD-F. The fluorescence intensities of the reaction medium of egg albumin with ABD-F were increased with increasing reaction time (Figure 6). Remarkable enhancement of fluorescence intensities in 0.5% SDS compared with those without SDS at any temperature was observed. It was shown that the labeling reaction of egg albumin was completed in 10 min at 60 "C in 0.1 M borate (pH 8.0) with 0.5% SDS. Without SDS, the reaction proceeded only up to a certain level. As shown in Figure 7, an excess amount of ABD-F (2500-fold) was also necessary for the completion of labeling of the egg albumin. These behaviors were different from the labeling of low-molecdar-weight thiols such as cysteine (Figure 4).
0
.//:
1 'O
min
60
Figure 7. Effect of ABD-F concentration on the reaction with egg albumin: (ABDF concentrations) (B) 0.25 m M (0)0.50 mM; (0)1.00 mM. The 0.1 pM egg albumin and ABD-F was reacted at 50 OC and in 0.1 M borate (pH 8.0, 2 mM EDTA 2Na).
Wavelength (nm)
Figure 8. Absorption spectra of native egg albumin, ABD-homocysteine and ABD-egg albumins: (0)ABDYSDS)-egg albumin (about 17 pM); (A)ABD1-egg albumin (about 20 pM); ('I S-ABD-homo) cysteine (about 70 pM); (B) egg albumin (about 23 pM). Absorption spectra were measured in 0.025 M phosphate (pH 6.86).
Absorption and Fluorescence Spectra of ABD-Egg Albumins and Determination of ABD Content in ABDEgg Albumins. Figure 8 shows the absorption spectra of S-ABD-homocysteine, native egg albumin and ABD1- and ABD4(SDS)-egg albumins. The absorption spectra from 300
ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
0
2o min
w a v e l a ~ t h(nm)
Figure 9. Excltation and emission spectra of S-ABD-homocystelne and ABD4(SDS)-egg albumin: (0) S-ABD-homocysteine; (-)
B
ABD4(SDS)-egg albumin. Fluorescence spectra were measured in 0.025 M phosphate (pH 6.86). nm to 500 nm of ABD-egg albumins were almost superimposable on that of S-ABD-homocysteine (Figure 8). SABD-cysteine was not used as a standard ABD adduct since it absorbs maximally at 375 nm. Therefore, e = 7800 at 385 nm of the authentic S-ABD-homocysteine in 0.025 M phosphate buffer (pH 6.86) was used as the unit of ABD moiety for the calculation of the ABD content in ABD-egg albumins. For determination of the amount of egg albumin in ABD-egg albumins, t = 36800 for 1 ABD labeled egg albumin at 280 nm estimated by the sum of 33 800 (of egg albumin) (20)plus 3000 (of S-ABD-homocysteine) at 280 nm was used. On the basis of these assumptions, the labeling number of ABD for ABD4(SDS)-egg albumin, ABD3.5(SDS)-egg albumin, ABD1.5-egg albumin, and ABDl-egg albumin were calculated as 4.2, 3.5, 1.5, and 1.1,respectively. The emission spectrum of ABD4-egg albumin was blue shifted about 30 nm compared with that of S-ABD-homocysteine (Figure 9), so that the fluorescence characteristics were not used for calculation of the labeling numbers. Acid Hydrolysis of ABD-Egg Albumins. Since ABD adducts were stable in strong acid and a t high temperature (Figure 3), ABD-egg albumins were hydrolyzed in 6 N HC1 a t 110 "C for 24 h. No other fluorescent peaks except for the peak corresponding to S-ABD-cysteine appeared on the HPLC chromatograms of the hydrolysate of ABD-egg albumins. a-Chymotryptic Digestion of ABD-Egg Albumins and Amino Acid Sequence Analysis of ABD-Labeled Peptide Fragments. Initially, the ABD4(SDS)-egg albumin was digested with a-chymotrypsin at a low enzyme-to-substrate ratio (1:100, by weight). About eight fluorescent peaks appeared on the chromatogram after 2 h and seven main peaks after 24 h digestion. For complete digestion an equal amount of a-chymotrypsin and the substrate was used in the next experiment. As depicted in Figure lOA, four main fluorescent peaks were observed after 24 h of digestion. There was no change in peak numbers on further digestion. On the other hand, only one peak was obtained from the ABD1-egg albumin after 24 h of digestion (Figure 10B). The digestion of ABD3.5(SDS)-egg albumin produced the same four main fluorescent peaks as no. 1-4 for ABD4(SDS)-egg albumin (Figure 10A) but the peak heights for no. 2, 3, and 4 were almost half that of peak no. 1. The same trend (peak no. 1 was largest) was also true for the ABD1.5-egg albumin. When the amino acid sequences of the ABD-labeled peptides isolated from the enzymic digestion of ABD-egg albumins were subjected to the Edman degradation method, the S-ABD-cysteine PTH derivative which eluted between PTH-proline and PTH-tryptophan on the chromatogram under the HPLC condition as described in the Experimental Section was obtained (details will appear elsewhere). As shown in Table 111, each of these peptides (corresponding to peaks
I
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I
Chromatograms of ABD-labeled peptides obtained from a-chymotryptic digestion of ABD-egg albumins. ABD-egg albumin (500 pg) dissolved in 500 pL of 0.1 M borate (pH 8.0) was digested with a-chymotrypsin (500 pg) at 37 "C for 24 h: (A) ABD4(SDS)-egg albumin; (B) ABD1-egg albumin. HPLC conditions were as follows: column, pBondapak C,* (300 X 3.9 mm, i.d., 8-10 pm); gradient eluention from CH,CN-H,O (5:95) Containing 0.1 % TFA to CH,CN-H20 (80:20) Containing 0.1 % TFA over 00 min (0-100%); fluorescence 1 380 nm and A, 510 nm; flow rate, 1.0 mLlm1n. detection, , Figure 10.
Table 111. Amino Acid Sequences of ABD-Labeled Peptides Obtained from a-Chymotryptic Digestion of ABD4(SDS)-Egg Albumin fraction (peak no.)
amino acid sequence
residue no.
1 2 3 4
Cys-Ile-Lys Gly-Arg-Cys-Val-Ser-Pro Cys-Pro-Ile-Ala-Ile-Met Cys-Phe-Asp-Val-Phe
367-369 380-385 30-35 11-15
no. 1-4) contained one cysteine residue having different sequences which coincided with the primary structures of those fragments (no. 367-369,380-385,30-35, and 11-15) reported before (16,17).The sequence of the peptide corresponding to peak no. 1 for ABD1-egg albumin (Figure 10B) was the same as that for no. 1 for ABD4(SDS)-egg albumin (Figure 1OA). They were not derived from cystine residue (no. 73 and no. 120) (16,17), which means that any cleavage of S-S bond in protein did not occur. From the above results, it was demonstrated that all of the ABD-labeled peptides contain cysteine residues and the most reactive cysteine of egg albumin was the 367th residue near the N-terminus.
DISCUSSION ABD-F was found to react with egg albumin, a model protein for thiol containing proteins, under relatively drastic conditions for the quantitative labeling of the protein (with the excess reagent of 2500 times a t pH 8.0 and at 60 "C for 1h in the presence of SDS) as compared with the conditions (pH 8.0 at 40 "C for 5 min) selected for the complete reaction of the low molecular weight thiols such as cysteine and glutathione (15). The necessity for such drastic conditions might be ascribed to the hindrance caused by the environment near the cysteine residues located in the large protein molecule. For quantitation or staining of proteins, total labeling with ABD-F might be required. In such cases, the reaction conditions with ABD-F mentioned above are recommended, since the quantitative labeling in the absence of SDS was ineffective even at the elevated temperatures or by prolongation of the reaction times. Although dassylaziridine, maleimide, bimane, and fluorescein isothiocyanate were reported to label selectively the
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 9, AUGUST 1985
cysteine residues in proteins, the clear evidence of the specific labeling was not presented (6-10). On the other hand, in this work, it was demonstrated that the reaction of ABD-F was specific to thiols of four cysteine residues of the protein according to the following evidence: (1)The absorption spectra of ABD-egg albumins were the same as that of S-ABDhomocysteine (Figure 8). (2) The excitation and emission spectra are similar to those of S-ABD-homocysteine (Figure 9), although a slight blue shift of the emission wavelength was observed. (3) The ABD-egg albumins released S-ABD-cysteine after their hydrolysis with 6 N HC1. (4)The four different ABD-labeled peptides and one ABD-labeled peptide obtained from a-chymotryptic digestion of the respective ABD-egg albumins (ABDI(SDS)- and ABD1-) contained four S-ABD-cysteine residues (no. 11, 30, 367, and 382) and one S-ABD-cysteine residue (no. 367), respectively, judging from sequence analysis by the Edman degradation method (21,22). Thus,ABD-F reacts specifically with thiols of cysteine residues to produce a fluorophore, but not with the S S group of cystine nor with the N-terminal and/or lysine t-amino groups present in the complex matrix of the protein. This feature of ABD-F is worthy to note, since under certain conditions, the maleimide-type reagents such as N-ethylmaleimide are reported to react with the amino acids other than cysteine such as lysine and histidine (23-25). ABD-F reacted selectively with one cysteine residue (no. 367) of the four residues of the egg albumin under the mild condition near the native state (40 "C at pH 8.0). This cysteine residue of egg albumin might be located around the hydrophilic region of the protein and therefore be easily attacked by the reagent while the remaining three residues might be located near the hydrophobic and/or sterically hindered region. The observation of the blue shift (about 30 nm) of the emission wavelength for ABD4(SDS)-egg albumin (Figure 9) has no bearing on the hydrophobicity of the three residual cysteines since the ABD moiety is not significantly affected by the hydrophobicity of its environment (15). Therefore, the mechanism of the selective labeling of the cysteine residue (no. 367) remains to be elucidated. Machida et al. reported approximately 0.6-0.7 mol of BIP-succinimide or DACsuccinimide moiety was incorporated per mol of egg albumin in labeling with BIPM (26) or DACM (27) without SDS, respectively. However, the selective labeling of the fixed cysteine residue was not mentioned. It may be that the rapid decomposition of BIPM or DACM (half lifetimes at 30 "C in phosphate buffer, pH 7.0 was 60 min or 115 min) (28) as compared with ABD-F (degradation lower than 5% at 60 "C and pH 10.0 for 2 h) makes it difficult to control the reaction conditions. As far as we know, this is the first finding to suggest that the fixed cysteine residue (no. 367) of the egg albumin is located in a different environment than the other three residues. When egg albumin was reacted at an elevated temperature such as 60 "C for 1h without SDS or with SDS for 5 min, the respective labeled ABD content in ABD1.5-egg albumin or ABDB.B(SDS)-egg albumin calculated by the absorption method was 1.5 mol/mol or 3.5 mol/mol of egg albumin. They released with a-chymotryptic digestion one S-ABD-cysteine (no. 367) containing peptide and a less amount of almost equal ratio of three other cysteine (no. 11,30, and 382) containing peptides judging from their HPLC chromatograms. Therefore, the selective labeling among the remaining three cysteine residues was not observed both in the absence of SDS in the reaction medium at the elevated temperature (40-60 "C) and in the presence of SDS in the shorter period (1 h to 5 min). Thus, under the present state of our experimental conditions, the differentiation of the remaining three cysteine residues is not possible.
Since the ABD moiety has a longer fluorescence maximum wavelength (A, 500 nm) as compared with other fluorophores such as that derived from BIPM (26), the intrinsic fluorescences of tryptophan (Aern 365 nm, A, 285 nm) and tyrosine (A,, 310 nm, A,, 275 nm) (29) residues in proteins do not interfere with detection of the ABD label. Therefore, based on the fluorescence of ABD moiety, the S-ABD-labeled peptides were selectively and sensitively (picomole level, data not shown) detected during HPLC in the presence of the many other non-cysteine-containingpeptides. This high sensitivity would permit the isolation of cysteine-containing peptides from nanogram levels of the original protein, if dilution in each sampling step is reduced. ABD-labeled peptides isolated on the basis of fluorescence could be subjected to the sensitive amino acid composition analysis with use of 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) (30,31) or o-phthalaldehyde (OPA) (32, 33) after 6 N HC1 hydrolysis. However, in the present experiment, they were sequenced directly by the Edman degradation method owing to the easy detection of PTH S-ABD-cysteine after degradation. In conclusion, ABD-F is useful not only for the sensitive detection of low molecular weight thiols but also for the specific and selective labeling and characterization of cysteine residues of proteins, and the isolation of trace levels of cysteine containing peptides and proteins.
ACKNOWLEDGMENT The authors thank T. Nakajima, University of Tokyo, for his valuable suggestions and discussion. Thanks are also due to A. Mukunoki and Y. Sengoku of Japan Scientific Instrument Co. for the amino acids sequence analysis, Y. Watanabe of Chugai Pharmaceutical for NMR measurement, and C. K. Lim of Clinical Research Center for his kind suggestion on the preparation of the manuscript. Registry No. ABD-F,91366-65-3;S-ABD-cysteine,91366-71-1; S-ABD-homocysteine,91366-66-4; CysIleLys, 96760-54-2; GlyArgCysValSerPro,96760-55-3; CysProIleAlaIleMet, 96791-07-0; CysPheAspValPhe, 96760-56-4; L-cysteine, 52-90-4. LITERATURE CITED Axelsson, K.; Mannervik, B. FEBS Lett. 1983, 752, 114-1 18. Francis, G. L.; Bailard, F. J. Biochem. J . 1980, 186, 581-590. Francis, G. L.; Ballard, F. J. Biochem. J . 1980, 186, 571-579. Macdonald, M. J. Biochim. Biophys. Acta 1980, 615, 223-238. Shimazu, T.; Tokutake, S.; Usaml, M. J . Biol. Chem. 1978, 253, 7378-7382. Scouten, W. H.; Lubcher, R.; Baughman, W. Biochim. Biophys. Acta 1974, 336, 421-426. Yamamoto, K.; Sekine, T.; Kanaoka, Y. Anal. Biochem. 1977, 79, 83-94. Klasen, E. C. Anal. Biochem. 1982, 121, 230-233. Kosower, N. S.; Kosower, E. M.; Newton, G. L.;Ranney, H. M. R o c . Natl. Acad. Sci. U . S . A . 1979, 76, 3382-3386. Wilderspin, A. F.; Green, N. M. Anal. Blochem. 1983, 732, 449-455. Chang, J.-Y.; Knecht, R.; Braun, D. G. Biochem. J . 1983, 211, 163-171. Dodt, J.; Muller, H.-P.; Seemtiller, U.; Chang, J.-Y. FEBS Lett. 1984, 165, 180-184. Egorov, T. A.; Sevenson, A.; Rqden, L.; Carlsson, J. R o c . Natl. Acad. SCi. U . S . A . 1975, 72, 3029-3033. Svenson, A.; Carlsson. J.; Eaker, D. FEBS Lett. 1977, 73, 171-174. Toyo'oka, T.; Imai, K. Anal. Chem. 1984. 56, 2461-2464. Nisbet, A. D.; Saundty, R. H.; Moir, A. J. G.; Fotherglli, L. A.; Fothergill, J. E. Eur. J . Biochem. 1981, 115, 335-345. McReynolds, L.; O'Malley, B. W.; Nisbet, A. D.;Fotherglll, J. E.; Givol, D.: Fleids. S.: Robertson. M.: Brownlee. G. G. Nature (London) 1978, 273, 723-728. Nunno. L. D.; Florio, S.; Todesco, P. E. J . Chem. SOC. C 1970, 1433-1 434. Toyo'oka, T.; Imal, K. Analyst (London) 1984, 109, 1003-1007. Cunningham, L. W.; Nuenke, J. J . Biol. Chem. 1959, 2 3 4 , 1447-1 451. Muramoto. K.: Kawauchi, H.; Tuzimura, K. Agric. Biol. Chem. 1978, 42, 1559-1563. Muramoto, K.; Kamlya, H.; Kawauchl, H. Anal. Biochem. 1984, 141, 446-450. Smyth, D. G.; Nagamatsu, A,; Fruton, J. S. J . Am. Chem. SOC. 1980, 82, 4600-4604. Guidottl, G.; Konigsberg, W. J . Biol. Chem. 1984, 239, 1474-1484.
Anal. Chem. 1985, 57, 1937-1941 (25) Brewer, C. F.; Riehm, J. P. Anal. Biochem. 1967, 78, 248-255. (26) Machida, M.; Sekine, T.; Kanaoka, Y. Chem. fharm. Bull. 1974, 22, 2642-2649. (27) Machlda, M.; Machida, M. I.; Sekine, T.; Kanaoka, Y. Chem. fharm. Bull. 1977, 25, 1678-1684. (28) Machida, M.; Machida, M. 1.; Kanaoka, Y. Chem. fharm. Bull. 1977, 25, 2739-2743. (29) Dnggan, D. E.; Bowman, R. L.; Brodie, B. 6.; Udenfriend, S. Arch. Blochlm. Blophys. 1957, 68, 1-14. (30) Imai, K.;Watanabe, Y. Anal. Chlm. Acta 1981, 130, 377-383.
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(31) Watanabe, Y.; Imai, K. J. Chromatogr. 1982, 239, 723-732. (32) Benson, J. R.; Hare, P. E. R o c . Natl. Acad. Sci. U . S . A . 1975, 72, 6 19-622. (33) Jones, B. N.; Gilligan, J. P. J. Chromatogr. 1983, 266, 471-482.
RECEIVED for review March 15,1985. Accepted April 24,1985. Presented in part at the 105th Annual Meeting of the Pharmaceutical Society of Japan, Kanazawa, April 3-5,1985.
Determination of Trimethylselenonium Ion in Urine by Ion-Exchange Chromatography and Molecular Neutron Activation Analysis Alan J. Blotcky and Gregory T. Hansen Medical Research, V.A. Medical Center, Omaha, Nebraska 68105
Laura R. Opelanio-Buencaminoand Edward P. Rack* Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304
A method has been developed for the determlnatlon of the trlmethylselenonlum Ion (TMSe) In urine by anion exchangecation exchange chromatography, seiectlveiy capturing the TMSe on the cation exchange resin. TMSe recoverles are 91.9 f 7.6%. The TMSe fractlon is lrradlated wlth neutrons and radloassayed for 77mSeactivity. Varlous experlmental procedures Including acld hydrolysls, nltrlc acld digestlon, and HPLC were evaluated In order to optlmlze the determination.
The importance of trace selenium in living biological systems has been demonstrated in the literature (1-5). It has been shown that selenium is not only a vital micronutrient but is also a toxic agent at excessive levels. There is a narrow range of selenium intake that is consistent with health; outside of this narrow range, deficiency diseases and toxicity occur. Because of its biological importance various analytical procedures have been developed for analysis of microquantities of elemental selenium in serum and tissue (6,7). For urine these include atomic absorption spectrometry, solution absorption spectrometry, solution fluorescence spectrometry, volumetry, and neutron activation analyses (6-8). There is a paucity of the number of total selenium urine values in normal and disease states. Valentine et al. (8)found selenium urine concentrations with a mean value of 79.3 f 38.7 pg L-l for a sample size of 35 subjects, They suggest that selenium urine values correlate well with selenium water intake while serum values do not. The most promising technique for total selenium in urine appears to be neutron activation analysis (NAA) employing 75Se = 120 days), %e = 18 min) (9,lo),and 77mSe(Tl/z = 17.6 s) (11,12).The use of the 77mSe isotope is advantageous in that a large number of samples can be analyzed routinely if a nondestructive technique is employed. The metabolism of selenium in living organisms is undoubtably quite complex and the form of selenium which occurs within the living system depends on the form supplied (13).The possible metabolic interrelationship between organic
and inorganic forms of selenium has been described in the literature (14). The biological mechanisms mostly involve reduction and methylation. While little is known about the pathways by which the different forms of selenium are metabolized to trimethylselenonium (TMSe), they must involve a detoxification mechanism (15-19),TMSe is an important urinary metabolite at doses of selenite insufficient to trigger the respiratory excretion of dimethyl selenide (DMSe). Previous techniques to measure TMSe levels in urine involve the use of the radiotracer 75Se(9,15-19). Its use is limited because of the relatively long biological half-life of the selenium isotope and the associated issues of radiation exposure as well as constraints associated with handling of radioactivity. Several methods have been developed for the determination of total selenium and TMSe in urine. Nahapetian (19)employed a modification of the procedure described by Janghorbani (10) for the total determination of urine selenium. The selenium underwent several wet oxidation steps to Se(1V) with subsequent precipitation with ammonium pyrrolidinecarbodithioate (APDC) and its content measured either by fluorometric means or by neutron activation analysis. Palmer et al. (17) were the first to identify TMSe as a major excretory product in urine. These authors employed a cation-exchange-paper chromatography method for the separation and identification of 75Se-labeledTMSe. Nahapetian (18)employed either a modification of the Palmer method (16,17) for 75Se-labeled TMSe or anion exchange-cation exchange chromatography with subsequent wet oxidation conversion to Se(1V) and APDC precipitation. It was our intent to simplify the procedures for total selenium and TMSe determination in urine employing ITmSeactivation. A molecular neutron activation analysis procedure (MoNAA) such as that developed by this laboratory for the determination of iodoamino acids and hormonal iodine (20) and chlorinated pesticides (21) in a urine matrix has definite advantages over radiometric and fluorometric techniques. By use of 77mSeactivation the parts per billion (ppb) range can be readily attained. The major purpose of this study is to evaluate various separation procedures such as ion exchange
0003-2700/85/0357-1937$01.50/0 0 1985 American Chemical Society
